upqm 90a .0 - 5 J-zx. mm 91:1. .1 .1 1:5) .1} 5 a; 1.1!: I 11.... .la‘. .. .313! 1.! 1: . :zlarrl .2141?! i.) . .0 2:. 1. it’s Sill‘l 1 1259.5: .mmnmfiinfitmf yawn it! nhzmpu§é . L fir... r. 2. ‘Mwfim um. I... an; 2..., .3: h. a. v»: . A {$.an ..\’Ix X .0: z . it! 5.53.... ‘ .. 1.1.2:..ka o z?! .. 3...... 3.2.. so: Mfihj. hwmafihflfi. up... .53.... THESlS p N LIBRARIES ns'lll‘lllllllll MICHIGAN STATE UNIVE\ \ \ l \ \ll \l\\\\\\\l\l\\\\l1\l ll 312930 561 1084 l LIBRARY ‘ Michigan State University This is to certify that the dissertation entitled Subcellular Localization of PGH Synthase-l and -2, and Cytosolic Phospholipase A2 presented by Martha K. Regier has been accepted towards fulfillment of the requirements for Ph. D. degree in Biochemistry 2/1/14; 1, fix Major professor Date Ma’y/Z/QZS/ MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE II RETURN BOX to remove thie checkout from your record. TO AVOID F INES return on or before dete due. DATE DUE DATE DUE DATE DUE it’lfi.’ g g 7131 l I MSU leAn Affirmative Action/Emmi Opportnnhy lnetituion Wane-9.1 SUBCELLULAR LOCALIZATION OF PGI'I SYNTHASE-l AND -2, AND CYTOSOLIC PHOSPHOLIPASE A2 By Martha K. Regier A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1995 ABSTRACT SUBCELLULAR LOCALIZATION OF PGH SYNTHASE-l AND -2, AND CYTOSOLIC PHOSPHOLIPASE A2 By Martha Kay Regier PGH synthase-l and -2 catalyze the conversion of arachidonic acid to prostaglandin Hz, the precursor for synthesis of biologically active prostanoids. One source of substrate arachidonic acid is provided by cytosolic phospholipase A2 hydrolysis of membrane phospholipids. Using immunocytochemistry and confocal microscopy we have investigated several aspects of PGH synthase—l and -2 and cytosolic phospholipase A2 subcellular localization. The C-terminal sequence of PGH synthase—l and-2 from a variety of species is -P/STEL, which is similar to the C-terminal sequences of a number of proteins which are localized to the endoplasmic reticulum by the -I O OUJ O O O O) [x g o; o o c or or (D to CD to In In ACTIVITY (0 .12 _’ 4 _‘ w , cox 0 100 20:1 17:9 0 24:1 16:5 P0X 0 100 16:5 9:4 0 10:1 13:2 72— - _ - _ — 63— — Figure 9. Enzyme activity and expression of ovine PGH synthases-1 having carboxyl terminal mutations. Native and mutant ovine PGH synthases-1 were expressed in cos-1 cells, and microsomes were prepared for assays of cyclooxygenase and peroxidase activities (upper panel) and SDS PAGE/ Western blotting (lower panel) as described in the text. Cyclooxygenase (COX) and peroxidase (POX) activities are expressed as a percentage of native enzyme activities. Cyclooxygenase activities were determined from two to seven separate transfections; peroxidase activities from one to three separate transfections. In different experiments, cyclooxygenase and peroxidase activities of native PGH synthase ranged from 213 to 269 nmol 20:4/ min/ mg and 274 to 344 nmol HzOz/min/ mg, respecitvely. The anti-peptide antibodies used for Western blotting were directed against the N-terminus of ovine PGH synthase-1 (89). Data are expressed +/- SEM. 50 permeabilized cos-1 cells transfected with native PGH synthase-1 or with any of the PGH synthase mutants (Figure 10). In permeabilized cells, intracellular staining patterns characteristic of the ER and N B were observed for both native and mutant PGH synthases (Figure 10). Sham-transfected cos-1 cells showed no detectable PGH synthase-1 (data not shown). The results presented in Figure 10 are for cells stained approximately 40 hours post- transfection. Overexpression of PGH synthase by transient transfection can significantly disrupt the morphology of cos-1 cells and result in areas of very intense immunofluorescence. To determine if subtle changes in localization were discernible with cos-1 cells exhibiting less intense staining and a more characteristic morphology, immunocytochemistry was also performed at earlier times (2430 hours) following. transfections. However, the immunofluorescence staining patterns observed at both earlier and later time points were the same, demonstrating the same ER and NE localization (data not shown) for native ovine PGH synthase-1 and the A597 PGH synthase-1 mutant. No cell surface staining was observed with any of the C-terminal PGH synthase-1 mutants, although the epitope (A25-C35) would be expected to be on the outside of the cell were any of the mutants not retained in the. ER (89). However, if PGH synthase-1 were relatively loosely associated with membranes, mutation of a functional retention signal would result in secretion of the protein. To determine if C-terminal mutations caused secretion of any of the PGH synthase-1 mutants, the media and microsomes from cos-1 cells expressing native PGH synthase-1 and the L600R and A597 mutant PGH synthases -1 were assayed for PGH synthase-1 protein by Western blot analysis. Although native PGH synthase-1 and the L600R 51 Figure 10. Subcellular localization of native and mutant ovine PGH synthases-1. Cos-1 cells were transfected with ovine PGH synthase-1 or the E5990 or A597 mutant PGH synthases -1. The subcellular locations of the native and mutant PGH synthases were determined by indirect immunofluorescence using purified peptide-specific rabbit antibodies generated against the N-terminus of ovine PGH synthase-1 (89) and FITC- conjugated goat anti-rabbit IgG. Staining of the ER and other internal membranes was performed using permeabilized cells (treated with saponin); staining of the cell surface was performed using non-permeabilized cells. Photomicroscopy was performed on a Leitz Orthoplan fluorescence microscope at 400x magnification with 2-minute exposures using Tri-X Pan 400 film. 52 NON-PERMEABILIZED PERMEABLIZED L600 E5990 FIGURE 10 53 and A597 mutant PGH synthases -1 were present in microsomes, they were not detectable in culture media (Figure 11). Thus, mutation of the putative ER retention signal does not result in secretion of mutant PGH synthase-1. These data, along with the results of immunocytochemistry, lead us to conclude that C-terminal mutations of the -PTEL sequence do not cause a change in the subcellular location of PGH synthase. The physico-chemical properties of the L600V PGH synthase-1 (mutant differed from those obtained with our other mutants. N o cyclooxygenase or peroxidase activity was detected with the L600V mutant. Subcloning and sequencing of a second L600V PGH synthase-1 mutant cDNA was repeated, and the new construct was transfected in cos-1 cells. Again, no enzyme activities were detected with microsomes prepared from cells transfected with this second L600V mutant construct. Furthermore, Western blot analysis showed that the L600V PGH synthase-1 protein migrates with an Mr = 63,000, an apparent decrease of 9 kDa compared with native PGH synthase-1 (Mr = 72,000; Figure 9). Initially, we suspected that the abnormal size of the L600V PGH synthase-1 was caused by proteolytic degradation of misfolded protein. However, when peptide-directed antibodies specific for either the N - or C-termini of ovine PGH synthase-l (89) were used in Western blot analysis of L600V PGH synthase microsomes, each antibody reacted with the 63 kDa L600V protein (data not shown). Thus, the smaller size of the L600V mutant is not attributable to proteolytic degradation from either of the protein termini. Furthermore, the decrease in the apparent molecular weight of L600V PGH synthase-1 was not caused by improper N-glycosylation. Endoglycosidase H treatment of microsomes prepared from cos-1 cells expressing either the native or the L600V mutant PGH synthase-l caused a decrease of 6 kDa for each protein (Figure 12). As discussed below, we suspect MEDIUM MICROSOMES C K [ o E? o r~ I I o i? o r~ l o E c or o E c or to to in ca .‘ on If) .r g .1 <1 .1 E .1 <1 72 Figure 11. Western blotting of PGH synthase-1 in microsomes and media from cos-1 cells transiently expressing native ovine PGH synthase-1, L600R PGH synthase-1, or A597 PGH synthase-1. Native ovine PGH synthase-1 or the L600R or A597 mutant PGH synthases -1 were expressed in cos-1 cells. Microsomes were isolated from the transfected cells; in addition, the media bathing the cells were subjected to immunoaffinity column chromatography as described in the text. Microsomal preparations and samples of column eluates were prepared in equal volumes and 50 ul of each were subjected to Western Blot analysis. 55 L6 0 0 L 6 0 0 V (NATIVE) I l r l _ + _ + 72 66 63 57 - III I l Figure 12. Endoglycosidase H treatment of native PGH synthase-1 and L600V mutant PGH synthase-1. Ovine PGH synthase-1 and the L600V mutant PGH synthase-1 were each expressed by transient transfection of cos-1 cells, and microsomes were prepared and treated with (+) or without (-) endoglycosidase H and subjected to Western blot analyses as described in the text. 56 that expression of the cDNA encoding the L600V mutant in the pSVT7 expression vector must lead to aberrant mRN A splicing. 57 D' . Earlier studies have indicated that PGH synthases -1 and -2 are membrane-associated proteins found in the ER and the NE (89,91,92). Our experiments were designed to test the hypothesis that the C-terminal tetrapeptide, -PTEL, of ovine PGH synthase-1 is required to target the enzyme to the ER. This hypothesis was based on the sequence similarities between the C-terminal tetrapeptides of PGH synthases and the -KDEL ER retention signals of other luminal ER proteins (Figure 8). Functional ER retention signals of a number of resident ER proteins consist of at least four residues at the C-terminus with invariant penultimate glutamate (E) and C-terminal leucine (L) residues (119). The other two residues vary, depending on the species and the protein. For example, -KDEL and -SDEL are ER retention signals for immunogloban heavy chain binding protein (BiP) from rat (123) and P. falciparium (150), respectively; -HTEL is the ER retention sequence for rabbit liver esterase (128), a soluble protein in the ER lumen; and -HDEL is a functional ER retention sequence for Sec20p (122), a type II integral ER membrane protein from S. cerevisiae. A C-terminal -XXEL sequence is found in all PGH synthases -1 and -2 sequenced to date (Figure 8). This sequence conservation suggests that these residues play a role in the function of PGH synthases. Proteins translocated into the lumen of the ER that lack signals for subcellular organelle targeting are transported to the plasma membrane by vesicles in the secretory pathway; membrane-associated proteins are retained within the plasma membrane, while soluble proteins are released from the cell when the vesicular membrane fuses with the plasma membrane. Therefore, we expected that disruption of a putative ER retention signal would result in the movement of ovine PGH synthase-1 from the ER 58 membrane to the plasma membrane. The converse has been reported by Tang et al. (131) with dipeptidyl peptidase IV (DPPIV), a type II integral plasma membrane protein. Addition of -KDEL to the C-terminus of DPPIV resulted in a change of localization from the plasma membrane to the ER membrane. Mutations previously shown to disrupt functional ER retention signals (123,125,127,128,131) were introduced at the C-terminus of ovine PGH synthase-1. It has been demonstrated repeatedly that mutation of either glutamate or leucine of -KDEL retention signals results in loss of retention. Even a seemingly conservative change from leucine to valine is not tolerated in the consensus sequence. However, cos-1 cells transfected with PGH synthases -1 having various mutations in the -PTEL sequences Showed no staining of the plasma membrane. Furthermore, media from cos-1 