- Email: [email protected]
Prostaglandin F2α stimulates tyrosine phosphorylation of phospholipase C-γ1
BBRC Biochemical and Biophysical Research Communications 297 (2002) 1102–1107 www.academicpress.com
Prostaglandin F2a stimulates tyrosine phosphorylation of phospholipase C-c1 Shahid Husaina,* and Farahdiba Jafrib a
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA b Department of Clinical Pharmacy, Medical College of Georgia, Augusta, GA 30912, USA Received 4 September 2002
Abstract In this study, we investigated the ability of prostaglandin F2a (PGF2a ) to induce tyrosine phosphorylation of phospholipase C-c1 (PLC-c1) in cat iris sphincter smooth muscle (CISM) cells. PGF2a ð1 lMÞ-stimulated PLC-c1 tyrosine phosphorylation in a time- and dose-dependent manner with a maximum increase of 3-fold at 0.5 min. The protein tyrosine kinase inhibitors, genistein, and tyrphostin A-25, blocked the stimulatory effects of PGF2a , suggesting involvement of protein tyrosine kinase activity in the physiological actions of the PGF2a . Furthermore, PGF2a -induced p42/p44 MAP kinase activation was also completely blocked by protein tyrosine kinase inhibitors. In summary, these findings show that PGF2a stimulates tyrosine phosphorylation of PLC-c1 in CISM cells and indicate that PGF2a -stimulated tyrosine phosphorylation is responsible for an early signal transduction event. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Phospholipase C-c1; Tyrosine phosphorylation; Prostaglandin F2a ; Non-vascular smooth muscle
Prostaglandins (PG) exert a broad range of physiological and pharmacological actions in a wide variety of tissues through interaction with specific cell-surface receptors. All PG receptors identified to date are seventransmembrane proteins that couple to specific Gproteins mediating the formation of cAMP or inositol phosphate/diacylglycerol second messengers [1]. In the eye, prostaglandin F2a ðPGF2a Þ and its analog latanoprost (PhXA41) mediate, through prostaglandin F2a (FP) receptors, a broad range of biological effects including smooth muscle contraction and reduction of intraocular pressure (IOP) in glaucoma patients [2]. FP receptors activation in a number of cell types has been shown to result in the generation of inositol phosphates via phospholipase C (PLC) activation with a subsequent mobilization of intracellular Ca2þ [3] and activation of a number of protein kinases such as Ca2þ -calmodulin-dependent protein kinase II, myosin light chain kinase, protein kinase C, and mitogen-activated protein (MAP) kinase [4,5].
*
Corresponding author. Fax: 1-706-721-6608. E-mail address: [email protected] (S. Husain).
Three families of mammalian PLC isozymes designated b, c, and d have been described based on their molecular structure and mechanisms of regulation [6]. The PLC-b group has been shown to be regulated by the Gq-class of heterotrimeric GTP-binding proteins [7,8]. The PLC-c family of isozymes appears to be regulated by tyrosine phosphorylation [9]. The regulatory mechanisms of PLC-d has not yet been elucidated although they do not appear to be stimulated by G-proteins or tyrosine phosphorylation [6]. Little information is available concerning PGF2a -induced PLC-c1 phosphorylation and down-stream signaling pathways in ocular smooth muscle. Therefore, the purpose of the present study was to determine the effects of PGF2a on tyrosine phosphorylation of PLC-c1 and identification of down-stream signaling targets in cat iris sphincter smooth muscle (CISM) cells. We show that PGF2a induces transient phosphorylation of PLC-c1, which leads to the activation of mitogen-activated protein (MAP) kinases in CISM cells. Furthermore, PGF2a -induced phosphorylation of PLC-c1 and p42/p44 MAP kinase were completely inhibited in the presence of protein tyrosine kinase inhibitors.