cells expressing native or mutant PGH synthases -1 showed no detectable PGH synthase-1 by Western blot analysis. Native and mutant PGH synthases -1 were only detected in the ER and NE. Because C-terminal mutations did not alter the subcellular locations of PGH synthase-l and, based on assays of activity, the mutant proteins had near normal structures, we conclude that the -PTEL sequence iS not required to target PGH synthase-1 to the ER. The results obtained with the L600V mutant of PGH synthase-1 were surprising. Western blot analysis of two independent L600V mutants expressed in cos-1 cells showed that this mutation leads to an apparent loss of 9 kDa in molecular mass and a complete loss of enzyme activity. We were unable to account for the smaller mass of the L600V mutant based on either sensitivity to proteolytic digestion or lack of N-glycosylation. A decrease of 9 kDa in molecular mass corresponds to a loss of approximately 70 amino acids. Several reports have described PGH synthase splice variants (57,159), one of which encodes a protein lacking approximately 37 amino acids (159). We 59 speculate that the L600V mutation at the 3’ end of the PGH synthase cDNA coding region promotes upstream splicing events which result in a variant protein of smaller molecular weight being expressed. In summary, data from our experiments indicate that a mechanism other that the -KDEL retention pathway is responsible for retaining ovine PGH synthase-1 in the ER and NE. The protein structure of ovine PGH synthase-1 has been determined for residues 33-586. However, lack of crystalographic data for the C-terminal residues 587-600 suggests that the C- terminus is without definite secondary structure. X-ray crystal structure analysis has suggested that ovine PGH synthase-1 could associate with membranes via a group of planar amphipathic helices Situated to expose hydrophobic residues to the luminal Side of the ER membrane (36). Using this model to orient the molecule, we extrapolate that following residue 586, the unstructured C-terminal tail would project toward the ER membrane. Perhaps this toplogical arrangement renders the P/ TEL C-terminal peptide inaccessible for neccessary interactions of the KDEL retention system. It will be important to determine if the proposed membrane binding domain composed of planar amphipathic helices, or some other domain of PGH synthase-1, is responsible for targeting the enzyme to the ER 60 Acknowledgment This work was previously published in the Archives of Biochemistry and Biophysics as Regier, M. K., Otto, I. C., DeWitt, D. L., and Smith, W. L., (1995) ”Localization of Prostaglandin Endoperoxide Synthase-l to the Endoplasmic Reticulum and Nuclear Envelope Is Independent of Its C- Terminal Tetrapeptide -PTEL” Arch. Biochem. Biophys. 317, 457-463., and is used with permission. CHAPTER 3 LOCALIZATION OF PGH SYNTHASE-Z Abstract Polyclonal antisera specific for prostaglandin endoperoxide (PGH) synthases-1 and -2, respectively, were used to determine the subcellular locations of each PGH synthase isozyme in detergent-permeabilized mouse 3T3 fibroblasts by indirect immunocytofluorescence. Antiserum to PGH synthase-1 demonstrated a mottled pattern of cytoplasmic and perinuclear staining of both serum-starved and serum-stimulated 3T3 cells. This pattern of staining is consistent with the results of earlier studies which demonstrated that PGH synthase-1 is associated with the endoplasmic reticulum and NE of these cells. As expected, antibodies directed‘against a peptide unique to PGH synthase-2 failed to stain serum-starved cells, which lack appreciable levels of this second form of the enzyme. However, serum- stimulated 3T3 cells, which do express PGH synthase-2, showed the same pattern of staining with PGH synthase-2 antibodies as was observed with anti- PGH synthase-1 serum—mottled cytoplasmic staining and perinuclear staining. We conclude that the subcellular location of PGH synthase-2 is the same as PGH synthase-1 in murine 3T3 cells. Thus, the notable differences in the primary amino acid sequence—the signal peptide and the additional 18 amino acid C-terminal segment in PGH synthase-Z—do not cause a change in localization. Colocalization of PGH synthases-1 and -2 implies that the source of arachidonate substrate, the site of PGHZ and prostanoid formation, and the 61 62 mechanism of product transport from the inside to the outside of the cell are the same for these isozymes. 63 Introduction PGH synthase is found in virtually all mammalian tissues (3) and catalyzes the conversion of arachidonic acid to prostaglandin endoperoxide H2 (PGHz) in a two step process carried out by cyclooxygenase and peroxidase activities. Prostaglandins and thromboxane A2, the final products formed from the intermediate precursor PGH2, are molecules involved in a variety of physiological roles such as inflammation (160,161), fluid volume homeostasis (162), ovulation (163-165), and mitogenesis (166,167). The current understanding of PGH synthase structure and function is based on data from studies with PGH synthase-1, originally isolated from sheep seminal vesicles (52,53). PGH synthase-1 is a 72 kDa hemoprotein that is membrane-associated and N-glycosylated (35,54,55,168,169). The subcellular location of PGH-1 synthase was first determined by immunofluorescence in kidney tissue sections (90,170) to be in the endoplasmic reticulum and nuclear membrane. This result was later ’ confirmed by immunoelectron microscopy of cultured mouse fibroblasts (91). Subsequent studies have continued to support the notion that, in general, PGH synthase-1 is localized to the endoplasmic and nuclear membranes. However, two additional locations of PGH synthase-1 have been reported. First, in 1983, Smith et al. demonstrated the presence of plasma membrane- associated PGH synthase-1 in arterial vascular smooth muscle—the single tissue positive for surface PGH synthase-1 of thirty-five different smooth muscle layers tested (171). Second, Weller et al. (172) recently described the association of PGH synthase-1 with non-membranous, structurally distinct lipid bodies. This is particularly interesting because lipid bodies represent a pool of arachidonate distinct from the prototypical sources of membrane phospholipids. 64 Recently, a gene encoding a second form of the enzyme, PGH synthase-2, has been cloned from chicken (57), mouse (58,59), and human (61) sources. Efforts to characterize this isozyme have been undertaken by several investigators. PGH synthase-2 differs from PGH synthase-1 in several ways. Comparison of the primary amino acid sequences shows that the signal peptides are dissimilar (64), and that there is a unique, C-terminal segment of 18 amino acids which is present only in PGH synthase-2. PGH synthase-2 also has a unique pattern of expression. In quiescent NIH 3T3 cells, constitutive levels of PGH synthase-2 mRNA (38,58,173) and PGH synthase-2 protein (68) are not detectable by Northern blot or Western blot analysis, respectively. However, stimulation by serum and growth factors rapidly induces transcription (38,58,59,61,64,173) and expression of PGH synthase-2 (68). The induction is transient and protein levels return to near basal levels within 8 hours of initial stimulation (68). Experiments have shown that PGH synthase—2 is encoded by a distinct gene (61,174) and is differentially glycosylated to form a 70 and 72 kilodalton protein (84). PGH synthase-2 catalyzes cyclooxygenase and peroxidase reactions (174) and has a Km for arachidonic acid similar to that of PGH synthase-1 (41). Both isozymes possess a N-terminal signal sequence peptide which is cleaved during protein maturation (54,64), and each has a putative endoplasmic reticulum retention signal at the C-terminus (128,133). This paper investigates the previously unanswered question of how the subcellular location of PGH synthase-2 compares to that of PGH synthase-1. 65 MatcrialsanclMcthods Preparation of Antibodies. Isozyme-specific antisera for PGH synthase-2 was produced by immunizing rabbits with the synthetic 17-mer peptide, cys-tyr-ser-his-ser-arg- leu-asp-asp-iso-asn-pro-thr-val—leu-iso-lys, which was coupled to maleimide- activated keyhole limpet hemocyanin (KLH) (Pierce). This peptide corresponds to a unique region of PGH synthase-2 protein near the carboxy- terminus which is not present in PGH synthase-1; the amino-terminal cysteine was added to the synthetic peptide sequence to facilitate coupling to maleimide-activated KLH. Affinity-purified PGH synthase-2 antibodies were prepared according to Mumby et al. (175) after applying 10 mg of the synthetic peptide to a SulfoLink coupling gel (Pierce). Antisera to PGH synthase-1 was produced by immunizing rabbits with purified sheep vesicular gland PGH synthase (91). Cell Culture. NIH 3T3 mouse fibroblasts were cultured in Dulbecco's modified Eagle medium (DME) supplemented with 8% calf serum (Hyclone) and 2% fetal calf serum (Hyclone). After treatment with 0.25% trypsin, cells from a 60 to 80% confluent monolayer (100 X 20 mm plate) were diluted in 15 mls of serum- supplemented DME and pipetted onto sterile glass coverslips (22 X 22 mm) (Corning). Approximately 4 hours later, the coverslips were washed with PBS, and the adherent cells were grown in DME supplemented with 0.2% calf serum for 24 to 48 hours (serum-starved conditions). For growth under serum-stimulated conditions, starved cells were supplied with 16.7% fetal calf serum (1 ml fetal calf serum added to 5 ml of serum-starved medium) and incubated for 2 hours prior to immunofluorescence staining. Immunofluorescence Staining. Intracellular staining was performed at room temperature. Coverslips with cultured cells were washed in phosphate-buffered saline (PBS), fixed for 8 minutes in 2% formaldehyde in PBS, and washed in 10% calf serum in PBS. Coverslips were inverted onto 200 ml of a) PGH synthase-1 antiserum diluted 1:20 in PBS containing 0.2% saponin and 10% serum, or b) affinity-purified PGH synthase-2 antibodies (0.29 mg/ ml) diluted 1:20 in PBS containing 0.2% saponin and 10% serum, and the samples were incubated for 1 hour. After washing in PBS containing 10% calf serum, the coverslips were incubated for 1 hour with a 1:40 dilution of fluorescein isothiocyanate (FIT C) conjugated goat anti-rabbit IgG (whole molecule) (Sigma) in PBS containing 0.2% saponin and 10% serum. Coverslips were then washed in PBS containing 10% calf serum, rinsed with PBS, and mounted on slides with Slowfade bleaching retardant (Molecular Probes). This procedure is essentially as described by Lippincott et al. (176). For a negative staining control, the affinity-purified PGH synthase-2 antibodies were incubated with the 17—mer antigen for 30 minutes prior to dilution and use on coverslips. The final concentration of peptide was 10 uM; the final dilution of purified antiserum was 1:20. Non- immune serum for PGH synthase-l was obtained from a rabbit prior to immunization and diluted 1:20 in PBS before use. Staining for cell-surface antigens was performed as above with the following exceptions: a) staining was performed at 4' C; b) saponin was not added to the antibody solutions; and c) fixation was performed after incubation with the secondary antibody. Coverslips were incubated with PGH synthase-1 antisera or PGH synthase-2 antibodies for 1 hour. The samples were rinsed in PBS containing 10% calf serum, and incubated with FITC- labeled goat anti-rabbit IgG. The cells were fixed for 8 minutes with 2% 67 formaldehyde in PBS and mounted on slides with Slowfade. For a positive control, the same staining procedure was performed using rat anti-transferrin receptor