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 2 3 4 7 - 1
S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107
Materials and methods Materials Anti-PLC-c1 and anti-p42/p44 MAP kinase antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). [c-32 P]ATP (specific activity, 3000 Ci/mmol) was obtained from Amersham Life Science (Arlington Height, IL). PGF2a , genistein, and herbimycin A, were obtained from Calbiochem (La Jolla, CA). Mylein basic protein, protein A–Sepharose, and tyrphostin A-25 were purchased from Sigma Chemical, (St. Louis, MO). Fetal bovine serum was obtained from Hyclone (Logan, UT) and all other cell culture supplies were obtained from Cell gro (Herndon, VA). Methods Cell culture. Iris sphincter smooth muscle cells were isolated from 4- to 6-months-old cats as described previously [10]. Briefly, the eyes were enucleated immediately after the death of the animal and sphincter muscle was dissected out, further cleaned and cut into 1– 2 mm2 pieces. The explants were placed in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mg ml1 collagenase type IA, 10% fetal bovine serum (FBS) and 50 lg ml1 gentamicin and then incubated for 1–2 h at 37 °C with occasional gentle shaking. The cell suspension was centrifuged at 200g and resuspended in DMEM/F-12 supplemented with 10% (v/v) FBS and 100 U ml1 penicillin, 100 lg ml1 streptomycin, and 0.25 lg ml1 amphotericin B in 5% CO2 humidified atmosphere. The contaminating fibroblasts were removed as previously described [10]. After 3 days, one third of the culture medium was replaced with fresh medium. The smooth muscle cells were subcultured at a split ratio of 1:3 using 0.05% trypsin and 0.02% EDTA. The cells of 3–7 passages were used in this study. Immunoprecipitation of PLC-c1 and Western blotting. CISM cells were starved in serum-free DMEM for 24 h and treated with PGF2a for indicated time periods. The inhibitors were added 15 min prior to the addition of the PGF2a . After treatment, cells were lysed in 50 mM Tris– HCl buffer, pH 8.0, containing 100 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, 50 mM NaF, 1 mM Na3 VO4 , 5 mM PMSF, 10 lg ml1 leupeptin, and 50 lg ml1 aprotinin for 20 min on ice. Cell lysates were centrifuged at 2500g for 10 min and the supernatant was used either for immunoprecipitation of PLC-c1 or for immuno-detection of p42/p44 MAP kinase. PLC-c1 was isolated by immunoprecipitation using rabbit anti-PLC-c1 polyclonal antibodies for 2 h followed by an incubation for 12 h with protein A–Sepharose at 4 °C. The immunoprecipitates were washed and extracted with Laemmli buffer as described earlier [10]. The proteins were resolved on 10% SDS–polyacrylamide gels and then transferred to nitrocellulose membranes. The membranes were probed with anti-phospho-tyrosine, anti-PLC-c1, or anti-p42/p44 MAP kinase antibodies followed by incubation with secondary antibodies (HRP-conjugated goat anti-rabbit IgG at 1:3000 dilution) for 1 h at 20 °C as previously described [4,10]. For chemiluminescent detection, the membranes were treated with ECL reagent for 1 min and subsequently exposed to ECL hyperfilm for 1–2 min. Measurement of p42/p44 MAP kinase activity in CISM cells. MAP kinase activity was characterized by the in situ myelin basic protein (MBP) phosphorylation assay as described earlier [4,10]. Briefly, quiescent cells were stimulated with 1 lM PGF2a and scraped into ice-cold extraction buffer (20 mM b-glycero-phosphate, 20 mM NaF, 2 mM EDTA, 0.2 mM sodium vanadate, 1 mM PMSF, 25 lg ml1 leupeptin, 10 lg ml1 aprotinin, and 0.3% (v/v) b-mercaptoethanol, pH 7.5). The cell extracts were centrifuged at 10,000g for 10 min at 4 °C, and the supernatant was resolved on a 10% SDS–polyacrylamide gel co-polymerized with 0.5 mg ml1 MBP. After electrophoresis, the gels were washed with 50 mM Tris–HCl buffer, pH 8.0, containing 20% (v/v) propanol to remove SDS, then washed with denaturing buffer (50 mM Tris–HCl, pH 8.0, containing 6 M guanidine hydrochloride and 5 mM
1103
b-mercaptoethanol). The enzymes on the gel were then renatured by washing with 50 mM Tris–HCl buffer, pH 8.0, containing 0.04% (v/v) Tween 40 and 5 mM b-mercaptoethanol at 4 °C for 21 h. The gel was then preincubated with assay buffer containing 40 mM Hepes, pH 8.0, 10 mM MgCl2 , 2 mM dithiothreitol, and 0.1 mM EGTA at 30 °C for 30 min. The MAP kinase activity was determined by incubating the gel with 20 ml of the assay buffer, which contained 20 lM ATP and 100 lCi [c-32 P]ATP, at 30 °C for 1 h. After extensive washing in 5% (w/v) trichloroacetic acid containing 10 mM sodium pyrophosphate, the gel was dried and autoradiographed at )70 °C.