monoclonal (IgG) hybridoma (ATCC, TTB 219) supernatant (0.050 mg/ ml) and FITC-conjugated goat anti-rat IgG (BMB). Approximately 8 mg of anti-transferrin receptor IgG per coverslip and a 1:40 dilution of anti-rat IgG in PBS were used as primary and secondary antibodies, respectively. Confocal MicroscOpy. A Meridian Instruments Insight bilateral scanning confocal microscope was used with an argon ion laser as the excitation source. A 100x objective lens and laser power of 30 to 50 mwatts were used for photography. Photographs were taken with Kodak Tri-X Pan 400 film with an exposure of 3 to 6 seconds. K II I D' . Subcellular Localization of PGH Synthase-I. PGH synthase-1 antiserum exhibited specificity for the form 1 isozyme under the denaturing conditions of Western blot analysis (68). However, the possibility of PGH synthase-1 antiserum cross-reacting with native confirmations of PGH synthase-2 could not be eliminated. Therefore, immunofluorescent staining for PGH synthase-l was performed using serum-starved cells which do not express detectable levels of PGH synthase-2 as determined by Western blot analysis (68). (The pattern of PGH synthase—1 staining observed in serum-starved cells was identical to the PGH synthase-1 pattern observed in serum-stimulated cells (data not shown).) Serum-starved 3T3 cells were fixed with formaldehyde and then incubated sequentially in the presence of 0.2% saponin with antiserum to PGH synthase-1 and then FlTC-labeled anti-rabbit IgG. As shown in Figure 13 A, cells stained using this procedure demonstrated immunofluorescent staining around the nucleus and mottled staining within the cytoplasm, a pattern of staining characteristic of antigens associated with the endoplasmic reticulum (177,178). As expected, when the staining procedure was performed using non-immune rabbit serum in place of the antiserum to PGH synthase-l, no immunofluorescent staining was observed (Figure 13 B). Staining of nonpermeabilized cells showed no plasma membrane staining for PGH synthase-1, while cell-surface staining was observed with a monoclonal antibody to the transferrin receptor, as expected (data not Shown). In an earlier study employing immunoelectron microsc0py and this same anti-PGH synthase-1 serum, PGH synthase immunoreactivity was found to be associated with the nuclear membrane and the endoplasmic reticulum of murine 3T3 cells (91). Thus, the results of the present study employing 69 Figure 13. PGH synthase-1 immunofluorescence staining of mouse fibroblasts.‘ NIH 3T3 cells were seeded onto coverslips (approximately 100 cells/coverslip) and starved for 24-48 hours in DME containing 0.2% calf serum. After 8 minutes of fixation with 2% formaldehyde in PBS, the coverslips were rinsed with PBS containing 10% calf serum and incubated 1 hour with a) PGH synthase-1 antiserum, or b) non-immune serum diluted in PBS containing 0.2% saponin and 10% calf serum. The coverslips were then incubated for 1 hour with FITC-conjugated goat anti-rabbit IgG diluted in the same solution as above. Coverslips were rinsed with PBS and mounted using a bleaching retardant compound. Immunofluorescence was photographed using the same exposure time in each case. Magnification, X 330. 70 __—_____J FIGURE 13 71 confocal microscopy are in accord with those obtained by immunoelectron microscopy. Subcellular Localization of PGH Synthase-Z. A rabbit antiserum was prepared against a C-terminal peptide segment unique to murine PGH synthase-2, and the antibodies to this peptide were purified by affinity chromatography. In Western blot analysis, these antibodies were shown to be specific for PGH synthase-2 (68). Expression of PGH synthase-2 is a transient phenomenon initiated by serum stimulation (38,58,59,61,64,173). Serum-starved NIH 3T3 cells fail to express detectable levels of PGH synthase-2 as determined by Western blot analysis (68). However, following treatment with fetal calf serum, PGH synthase-2 levels are detectable after 1 hour, peak after 2 hours, and return to near basal levels after 8 hours (68). Therefore, to localize PGH synthase-2, serum-starved NIH 3T3 cells were stimulated with fetal calf serum for 2 hours and then subjected to immunofluorescent staining using the PGH synthase-2 antibodies. As shown in Figure 14 A, both perinuclear and mottled cytoplasmic staining were observed. Moreover, when the purified anti-peptide serum was preincubated with the peptide itself prior to staining stimulated cells, no staining was observed (Figure 14 B). As expected, no staining was observed using PGH synthase-2 antibodies with serum-starved cells (Figure 14 C). No plasma membrane immunofluorescence was observed using nonpermeabilized cells and PGH synthase-2 antibodies (data not shown). These results indicate that PGH synthase-2 expressed in serum-stimulated NIH 3T3 cells is associated with the NE and the endoplasmic reticulum. Thus, PGH synthase—2 is found in association with the same membranes as PGH synthase-1 in murine fibroblasts. Further experiments to determine the Figure 14. PGH synthase-2 immunofluorescence staining of mouse fibroblasts. NIH 3T3 cells were seeded onto coverslips (approximately 100 cells/coverslip) and starved for 24-48 hours in DME containing 0.2% calf serum. Cells were stimulated for 2 hours by the addition of fetal calf serum to a final concentration of 16.7% (A, B), or cells remained unstimulated (C). After fixation for 8 minutes with 2% formaldehyde in PBS, coverslips were rinsed with PBS and incubated for 1 hour with purified antibodies diluted in PBS containing 0.2% saponin and 10% calf serum: A, C) PGH synthase-2 antibodies, B) PGH synthase-2 antibodies preincubated with the peptide against which the antibody was generated (10 M final concentration). The coverslips were then incubated for 1 hour with FITC-conjugated goat anti- I rabbit IgG diluted in the same solution as above. Coverslips were rinsed in PBS and mounted using a bleaching retardant compound. Immunofluorescence was photographed using the same exposure time in each case. Magnification, X 330. 73 FIGURE 14 74 FIGURE 14 (cont’d) 75 extent of colocalization of the isozymes could investigate PGH synthase-2 localization in arterial smooth muscle, the only tissue reported to express PGH synthase-1 at the cell surface (171). Additionally, studies to determine if PGH synthase-2, like PGH synthase-1, is associated with lipid bodies are important to extend our understanding of the relationship of these enzymes. In summary, the data presented here suggest that subcellular locations of PGH synthase-1 and -2 are the same in murine 3T3 cells. From this, we can conclude that the obvious differences in primary amino acid sequences of the signal peptides and the unique C-terminal 18 amino acid segment in PGH synthase-2 do not cause a change in location. Colocalization implies that the source of arachidonate substrate and the site of PGHZ and prostanoid formation is the same for PGH synthase-1 and -2; and further, that the mechanism of prostanoid transport from the inside to the outside of the cell are the same for both isozymes. 76 Acknoulcdgmcnt This work was previously published in the Archives of Biochemistry and Biophysics as Regier, M. K., DeWitt, D. L., Schindler, M. S., and Smith, W. L., (1993) ”Subcellular Localization of Prostagladin Endoperoxide Synthase-Z in Murine 3T3 Cells” Archiv. Biochem. Biophys. 301, 439-444., and is used with permission. This work was supported in part by U.S.P.H.S. NIH Grants DK42509 (WIS), DK22042 (WLS), GM40713 (DLD), and Training Grant HL0740 (MKR). We thank]. Wang for the generous gift of FlTC-labeled goat anti-rat IgG and transferrin receptor antibodies, and P. Voss for valuable assistance with staining procedures. CHAPTER 4 INVEST'IGATTON OF THE PGHS-2 CASSETTE Introduction The work presented in chapter 2 showed that PGH synthase-1 and -2 are both localized to the ER and NE in murine NIH 3T3 fibroblasts. The isozymes are present on the same intracellular membranes, however, PGH synthase-2 immunofluorescence appears more intense on the NE (Figure 15) (93,179). Intense fluorescence staining of the perinuclear ring, more intense than the staining in the ER, is consistently observed in PGH synthase-2 stained cells, whereas the fluorescence intensity of the perinuclear ring and ER in PGH synthase-1 stained cells is similar. This observation suggests that distribution of the isozymes is different in these membrane systems. Based on this observation the subcellular localization of murine PGH synthase-1 and -2 in 3T3 fibroblasts was re-examined using quantitative confocal immunofluorescence imaging microscopy (93). This work was designed to quantify the observed difference in N E localization of PGH synthase isozymes. The fluorescence intensity in the N E and ER was determined for each isozyme and compared. The fluorescence intensity of PGH synthase-2 staining of the NE was twice as concentrated as that of the ER In constrast, PGH synthase-1 fluorescence intentsity was approximately the same in the NE and the ER The same results were obtained in both human and bovine endothelial cells and in experiments using a second PGH 78 murine PGH synthase-1 murine PGH synthase-2 murine PGH synthase-2 Figure 15. Subcellular localization of PGH synthase-1 and -2 in serum stimulated murine NIH 3T3 fibroblast. Murine NIH 3T3 fibroblasts were stimulated with serum for 2 hours and then processed for immunocytochemistry using purified isozyme-specific antisera as follows: A, anti-Leu274-Ala288 murine PGHS-l; B, anti-Ser582-Ly5598 murine PGHS-Z; C, anti-Gln569-Asn580 murine PGHS-Z. 79 synthase-2 antiserum (93). Therefore, the localization of PGH synthase-2 is quantitatively different from PGH synthase-1. PGH synthase-2 is preferentially distributed, targeted, to the NE, while PGH synthase-1 is uniformly distributed in the ER and NE. We reasoned that unique localization of PGH synthase-2 could be maintained by a unique protein structural domain of the second isozyme. As discussed in Chapter 1, the amino acid sequences of PGH synthase-1 and -2 are similar in most of the protein. However, one region of sequence divergence is found at the C-termini. Alignment of the murine isozyme sequences shows a 18 amino acid sequence at the PGH synthase C-terminus (PGHS-2 cassette) which is not found in PGH synthase-1 (Figure 16). The objective of this work was to determine whether the PGHS-2 cassette is involved in targeting the enzyme to the NE. Figure 16. Comparison of the deduced amino acid sequences of murine PGH synthase-1 and murine PGH synthase-2. Murine PGH synthase-1 sequence in shown in the upper line, murine PGH synthase-2 in the lower line. The amino acid numbering of each isozyme is shown at the side. The signal peptides are in bold type. The sites of glycosylation are noted by an open triangle. The serine acetylated by aspirin is marked with circle. The residues important in catalysis are in bold type. The PGHS—Z cassette (Ala581-LysS98) near the C-terminus of PGH synthase—2 is in bold type. Epitopes used to generate isozyme specific antisera are underlined: GlnS69-Asn580 and SerS82- Ly5598 for PGH synthase-2; Leu274-Ala288 for PGH synthase-1. 81 1 MSRRSLSLHFPLLLLLLLPPTPSVLLADPGVPSPVNPCCYYPCQNQGVCV 50 1 51 34 101 84 150 134 200 184 250 234 300 284 350 334 400 384 450 434 500 484 550 534 587 584 ................. itsgtvelelreétéeiiiléésNlééleéeét RFGLDNYQCDCTRTGYSGPRCTIPEIWTWLRNSLRPSPSFTHFLLTHGYW STGFDQiKCDCIRIGFYGERCITEEFLIRIKLLLKPTPRTVAYILIAFKG LWEFVNA.TEIREVLMRLVLTVRSNLIPSPPTYNSAHDYISWESFSRVSY oitillsxslilg£TARQ$llsléyllaélllilvatelxélltléltéi YTRILPSVPKDCPTPMGTKGKKQLPDVQLLAQQLLLRREFIPAPQGTNIL Ill ll.l:.|llllll.ll.l:|l| . : :.:l||ll|ll.lll.l:: YTRALPPVADDCPTPMGVKGNKELPDSKEVLEKVLLRREFIPDPQGSNMM FAFFAQHFTBQFFKTSGKMGPGFTKALGHGVDLGHIYGDNLERQYHLRLF Illllllllllllll. |.|llll::|||||||.llll:.l:||..|l|| FAFFAQHFTRQFFKTDHKRGPGFTRGLGHGVDLNHIYGETLDRQHKLRLF KDGKLKYQVLDGEVYPPSVEQASVLMRYPPGVPPERQMAVGQEVFGLLPG lllllllll::lll|ll.l.:. | I III :|.: |:||lI|I|l|:I| KDGKLKYQVIGGEVYPPTVKDTQVEMIYPPHIPENLQFAVGQEVFGLVPG LMLFSTIWLREHNRVCDLLKEEHPTWDDEQLFQTTRLILIGETIKIVIEE Il::.l|IllllllllI:ll:l||.l:|Il|lll.II|I|l|l|IIIII: LMMYATIWLREHNRVCDILKQEHPEWGDEQLFQTSRLILIGETIKIVIED YVQHLSGYFLQLKFDPELLFRAQFQYRNRIAMEFNHLYHWEPLMPNSFQV llllllll : .llllllllL IIII. IIII Ill lllllll: l: I YVQHLSGYHFKLKFDPELLFNQQFQYQNRIASEFNTLYHWBPLLPDTFNI V GSQEYSYEQFLFNTSMLVDYGVEALVDAFSRQRAGRIGGGRNFDYHVLHV llll: III: I. l: |:: |: HI L || lll: :llll l | EDQEYSFKQFLYNNSILLEHGLTQFVESFTRQIAGRVAGGRNVPIAVQAV AVDVIKESREMRLQPFNEYRKRFGLKPYTSFQELTGEKEMAAELEELYGD I . |.:llll:.|.:|ll|lll:||l|||l:||||||||llll..||:| AKASIDQSREMKYQSLNEYRKRFSLKPYTSFEELTGEKEMAAELKALYSD O IDALEFYPGLLLEKCQPNSIFGESMIEMGAPFSLKGLLGNPICSPEYWKP ||.:|:II:||:l|..|:.|Ill.|:|:lllllllll:ll||I||:|||l IDVMELYPALLVEKPRPDAIFGETMVELGAPFSLKGLMGNPICSPQYWKP STFGGDVGFNLVNTASLKKLVCLNTKTCPYVSFRVPDYPGDDGSVLV--- lllll: ||| Hllll I: l L I II: II | l : ....: STFGGEVGFKIINTASIQSLICNNVKGCPFTSFNVQDPQPTKTATINASA ............... RRSTEL 602 llllll SHSRLDDINPTVLIKRRSTEL 604 FRSLHUEI6 33 100 83 149 133 199 183 249 233 299 283 349 333 399 383 449 433 499 483 549 533 586 583 82 MatsflalmdMflhnds Materials Many of the reagents used are described in Chapter 2 and 3. Materials used for microinjection are cited here: CELLocate coverslips were from Eppendorf North America. LabTech mulit-well culture dishes were from Nunc. Borosilicate glass capillary tubing was from World Precision Instruments. Methods Many of the methods used are described in Chapter 2 and 3. The conditions for cell culture of NIH 3T3 fibroblasts are described in chapter 3; for cos-1 cells, in chapter 2. The methods for mutagenesis, transient transfection, and Western blot analysis are described in Chapter 2. Cyclooxygenase activity was determined by polarographic oxygen electrode and is described in Chapter 2. The immunocytochemistry protocol for subcellular localization of PGH synthases was performed by 2% formaldehyde fixation and 0.2% permeabilization and is described in chapter 2 and 3. Details pertaining to the experiments in Chapter 4 are described below. Mutagenesis Murine PGH synthase-2 cDNA was subcloned into the Not I restriction site of M13mp48, a M13mp19 derivative with a modified polylinker. Deletion mutagenesis was performed using a 35 base synthetic oligonucleotide mutagensis primer that encodes the residues flanking the PGHS-Z cassette (Figure 17). The resulting mutant which lacks the PGHS-Z cassette was designated as PGH synthase-ZAcassette. Mutagenesis was performed using a Muta-gene kit patterned after the method of Kunkel (152). Native and mutant murine PGH synthase-2 cDNAs were subcloned into the Not I 83 restriction site of the pSVLN expression plasmid, a derivative of pSVL (153) with an altered polylinker, and sequenced to verify the mutations (151). Ovine PGH synthase-1 cDNA was subcloned into the Sal I restriction site of the pSVT7 expression plasmid (153). Cell culture and transient transfection Cos-1 cells were transfected using the DEAE dextran/ chloroquine method. Murine PGH synthase-1 and -2 and PGH synthase-ZAcassette were transfected into cos-1 cells for preparation of microsomes or for immunocytochemistry. Transfections without plasmid (sham) were performed for negative controls. Cells for immunocytochemistry were cultured on CELLocate glass coverslips. Isa zyme-specific Antibodies: A number of isozyme-selective antisera were used for immunocytochemistry. Those antisera previously described were generated from isozyme-specific peptides from the following protein regions: Ala25- Cy535 of ovine PGH synthase-1 (89);Leu274-A1a288 of murine PGH synthase-l (93); and Ser582-LysS98 murine PGH synthase-2 (92). A second isozyme- specific antisera for murine PGH synthase-2 was produced by immunizing rabbits with the synthetic 13-mer peptide, Cys-Gln-Asp-Pro-Gln-Pro-Thr-Lys- Thr-Ala-Thr-Iso-Asn, which was coupled to maleimide-activated keyhole limpet hemocyanin (KLH) (Pierce). This peptide which corresponds to the amino acids (Gln569-Asn580) immediately upstream of the PGHS-Z cassette is not similar in sequence to the analogous residues in murine PGH synthase-1 (Figure 16). The amino-terminal cysteine was added to the synthetic peptide sequence to facilitate coupling to maleimide-activated KLH. Affinity-purified PGH synthase-2 antibodies were prepared according to Mumby et al. (175) after 84 applying 10 mg of the synthetic peptide to a SulfoLink coupling gel (Pierce). Purified antiserum is immunoreactive with both native murine PGH synthase-2 and PGH synthase-Z-Acassette, but is not immunoreactive with murine PGH synthase-1 as determined by Western blot analysis (Figure 18). Western Blot Analysis Microsomes were prepared from murine PGH synthase-1, murine PGH synthase-2, and PGH synthase-ZAcassette transfected cos-1 cells as described in chapter 3. Microsomes prepared from cells transfected with no DNA (sham) were used as controls. Microsomal protein preparations were subjected to Western blot analysis (10 ug protein/ lane). Visualization of native and mutant protein was performed using murine PGH synthase-Z-specific antiserum and ECL kit reagents. K... determination Km values were determined by measuring cyclooxygenase activity using arachidonate ranging from 2 to 100 M . The Km value was calculated from Lineweaver-Burk analysis. Prostaglandin Synthesis Prostaglandin product formation from [14C] arachidonate by transfected cos-1 cells was performed as previously described (37). Cos-1 cells transfected with murine PGH synthase-l, murine PGH synthase-2, PGH synthase-2- Acassette, or without plasmid (sham) were harvested in ice—cold PBS by scraping the culture dish with a spatula. Cells were resuspended in DMEM and incubated with [MC] arachidonate at 37° C for 40 min. The cells were collected by centrifugation, and arachidonate products were isolated from the 85 supernatant by chloroform extraction following acidification. The lipid products were separated by thin-layer chromatography in benzenezdioxanezacetic acid: formic acid (82:14:1:1; v:v:v), and visualized by exposure to X-ray film for 40 hours. Radioactive bands were identfied by comparison with prostaglandin standards. Microinjection Cos-1 cells and 3T3 fibroblasts were cultured on CELLocate glass coverslips in 100 x 20 mm tissue culture dishes, and maintained in DMEM supplemented with 8% fetal calf serum, 2% bovine calf serum for cos-1 cells and 3T3 fibroblasts, or 0.2% bovine calf serum for quiescent 3T3 fibroblasts. CELLocate coverslips are glass coverslips etched with a 175 um alphanumeric grid to enable precise relocation of individual (rnicroinjected) cells. After cell attachment, coverslips were transferred to multi-well glass slides, LabTech chambers, and covered with 1.5 ml of appropriate media. Cells were at subconfluent densities at the time of injection. Nuclei were injected with cesium chloride-purified plasmid DNA at 1 mg/ ml in PBS containing 1 mM MgC12. Microinjection was performed using an Insight bilateral scanning ‘ confocal microscope (Meridian Instruments, Okemos, MI). The micropipette was mounted on the Insight confocal microscope with an IMTZ-SFY inject attachment, and maneuvered with a micromanipulator (N arishige). This system is designed for vertical injection of samples, a micropipette angle that minimizes interference of the phase contrast optics used to view the injection. Injection of plasmid solutions was controled by hydraulic pressure using a glass syringe and tubing connected to the micropipette. Cells were viewed with 20X objective for injections. Native and mutant PGH synthases 86 were expressed by direct microinjection of plasmid DNA into nuclei using the methods of Capecchi (180) as described by Wozniak (147). Micropipettes were pulled from thin-wall borosilicate glass capillaries with internal filament (1 mm outer diameter, 0.75 inner diameter) using a Sutter Instrument P-87 Brown-flaming micropipette puller. Successful microinjection was obtained with the following instrument parameter values: Heat, 715 ; Pull 70; Velocity, 90; Time, 250; and Pressure, 400. Fluorescence Confocal Microscopy An Insight Bilateral scanning confocal microscope (Meridian Instruments, Okemos, MI) was used with an argon ion laser as the excitation source (181). Laser power settings between 50 and 75% were used depending on intensity of staining. 87 Results Expression and catalytic activity of PGH synthase-Z-Acassette Site-directed deletion mutagenesis was performed to generate a murine PGH synthase-2 mutant lacking the PGI-IS-Z cassette (PGH synthase-2- Acassette) (Figure 17). Deletion of the PGI-IS-2 cassette removes 18 amino acids from the protein and eliminates a site of glycosylation at Asn580. Native murine PGH synthase-2 and mutant PGH synthase-ZAcassette cDN As were subcloned into the pSVL expression vector. To determine the effect of the mutations on the structural and catalytic properties of the enzyme, we performed Western blot analysis (Figure 18) and prostaglandin product analysis (Figure 19). Western blot analyses of cos-1 microsomal protein preparations showed that PGH synthase-ZAcassette had an altered electrophoretic mobility as compared with native PGH synthase-2 (74 and 72 kDa). PGH synthase-ZAcassette migrated at an apparent molecular mass of 68 kDa, which was consistent with the estimated loss of mass resulting from deletion of the PGHS-Z cassette (18 amino acids and one N-linked carbohydrate moiety). Deletion of the PGHS-Z cassette disrupts the glycosylation consensus sequence (Asn-Ala-Ser) of Asn580. Glycosylation of native PGH synthase-2 at this site is about 50% efficient, which results in two protein species that migrate as 74 and 72 kDa by SDS PAGE (84). PGH synthase-2Acassette migrates as a single 68 kDa species as expected in the absence of differential glycosylation at Asn580. Cos-1 cells transfected with native murine PGH synthase-1 and -2 and mutant PGH synthase-Z-Acassette were incubated with [14C] arachidonate, and prostaglandin products were separated by thin-layer chromatography. Cos-1 cells expressing PGH synthase-Z-Acassette generated prostaglandin products ‘Q Ikilhll unnnhnnnxxu f. pom rasmsucmvxccrrrsmvgnpoemm masmomm: rm. m rnsrosucxuvxccer'rsmgopgemmmnnsm WMWWWDYPGDDGW Pflflid Figure 17. Mutagenesis scheme for PGH synthase-ZAcassette. The C-terminal regions of murine PGH synthase-2 (PGHS-Z), PGH synthase-2Acassette (PGHS- 2Acassette), and murine PGH synthase-1 (PGHS-l) are aligned. The PGHS-Z cassette which was deleted in mutagenesis is marked in a stipled box. Residues corresponding to the oligonucleotide used in mutagenesis are marked with overlined asterisks. Identical residues in murine PGH synthase- 1 and PGH synthase-ZAcassette are underlined. 74— 72— 68— Figure 18. Isozyme-specific immunoreactivity of purified anti-GlnS69- Asn580 murine PGH synthase-2 antisera, and expression of PGH synthase- 2Acassette. Isozyme-specificity of antisera generated against residues Gln-569- Asn580 of murine PGH synthase-2 was tested by Western blot analysis. Cos-1 cells were transiently transfected with pSVLN expression plasmids encoding PGH synthase cDNAs. Microsomal protein was subjected to Western blot analysis using purified antisera generated against residues G1n-569-Asn580 of murine PGH synthase-2. Microsomal samples (10 pg protein/ lane) from transiently transfected cos-1 cells are as follows: transfected without plasmid, Lane 1; murine PGH synthase—1, lane 2; murine PGH synthase-2, lane 3; and PGH synthase-ZAcassette, lane 4. 9O (PGEZ, PGF2a, PGD2) comparable to those observed with native enzymes (Figure 19), indicating that PGH synthase-Z-Acassette retains cyclooxygenase and peroxidase activity. To further investigate the effect of PGHS-2 cassette deletion, the Km for native murine PGH synthase and PGH synthase-Z-Acassette were compared. Microsomes prepared from transfected cos-1 cells were used in cyclooxygenase activity measurements to determine the Km values for the native and mutant enzymes. The Km values with arachidonate were similar, 3.3 uM for PGH synthase-2-Acassette and 2.5 M for native murine PGH synthase-2. These values are also similar to that previously reported for native murine PGH synthase-2, 2.5 uM arachidonate (41) . We conclude that deletion of PGHS-Z cassette is permissible with relatively minor changes in the conformation of murine PGH synthase-2. Subcellular localization of PGH synthase-ZAcassette in transiently transfected cos-1 cells Immunocytochemistry was performed to determine whether deletion of the PGHS-2 cassette altered the preferential localization of PGH synthase-2 to the NE. Intracellular staining of cos-1 cells transfected with native murine PGH synthase—1 and -2 cDNAs and mutant PGH synthase-Z-Acassette cDNA was performed. In all cases staining was observed in the ER and NE (Figure 20). Quantitation of fluorescence intensity in the ER and NE was difficult because of the unusual morphology of the transfected cos-.1 cells. Often the cos—1 nucleus is asymmetrically positioned in the cytoplasm with a portion of the nuclear perimeter touching the plasma membrane. In previous studies using 3T3 fibroblasts, the nucleus is usually centered in the cytoplasm and fluorescence can be quantitated more easily. Quantitation was also difficult in 91 '— PGDZ PGFZa PGEZ Figure 19. Prostaglandins synthesis in cos-1 cells transiently transfected with native and mutant PGH synthases. Cos-1 cells were transiently transfected with pSVLN expression plasmids encoding PGH synthase cDNAs. Transfected cos-1 cells were harvested and incubated with [MC] arachidonate Prostaglandins products were separated by thin-layer chromatography. Lane 1, no plasmid, Lane-2 murine PGH synthase-1; Lane 3, murine PGH synthase- 2; Lane 4, PGH synthase-ZAcassette. Prostaglandin species, arachidonic acid (AA) and the origin (0) are identified on the right edge. 92 murine PGH synthase-1 murine PGH synthase-2 C murine PGH synthase-2 -Acassette Figure 20. Expression of native and mutant PGH synthases in transiently transfected cos-1 cells. Cos-1 cells were transiently transfected with pSVLN expression plasmids encoding PGH synthase cDNAs and stained with anti- PGH synthase antisera: A, murine PGH synthase-1 stained with anti-Leu274- Ala288 PGHS-l; B, murine-PGH synthase-2 and C, PGH synthase-2Acassette stained with anti-GlnS69-Asn580 PGHS-Z. 93 cos-1 cells due to areas of intense cytoplasmic immunofluorescence (Figure 20). The ratio of NE to ER fluorescence intensities (N E/ ER ratio) determined for native murine PGH synthase—1 and -2 and PGH synthase-Z-Acassette in transfected cos-1 cells were 1.4, 1.2, and 1.0 respectively. The PGH synthase-1 and -2 NE/ER ratios do not agree With those observed in 3T3 cells (PGH synthase-1, approximately 1.0, PGH synthase-2, approximately 2.0) (93) suggesting that localization of PGH synthase-2 overexpressed in cells may be aberrant. We conclude that transient transfection of cos-1 cells is not a suitable system for localization experiments regarding PGH synthase-2 NE targeting. To overcome this limitation, I next attempted to express these enzymes in 3T3 cells by microinjection of their cDNAs. Subcellular localization of PGH synthase-ZAcassette by 313 microinjection in cos-1 and 313 cells Localization experiments were performed by direct injection of plasmid cDNAs in cell nuclei. Preliminary microinjection experiments to establish the parameters of the protocol were conducted using COS-1 cells and native ovine PGH synthase-1 cDN A in the pSVT7 expression plasmid. Ovine PGH synthase-1 cDNA plasmid was microinjected in cos-1 nuclei. The cells were then maintained in culture for various times, and then processed for immunofluorescence staining. Microinjected cells from 5, 10, 18, 24 and 48 hours post-injection were analyzed. Ovine PGH synthase-1 protein expressed from microinjected plasmid was localized to the ER and NE (Figure 21). The number of cells expressing microinjected plasmid was greatest for samples incubated 5 hours post-injection, and no expression was observed for cells cultured more than 24 hours. We speculate that the viability of microinjected 94 ovine PGH synthase-1 Figure 21. Subcellular localization of ovine PGH synthase-1 in microinjected cos-1 cells. Cos-1 cells were microinjected with ovine PGH synthase-1 and stained with anti-A1a25-Cys35 ovine PGHS-l. Cells from several experiments are shown. 95 cells is compromised, and after longer incubation periods microinjected cells die and detach from the coverslip. We conclude that incubation periods as short as 5 hours post-injection are sufficient for detectable expression of cDNAs in the pSVT7 expression plasmid. Unfortunately, microinjected cos-1 cells exhibited the same morphology transfected cos-1 cells, a morphology not conducive to quantitation. In addition, very few injected cells expressed the injected plasmid (see further discussion below). Therefore, I next attempted to microinject 3T3 cells, the cell line used for the quantified subcellular localization of the native murine PGH synthase isozymes. Microinjection of native ovine PGH synthase-1 in pSVT7 and native murine PGH synthase-2 in the pSVL expression plasmid was performed in growing and quiescent 3T3 fibroblasts nuclei. Injected cells were cultured for 5 hours and then processed for immunocytochemistry. Staining of ovine PGH synthase-1 and murine PGH synthase-2 was observed in the ER and NE (Figure 22). These experiments demonstrate that expression of microinjected pSVT7 and pSVL expression plasmids in growing and quiescent cells is detectable as early as 5 hours post-injection. Importantly, we demonstrate that these plasmids are expressed in quiescent 3T3 cells which is critical to our experimental design (see discussion below). Although expression of microinjected plasmids was achieved, the efficiency of expression was substandard. An average of one percent or fewer of injected cells were found to express PGH synthase. The most successful experiment yielded nine positively expressing cells out of approximately 500 cells injected. Many expressing cells appeared damaged or multinucleated (Figure 21, Figure 22). We conclude that our microinjection technique will need to be improved considerably to make this technique a feasible approach for localization studies. 96 ovine PGH synthase-1 murine PGH synthase-2 Figure 22. Subcellular localization of ovine PGH synthase-1 and murine PGH synthase-2 in microinjected quiescent murine NIH 3T3 fibroblasts. Quiescent NIH 3T3 cells were microinjected with (A) ovine PGH synthase-1, stained with anti-AlaZS-CySBS ovine PGHS-l and (B) murine PGH synthase-2, stained with anti-Gln569-Asn580 murine PGHS-Z. 97 D . . Although both PGH synthase isozymes are present in the ER and NE, recent work has shown that PGH synthase-2 is preferentially distributed in the NE (93). Intense staining of the perinuclear ring which is greater than the intensity of the ER has been shown for PGH synthase-2 in 3T3 fibroblasts, bovine arterial endothelial cells, and human umbilical vein endothelial cells (93). PGH synthase-1 staining in these cells is of similar intensity in the ER and NE. Staining intensities were quantitated and expressed as a ratio of NE to ER fluorescence intensities (N E / ER ratio) for PGH synthase-1 and-2. The PGH synthase-2 NE/ER ratio is approximately 2, whereas the NE/ER ratio for PGH synthase-1 is about 1. Because immunoreactivity, or fluorescence intensity, is roughly proportional to the mass of protein present, the N E/ ER ratio results suggest that PGH synthase-2 is concentrated in the NB. This is unlike PGH synthase-1 which is uniformly distributed in the two membrane systems. It seems likely that the unique localization pattern of PGH synthase-2 could be related to a unique structural domain in the protein. We were interested in identifying the putative protein determinant responsible for concentrating PGH synthase-2 in the NE. Murine PGH synthase-1 and -2 share significant similarity in protein sequence (Figure 16). However, at the C-terminus of PGH synthase-2 there is a unique sequence of 18 residues (Ala581-Ly5598). The peptide sequence abruptly interrupts the well conserved isozyme sequence alignment, and its sequence is not similar to any other reported protein sequence (GenBank sequences). This 18 amino acid sequence (PGHS-Z cassette) which is found only in PGH synthase-2 was identified as a candidate for a PGH synthase-2 NE targeting domain. Site directed mutagenesis was performed to delete the PGHS-Z cassette from murine PGH 98 synthase-2, and I have attempted to determine what effect this deletion has on PGH synthase-2 localization. No obvious structural or catalytic changes occured following deletion of the PGHS-2 cassette from PGH synthase-2. Cyclooxygenase activity, formation of prostaglandins, and affinity for arachidonate are similar for PGH synthase- 2-Acassette and native murine PGH synthase-2. Thus the PGHS—Z cassette is not involved in enzyme catalysis. Information inferred from the ovine PGH synthase—1 crystal structure supports this interpretation (36). The protein structure for the C-terminal tail of ovine PGH synthase-1, Arg586 to Leu600 (ovine PGH synthase-1 numbering), is absent from the crystal structure, which suggests that this region of ovine PGH synthase-1 is disordered. Together these results suggest that the PGHS-Z cassette, like the PGH synthase-1 C-terminal tail, does not interact significantly with other structural domains of the PGH synthase protein. Using cos-1 cells expressing native and mutant enzymes we were unable to localize murine PGH synthase-2 or PGH synthase-Z-Acassette. The NE/ ER ratio calculated for murine PGH synthase-1 and -2 and PGH synthase-2- Acassette in transfected cells did not agree with NE/ ER ratios of the native enzymes endogenously expressed in murine 3T3 fibroblasts. Two technical limitations precluded our use of the cos-1 system. First, high levels of overexpressed protein in cos-1 cells caused very intense staining in certain regions of the cytoplasm. Our method of NE/ ER ratio quantitation cannot accommodate such non uniform staining of cytoplasmic areas immediately surrounding the nucleus. Second, cos-1 nuclei were frequently positioned near the plasma membrane of the cell, rather than in the center of the cell; the NE was bordered by cytoplasm for less than the entire perimeter. Because accurate calculation of the NE/ER ratio is not feasible in cos-1 cells, we 99 conclude that transient transfection in this system is not suitable for our localization studies. It is also possible that cos-1 cells, which lack endogenous PGH synthase, lack the proper targeting machinery for differential distribution of PGH synthase-2, or that the targeting machinery is disrupted by overexpression or by the process of transfection. A strategy employing microinjection of PGH synthase cDN A expression plasmids in 3T3 fibroblasts was developed as an alternative approach for localization experiments. This system has several advantages: expression plasmids are introduced without transfection treatments, and 3T3 fibroblasts have demonstrated differential localization of PGH synthase-2 previously (93). We modeled our experiments after the work of Wozniak and Blobel in the determination of the targeting domain of gp210, a glycoprotein of the nuclear pore complex (147). In their experiments mutant cDN As carried in the pSVL expression plasmid were microinjected in 3T3 fibroblast, and localization was determined by immunocytochemistry. 3T3 fibroblasts constitutively express murine PGH synthase-1 and transiently express murine PGH synthase-2. Therefore, it was necessary to manipulate the experimental conditions so that protein expressed from microinjected plasmids could be distinguished from endogenous protein expression. The problem of endogenous murine PGH synthase-2 expression was addressed by culturing cells in low serum medium (0.2% serum); 3T3 fibroblasts grown under these conditions do not express PGH synthase-2. To circumvent the problem of endogenous murine PGH synthase-1 expression, ovine PGH synthase-1 was used in microinjection experiments; species- selective antisera were used to detect only the transfected ovine PGH synthase-1 . 100 A series of control experiments were performed to establish the technique of. microinjection and the proper microinjection conditions for our system. This work included developing micmpipettes appropriate for microinjection, determining the optimal time of expression following microinjection, and determining whether pSVT7 and pSVL expression plasmids are expressed in quiescent cells. Expression of microinjected plasmids was achieved for native ovine PGH synthase-1 and native murine PGH synthase-2 in cos-1 cells (Figure 21) and quiescent 3T3 fibroblasts (Figure 22). Importantly, this work demonstrates that our system of microinjection is a valid approach for studying the localization of PGH synthase in quiescent 3T3 fibroblasts. A significant limitation in these experiments was the very low percentage of injected cells found to express PGH synthase. In all experiments, one percent or fewer of the cells injected expressed protein. This compares with 20-50% expressing cells reported by others using a similar system (R. Wozniak, personal communication). In addition microinjections were successful only occasionally; in my final series of experiments, one out of four days injecting produced cells that expressed protein. Improvement in the efficiency of microinjection will be necessary to make this experimental approach practical for regular use. My experience suggests that the micropipettes‘and the method of solution injection are very important components of the system. It is critical that the micropipette is small enough to avoid excessive damage to the NE, and that the injection of plasmid solution be very carefully controlled to avoid injection of too large a volume (R. Wozniak, personal communication). Cell viability following injection is the bottleneck in this technique. Refining these two parameters is likely to dramatically improve microinjection efficiency. 101 In summary, the work presented here provides initial characterization of PGH synthaseAcassette. The mutant enzyme retains cyclooxygenase and peroxidase activities, and produces prostaglandins similar to native PGH synthase, and removal of the PGHS-Z cassette does not alter the enzyme affinity for substrate. These data demonstrate that the PGHS-Z cassette is not involved in catalysis. Subcellular localization studies demonstrate that pSVT7 and pSVL expression plasmids are expressed in quiescent 3T3 cells, 5 hours post-injection. Improvement of the microinjection system will be necessary to obtain sufficient sample sizes for NE/ ER ratio calculation. This system will be useful for assaying the PGHS-Z cassette and other domains of interest in localization of PGH synthase-2. CHAPTER 5 SUBCELLULAR LOCALIZATION OF CYTOSOLIC PHOSPHOLIPASE A2 Abstract Cytosolic phospholipase A2 (cPLA2) is induced by a wide variety of stimuli to release arachidonic acid, the precursor of the potent inflammatory mediators prostaglandins and leukotrienes. Specifically, cPLA2 releases arachidonic acid in response to agents that increase intracellular Ca2+. In vitro data has suggested that these agents induce a translocation of cPLA2 from the cytosol to the cell membrane, where its substrate is localized. Here, we use immunofluorescence to visualize the translocation of cPLA2 to distinct cellular membranes. In CHO cells that stably overexpress cPLA2, this enzyme translocates to the nuclear envelope upon stimulation with the calcium ionophore A23187. The pattern of staining observed in the cytoplasm suggests that cPLA2 may also translocate to the endoplasmic reticulum. We find no evidence for cPLA2 localization to the plasma membrane. This translocation is dependent on the calcium-dependent phospholipid binding (CaLB) domain, as a CaLB deletion mutant of cPLA2 (ACII) fails to translocate in response to Ca2+. In contrast, cPLA2 mutated at Ser505, the site of mitogen-activated protein (MAP) kinase phosphorylation, translocates normally. This observation, combined with the observed phosphorylation of ACII, establishes that these two mechanisms function independently to regulate cPLA2. The effect of these mutations on cPLA2 translocation was confirmed by subcellular fractionation. Each of these 102 103 mutations abolishes the ability of cPLA2 to release arachidonic acid, establishing that cPLA2-mediated arachidonic acid release is strongly dependent on both phosphorylation and translocation. These data help to clarify the mechanisms by which cPLA2 is regulated in intact cells and establish the nuclear envelope as a primary site for arachidonic acid production in the cell. 104 Introduction The 85 kD cytosolic phospholipase A2 (cPLA2), which selectively releases arachidonic acid from the sn-2 position of membrane phospholipids, is crucial to the initiation of the inflammatory response. cPLA2 activity is stimulated by a wide variety of agents, including the proinflammatory cytokines interleukin 1 (182,183) and tumor necrosis factor (184), macrophage colony-stimulating factor (184), thrombin (185,186), ATP (185), mitogens (29,187-189) and endothelin (190). The release of arachidonic acid is the rate- limiting step in the generation of prostaglandins and leukotrienes, the proinflammatory eicosanoids. Cleavage of arachidonyl-containing phospholipids also results in the release of lysophospholipid, the precursor of the inflammatory mediator platelet-activating factor (191). cPLA2 is expressed in many cell types. Many of these are associated with the inflammatory response, such as monocytes (184), neutrophils (192), and synovial fibroblasts (193). It is also expressed in a wide variety of tissues, including kidney, spleen, heart, lung, liver, testis and hippocampus (194). This diverse pattern of expression is consistent with accumulating evidence that in addition to its role in inflammation, cPLA2 also participates in the signaling of such diverse processes as platelet activation (21,186), tumor necrosis factor-induced cytotoxicity (195), and proliferation (187,189,196). cPLA2 activity is regulated both transcriptionally and post-translationally. Post-translational activation is thought to occur by two mechanisms. One mechanism involves agonist-induced MAP kinase phosphorylation of cPLA2, resulting in stimulation of its instrinsic enzymatic activity (22,197). The second involves the Ca2+-dependent translocation of cPLA2 from the soluble to the membrane fraction of cells (20,198,199), allowing cPLA2 access to its arachidonyl phospholipid substrate. As discussed below, the results 105 presented in this study establish that both mechanisms are critical for the stimulation of cPLA2-induced arachidonic acid release. In vitro studies have strongly suggested that the membrane-binding function of cPLA2 resides in its Ca2+-dependent phospholipid-binding (CaLB) domain (20,200), a region similar to the C11 domain of protein kinase C. This domain has been shown to be necessary and sufficient for Ca2+-dependent membrane binding (20,200). In this study we characterize the translocation of cPLA2 in intact cells and establish that this enzyme translocates to the nuclear envelope. Our data also suggests that cPLA2 translocates to the endoplasmic reticulum. Deleting the CaLB domain abolishes the ability of cPLA2 to translocate to these membranes, whereas a mutation at the MAP kinase phosphorylation site has no effect on translocation. Either of these mutations prevents cPLA2-induced arachidonic acid release. These data, together with recent reports localizing several arachidonic acid-metabolizing enzymes to the nuclear envelope and endoplasmic reticulum (92,201-203), establish these membranes as important sites of arachidonic acid production and metabolism in the cell. 106 MW Cell Culture and Antibodies CHO cells were maintained as previously described (185). Monoclonal antibody 1.1.1 (22) was raised against human cPLA2 purified from E5-CHO cells and was used for staining of parental and cPLA2-overexpressing CHO cells. Polyclonal antibody 7905, generated against cPLA2 produced in Escherichia coli, was used for immunoblotting (20). PGHS-l-specific ‘ polyclonal antibody was generated against the N-terminus of ovine PGHS—l, residues Ala-25 to Cys-35 (89). E-selectin monoclonal antibody was generated against soluble human E-selectin (204). Immunofluorescence Staining Cells grown on coverslips were rinsed with serum-free media prior to a 2 min treatment with 2 uM calcium ionophore A23187 (Sigma). Treated and untreated cells were washed twice briefly with TBS containing 0.01% Triton X-100. During all washes and incubations throughout this procedure, all A23187-treated cells were incubated in the presence of 1 mM CaC12, to lessen the chance that translocated cPLA2 would detach from membranes. Cells were fixed for 2 min at room temperature in a 50:50 mixture of acetone and methanol. After 2 brief washes, blocking was performed in TBS / 20% goat serum for 1 hr at 37'C. Cells were incubated with primary antibody (150 mg/ ml cPLA2 antibody, 100 mg/ ml E-selectin antibody, or a 1:20 dilution of PGHS—l antibody) for 1 hr at 37' and washed once briefly followed by 2 x 10 min washes. Second antibody incubation was performed for 1 hr at 37'C using a 1:100 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse or -rabbit antibody and washed as above. After a brief wash with 107 water, coverslips were inverted onto slides with Slowfade bleaching retardant (Molecular Probes). Fluorescence Confocal Microscopy Subcellular localization of cPLA2 was visualized by fluorescence confocal microscopy. An Insight Bilateral scanning confocal microscope (Meridian Instruments, Okemos, MI) was used with an argon ion laser as the excitation source (181). The laser was used at a power ranging from 25-75 mW, depending on the level of fluorescence intensity. All images were photographed with a 100x objective. Untreated and A23187-treated samples for a given cell line were analyzed using identical instrument settings. Fractionation and Immunoblotting Cells were starved overnight in serum-free media and treated with 2 uM A23187. After washing with TBS, cells were collected by scraping and lysed in 1 ml lysis buffer (20 mM Hepes, pH 7.4/ 1 mM EGTA/ 0.34 M sucrose/ 10 ug/ ml leupeptin/ 2 mM PMSF/ 5 mM DTT) by Parr bombing, 600 psi for 5 min. Lysates were centrifuged for 1 hr at 100,000 x g and 5 mg of protein from supernatant and pellet fractions was electrophoresed by SDS PAGE. Proteins were transferred to nitrocellulose, blotted with anti-cPLA2 polyclonal antibody 7905 (20), and developed by chemiluminescence after incubation with I-IRP-conjugated protein A (Amersham). Arachidonic Acid Release Cells were plated onto 12-well cluster dishes (Costar) at 1.5 x 105 cells per well in growth medium. After a 20 hr incubation, the medium was removed and replaced with 0.5 ml of a medium (GIBCO) containing 0.5 uCi of 108 [3H]arachidonic acid (100 Ci / mmol; New England Nuclear) and incubated for another 20 hr at 37'C. The cells were then washed three times with medium containing 0.1% bovine serum albumin and incubated with A23187 for 10 min. The medium was removed, and radioactivity was determined by scintillation counting. 109 Results Ca2+ Ionophore Induces cPLA2 Translocation to the Nuclear Envelope cPLA2 can be induced to associate with natural membranes in the presence of physiologically relevant Ca2+ levels in vitro (20,198). This Ca2+-dependent translocation is thought to reflect a translocation that occurs in viva when cells are stimulated with agents that increase intracellular Ca2+. As the translocation of cPLA2 in response to Ca2+ has not yet been characterized in intact cells, the membrane site(s) of arachidonic acid release by cPLA2 has not been identified. To address this issue, indirect immunofluorescence was performed using E5-4, a CHO cell line that overexpresses human cPLA2 (185) (Figure 23 A). In resting cells, cPLA2 was found distributed throughout the cytoplasm. In contrast, after treatment with the Ca2+ ionophore A23187, significant cPLA2 staining appeared in a discrete ring surrounding the nucleus. This suggests that an increase in cytosolic Ca2+ induces a translocation of cPLA2 from the cytosol to the nuclear envelope. Interestingly, staining of A23187-treated E5-4 cells was consistently brighter than that seen in untreated cells (quantitated in Figure 23 B). This is most likely due to a greater loss of soluble cPLA2 than membrane-bound cPLA2 throughout the staining procedure. A similar interpretation was offered for the results observed upon immunogold labeling of the arachidonic acid- metabolizing enzyme 5-lipoxygenase (5-LO), which also translocates to the nuclear envelope in response to A23187 (202). In that study, 5-LO was undetectable in unstimulated cells but was apparent at the nuclear envelope after ionophore treatment. Alternatively, binding of cPLA2 to membranes may result in a better exposure of the monoclonal antibody epitope, resulting in more efficient antibody binding. In either case, the observed increase in cytoplasmic staining intensity suggests that cPLA2 may translocate not only to 110 Figure 23. Differential localization of cPLA2 in untreated and Ca“ ionophore-stimulated cells. CI-IO cells overexpressing wildtype cPLA2 were stimulated with 2 uM CaZ+ ionophore (+A23187) for 2 min or left untreated (-A23187). (A) Immunofluorescence was performed using a monoclonal antibody specific for cPLA2 and a fluorescein-conjugated second antibody. Confocal microscopy reveals a translocation of cPLA2 to the nuclear envelope (magnification, X330). As discussed in the text, punctate staining was observed in the cytoplasm in some experiments, consistent with translocation to the endoplasmic reticulum. (B) Quantitative fluorescence imaging of cytoplasm and nuclear envelope. The intensity of immunofluorescent staining was quantified by measuring fluorescence before and after ionophore treatment in equivalent areas of the cytoplasm and nuclear enve10pe. 111 - A23187 + A23187 ACII S on N U! 550 Fluorescence Intensity (arbitrary units) N \3 U't ER + NE ER + NE Y - A23187 + A23187 FIGURE 23 112 the nuclear envelope but also to a cytoplasmic membrane structure, most likely the endoplasmic reticulum. Indeed, in some experiments ionophore treatment resulted in an increase in punctate cytoplasmic fluorescence characteristic of endoplasmic reticulum staining. The plasma membrane was consistently found to be devoid of cPLA2 staining. To ensure that fixation had not disrupted the integrity of the plasma membrane, CHO cells overexpressing the plasma membrane protein E- selectin were stained. As shown in Figure 24, E-selectin antibody was clearly able to label the plasma membrane. No labeling of the nuclear envelope was observed in these cells. As a control for the specificity of the anti-cPLA2 monoclonal antibody, immunofluorescence was also performed on the parental CHO cells used for these studies. Only faint staining was observed (Figure 24), similar to that seen when the monoclonal antibody was eliminated from the staining protocol (data not shown). The lack of endogenous cPLA2 staining is likely to be due both to the low level of cPLA2 expressed in these cells and the failure of this antibody to recognize murine (and by inference, hamster) cPLA2 efficiently. Figure 24 also shows staining of CHO cells overexpressing prostaglandin-endoperoxide synthase-1 (PGHS-l), which metabolizes arachidonic acid to prostaglandin. Similar staining results were obtained with a CHO line expressing the isoform PGHS-Z (not shown). Both PGHS isoforms and have been localized to the endoplasmic reticulum and nuclear envelope (92). As expected, neither E-selectin nor PGHS-l staining patterns were affected by ionophore treatment. CaLB, but not Ser505 Phosphorylation, is Required for Translocation In cell-free systems, the CaLB domain is required for the association of cPLA2 with membranes (200). To confirm the importance of the CaLB 113 - A23187 +A23|87 E5-4 E-selectin PGH synthase-l Figure 24. Confocal microscopy of CHO cells overexpressing various proteins. Indirect immunofluorescence was performed on untransfected CHO cells and stable CHO cell lines expressing cPLA2, E-selectin, and PGHS-l, with (+) and without (-) A23187 treatment. 114 domain for cPLA2 translocation in intact cells, immunofluorescence was performed on a CHO line (ACII) that overexpresses cPLA2 lacking the CaLB domain (amino acids 1-134) (200). As shown in Figure 25 A, no increase in nuclear envelope staining was observed, indicating that this truncated cPLA2 is unable to translocate in response to ionophore. Consistent with this result, ionophore did not induce an increase in staining intensity in these cells (Figure 25 B). The effect of a mutation at the MAP kinase phosphorylation site, Ser505, was also investigated. MAP kinase phosphorylation at this residue has previously been shown to increase the intrinsic enzymatic activity of cPLA2 (22,197). To determine whether this phosphorylation was involved in regulating translocation, immunofluorescence staining of CHO cells expressing SA505-cPLA2, containing a serine-to-alanine mutation at this residue, was performed. The translocation of SA505-cPLA2 in response to ionophore was indistinguishable from that observed with wildtype cPLA2 (Figure 26). The increase in staining intensity seen with wildtype cPLA2 upon inophore treatment was also observed in the SA505 line, further supporting the ability of this mutant to translocate to the membrane. To confirm that deletion of the CaLB domain, but not the mutation of Ser- 505, abolished cPLA2 translocation, fractionation was performed with cells expressing these mutants. After ionophore treatment, cell lysates were centrifuged at 100,000 x g and the soluble and particulate fractions immunoblotted for cPLA2. As shown in Figure 27, both cPLA2 and SA505 redistributed to the particulate fraction upon ionophore treatment, whereas ACII did not. The insert shown below the ACII portion of the figure represents a longer exposure of the blot, revealing the translocation of endogenous cPLA2 in the ACII line. These data establish that phosphorylation at Ser505 is not required for cPLA2 translocation. 115 Figure 25. Translocation of cPLA2 is dependent on the CaLB domain. (A) ACII cells, which stably express cPLA2 lacking the CaLB domain, show similar staining in untreated H and ionophore stimulated (+) cells. Staining and visualization are the same as described in the legend to Figure 23. (B) Quantification of fluorescence intensity. Staining intensity was measured as described in Figure 23 B. 116 - A23187 + A23187 ACII 1100 Fluorescence Intensity (arbitrary units) LII (J: o 275‘ ER+ NE Cyto -A23187 +A23 187 FIGURE 25 117 _ A23187 + A23187 Figure 26. Translocation of SA505-cPLA2. Cells stably overexpressing SA505- cPLA2, which contains a serine-to-alanine mutation at the Ser505 MAP kinase phosphorylation site, were probed with antibody to cPLA2. Indirect immunofluorescence was performed as described in the legend to Figure 23. As seen with wildtype cPLA2 (Figure 23), ionophore (+A23187) induces the translocation of SA505-cPLA2 to the nuclear membrane as well as an increase in overall fluorescent staining intensity. 118 WT ACII SA505 A23187: - + - + — 'I- S P S P S P S P S P S P 116- .3 - - - J CPLA2 86- .. :I A011 58 - .h- -.. - -' t - - CHO cPLA2 Figure 27. Fractionation of lysates from wildtype, ACII and SA505 cells. Cells overexpressing each cPLA2 construct were stimulated with 2 uM ionophore for 10 min (+) or left untreated (-). Lysates were spun at 100,000 x g, and 5 ug of protein from the supernatant (S) and pellet (P) fractions electrophoresed on SDS PAGE. Proteins were transferred to nitrocellulose and immunoblotted for cPLA2. Development was by chemiluminescence. The positions of full length cPLA2 and ACII are indicated. The insert below the ACII portion of the blot represents a longer exposure of these lanes. Endogenous cPLA2 can be seen to translocate normally in the ACII line. * indicates a cross-reactive species of unknown origin. 119 Conversely, translocation is not required for Ser505 phosphorylation, as evidenced by the presence of a cPLA2 doublet in the ACII but not the SA505 line. The upper band of the cPLA2 doublet has previously been established to be a result of phosphorylation at Ser505 (22). These data establish that translocation and phosphorylation occur independently of each another. Both Translocation and Ser505 Phosphorylation are Required for Stimulation of Arachidonic Acid Release In order to investigate the relative importance of translocation and Ser505 phosphorylation for cPLA2-mediated arachidonic acid release, the amount of arachidonic acid liberated upon A23187 treatment was measured in 1354, ACII, SA505 and parental CHO cells (Figure 28). The level of arachidonic acid released from stimulated E5-4 cells was consistently at least 2 to 3 times greater than that released from parental CHO cells. As shown in Figure 28 A, cells expressing ACII did not show enhanced arachidonic acid release in response to A23187 relative to CHO cells. Similarly, SA505-expressing cells showed no increase in arachidonic acid release over the parental line (Figure 28 A). The observation that each of these mutations, separately, can prevent arachidonic acid release establishes that both translocation and Ser505 phosphorylation are indispensable to cPLA2 function in intact cells. 120 350 § 250 § 150 cpm released (x102) _ ‘68 0 'CH6’ ' 554+ ' AC1? A23187 Treatment E 120 _. '8 8 cpm released (x102) 8 8 N C 0 - + - + - + cuo its-4 SA505 A23187 Trcauncnt Figure 28. Effect of ACII and SA505 mutations on arachidonic acid release. Cells were labeled with [3H] arachidonic acid for 20 hr. After washing, cells were stimulated with 2 uM A23187 (+) or left untreated (-) for 10 min, and radioactivity was measured in the media by scintillation counting. The levels of arachidonic acid released from ACII (A) and SA505 (B) are compared with parental CHO cells and E5-4. 121 12' . Cellular Localization of cPLA2 The data presented here establish that in intact cells, cPLA2 is induced to translocate to membranes in response to a rise in intracellular Ca2+. Specifically, cPLA2 binds to the nuclear envelope. The increase in cytOplasmic staining intensity seen upon ionophore treatment suggests that cPLA2 also binds to a cytoplasmic membrane structure. The identity of this cytoplasmic membrane is not yet known, although we suggest that cPLA2 binds both the nuclear envelope and the endoplasmic reticulum, consistent with the localization of PGHS—I and-2 (92). 5-lipoxygenase, which metabolizes arachidonic acid to leukotrienes, as well as its activating protein, FLAP, are localized to the nuclear envelope (202). These results, taken together, implicate the nuclear envelope and endoplasmic reticulum as the primary sites for arachidonic acid production and metabolism in the cell. Consistent with this, we note a complete absence of cPLA2 staining at the plasma membrane. Translocation of cPLA2 is abolished upon deletion of the CaLB domain but not upon mutation of Ser505. This suggests that translocation and phosphorylation regulate cPLA2 independently, a conclusion supported by the observation that ACII is phosphorylated in CHO cells (as evidenced by the doublet in Figure 27). Both translocation and phosphorylation are critical for A23187-induced arachidonic acid release. The observation that these regulatory mechanisms function independently strongly supports a model in which phosphorylation at Ser505 serves primarily to activate the enzymatic activity of cPLA2 (22,197), and translocation allows access of the enzyme to its substrate. 122 This discussion focuses on the localization of cPLA2 to the nuclear envelope and endoplasmic reticulum, although the identity of the cytoplasmic membrane(s) to which cPLA2 binds has not yet been unequivocally established. How cPLA2 is selectively localized to these sites is not known. The translocation of cPLA2 to the nuclear envelope is consistent with previous data indicating that arachidonic acid is preferentially released from the nuclear envelope, as observed in pulse-chase experiments using [14C]arachidonate-labeled HSDM1 C1 cells stimulated with bradykinin (205). Interestingly, electron microscopic studies using [3H]arachidonic acid have shown that the nuclear membrane is the preferred site of initial arachidonic acid incorporation in these cells (206). Arachidonic acid is also incorporated rapidly into the endoplasmic reticulum; transit to the plasma membrane is slow. This study shows that although arachidonic acid is preferentially incorporated at particular sites, it becomes evenly distributed throughout the cell. Thus a correlation exists between the cellular localization of cPLA2 and the sites at which arachidonic acid is initially incorporated into the cell. However, the significance of this correlation is not known. Several arachidonic acid-independent mechanisms could also explain the localization of cPLA2. A ”docking protein” might bind cPLA2 at the nuclear membrane and endoplasmic reticulum, although it is known that such a protein is not essential, since Ca2+ induces cPLA2 membrane binding in the absence of protein (200). It is unlikely that a spatially-localized release of Ca2+ is responsible for the targeting of cPLA2, since our experiments revealed no I translocation of cPLA2 to the plasma membrane with A23187, whose effects are thought to include the triggering of a Ca2+ influx across the plasma membrane. Another possibility is that some feature of the plasma membrane excludes cPLA2, and cPLA2 simply translocates to all cellular membranes 123 which it is capable of binding. Interestingly, the plasma membrane is known to be enriched in sphingolipids compared to the nuclear envelope and endoplasmic reticulum. It is conceivable that the increase in phospholipid packing density that results from a high sphingolipid content may prevent cPLA2 binding. Consistent with this idea, Leslie and Channon (198) have shown that both the activity and calcium sensitivity of cPLA2 are inhibited by the increased substrate packing density induced by sphingolipids. Clearly, a better understanding of the lipid and protein components of distinct cellular membranes will greatly facilitate our understanding of the mechanism(s) governing the localization of cPLA2. Coupling of cPLA2 Induced Arachidonic Acid Release to PGHS As discussed above, the localization of cPLA2 is similar to that reported for PGHS-1 and -2, which metabolize arachidonic acid to prostaglandins. PGHS-1 is expressed constitutively and is thought to perform certain physiological ”housekeeping” functions. PGHS-2, whose expression is induced by cytokines, has been implicated in inflammation (48,49). It is tempting to speculate that cPLA2-mediated release of arachidonic acid couples primarily to PGHS-2, since both enzymes are inducible by a wide variety of agonists. Indeed, antisense oligonucleotide inhibition of cPLA2 has been shown to decrease the level of endotoxin-stimulated PGE2 in monocytes (without affecting PGHS-2 activity) (207). This is significant because PGE2 release in endotoxin-stimulated monocytes has been shown to be dependent on PGHS-2 activity even in the presence of PGHS-1 (32), suggesting a coupling between cPLA2 and PGHS-2. Studies in bone marrow-derived mast cells demonstrate the coupling of PGHS-1 and -2 to different stimuli (31), further supporting the idea that the PGHS isoforms participate in different signaling pathways and 124 may metabolize arachidonic acid from distinct pools. Experiments using an antisense oligonucleotide to the group II PLA2 suggest that in macr0phages stimulated with endotoxin and platelet-activating factor, different PLA2 enzymes participate in different phases of arachidonic acid release (208). These data, taken together, suggest that different stimuli may induce distinct PLA2 enzymes, which may themselves each couple to a specific PGHS isoform. The presence of cPLA2 in the nuclear envelope and (most likely) the endoplasmic reticulum correlates with the localization of both PGHS-1 and PGHS-2 However, a recent study that more closely examines the localization of these isoforms reveals that their cellular distributions overlap but are not identical. PGHS-1 was shown to be equally distributed in the endoplasmic reticulum and nuclear envelope, whereas PGHS—2 was twice as concentrated in the nuclear envelope as in the endoplasmic reticulum (93). Localization of the cyclooxygenase/ peroxidase activity of these isoforms using a histofluorescent staining method revealed an even more striking difference. The fluorescent product formed from PHGS-l activity was found in the cytoplasm, whereas that formed from PGHS-2 was detected in both the cytoplasm and within the nucleus. The colocalization of cPLA2 protein and PGHS-2 activity at the nuclear membrane is consistent with a coupling between these enzymes. However, as cPLA2 also appears to translocate to the endoplasmic reticulum, where both PGHS isoforms are located, a role in releasing arachidonic acid to PGHS-1 is also possible. Although the mechanisms of arachidonic acid transfer between the enzymes in this cascade have not yet been elucidated in detail, the colocalization of cPLA2 with PGHS and 5-LO is certain to ensure the efficient utilization of arachidonic acid upon 125 agonist stimulation, consistent with a central role for cPLA2 in the agonist— induced biosynthesis of prostaglandins and leukotrienes. 126 Acknowledgment The work in this chapter was conducted in collaboration with A. Schievella and L-L. Lin at Genetics Institute, Cambridge, Massachusetts. This chapter was written as a manuscript in preparation for publication. We are grateful to James Clark, Eric Nalefski, John Knopf and David DeWitt for helpful discussions. We would also like to thank Edwin de Feijter (Meridian Instruments, Okemos, MI) for excellent technical assistance in confocal microscopy and image processing. We also thank Jennifer Chen for the PGHS-1 cell line, Dina Martin for the ACII line, and Mary Shaffer for the E-selectin line. This work was supported in part by United States Public Health Service National Institutes of Health Grants DK22042 and DK20945 (WLS). CONCLUSION There is an increasing number of observations which suggest, when they are considered together, that PGH synthase isozymes have distinct biological roles. One observation is the unique patterns of isozyme expression. PGH synthase-1 is present constitutively which affords the cell a pathway for immediate synthesis of prostaglandins in response to hormonal signals associated with maintenance of physiological homeostasis. PGH synthase-2 prostaglandin synthesis is possible only after induction of the enzyme by an appropriate stimulus. Many of the extracellular signals which induce PGH synthase-2 are stimuli of mitogenesis or inflammation. A second observation is that prostaglandin biosynthetic capacity increases only 1.5-2 fold after induction of PGH synthase-2 (30,69,74) . Furthermore, the kinetic properties of PGH synthase-1 and -2 are very similar. It seems unlikely that PGH synthase-2, which has a similiar affinity for substrate, is induced soley to supplement PGH synthase-1 synthesis of PGHZ. Functional separation of isozymes is likely to occur at many levels. Evidence suggests that arachidonate is stored in isozyme-specific pools (31,32). In fibroblasts and macrophages, phorbol ester and LPS treatment induce PGH synthase-2 expression and mobilize archidonate. However, constitutive PGH synthase-1 does not utilize this supply of arachidonate for substrate (32). This suggests that PGH synthase-1 utilizes a unique pool of arachidonate which is separate from the pool mobilized by PGH synthase-2 inducers. In bone 127 128 marrow-derived mast cells prostaglandin synthesis from endogenous arachidonate occurs by two paths: a cytokine/PGH synthase-2 pathway and a IgE/ PGH synthase—1 pathway (31). It is appears that these pathways are segregated by specific coupling of a specific agonist with a specific arachidonate pool. Arachidonate pools may result from different phospholipase activities such as cytosolic PLA2 and secretory PLA2, or different sources such as LDL (27,28), lipid bodies (172), and membranes. The co-localization of cPLA2 and PGH synthase-1 and -2 to the ER and NE demonstrates cPLA2 is in a suitable cellular location to provide arachidonate to PGH synthase isozymes. Whether cPLA2 arachidonate release is channeled specifically to one of the isozymes remains to be seen. Differential subcellular localization is another potential mechanism for functional separation of PGH synthase isozymes. While PGH synthase isozymes are localized to the same membrane systems, concentration of PGH synthase-2 at the NE is unique (93). It has also been shown that PGH synthase enzyme activity is uniquely distributed. PGH synthase-2 products are concentrated in the nucleus, while PGH synthase-1 products are mostly in the cytoplasm (93). Taken together this suggests that the biosynthetic pathways are separated by subcellular distribution of enzyme and products. Whether it be different substrate pools, stimulus coupling, and/ or localization of PGH synthase protein, it is expected that isozyme segregation is in some way related to protein structure. There are several regions of protein structural distinction between the isozymes. PGH synthase-2 has a unique 18 amino acid sequence near the C-terminus. We report here a mutant murine PGH synthase-2 that lacks this region, PGH synthase-2Acassette, which is catalytically similar to the native enzyme. 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