Results and discussion Results Immunochemical identification of PLC-c1 We examined the presence of PLC-c1 in cat iris sphincter smooth muscle (CISM) cells by polyclonal antibodies specific for PLC-c1 isoform. The anti-PLCc1 antibodies reacted with a 145-kDa protein. As shown in Fig. 1, PLC-c1 is predominantly present in cytosolic fraction. Cytosolic and membrane fractions were obtained by centrifugation at 100,000g. The specificity of PLC-c1 band was confirmed by loss of the immunoreactive band upon incubation with the appropriate blocking peptide (i.e., an antigenic peptide that was used to produce PLC-c1 antibodies). PLC-c1 band was completely disappeared when 1:1 ratio of antibodies and blocking peptides were used, suggesting that 145-kDa band correspond to PLC-c1 isoform. Time course study of PGF2a -induced tyrosine phosphorylation of PLC-c1 To investigate the effects of PGF2a on PLC-c1 phosphorylation, PLC-c1 was immunoprecipitated from crude homogenates of PGF2a treated and untreated (control) CISM cells with polyclonal antibodies specific to PLC-c1. PGF2a -stimulated tyrosine phosphorylation of PLC-c1 in CISM cells was analyzed by two different protocols. First, anti-PLC-c1 antibodies were used to immunoprecipitate the PLC-c1 from PGF2a treated and
Fig. 1. Immunochemical indentification of PLC-c1 in CISM cells. Western blot analysis of PLC-c1 in cytosol (3), membrane (2), and total homogenate (1) of CISM cells. Appropriate amounts (10–15 lg protein) were subjected to 10% SDS–PAGE. Proteins were transferred to nitrocellulose membranes and immunoblotted with antibodies specific to PLC-c1 as described in Materials and methods. Results given are from a representative experiment of three separate experiments.
1104
S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107
probed with anti-PLC-c1 antibodies. These antibodies selectively recognized one band of 145 kDa and all the samples showed a comparable amounts of immunoprecipitated PLC-c1 in these cells (Fig. 2B).
Fig. 2. Time course study for PGF2a -stimulated tyrosine phosphorylation of PLC-c1. Starved CISM cells were treated for the indicated times periods with 1 lM PGF2a and harvested with lysis buffer as described in Materials and methods. In panel A, proteins were immunoprecipitated with an anti-PLC-c1 antibodies and developed with anti-phospho-tyrosine antibodies. Panel B shows Western blot analysis of immunoprecipitates that were probed with anti-PLC-c1 antibodies. Results given are from one experiment that is a representative of five separate experiments (n ¼ 5).
untreated homogenates. Western blot analysis of PLCc1 immunoprecipitates with anti-phospho-tyrosine antibodies showed an increase in tyrosine phosphorylation of PLC-c1 by 3-fold relative to control levels (Fig. 2A). The peak response was seen at 0.5 min and returned to near control levels by 5 min. Similar results were seen when tyrosine-phosphorylated protein were immunoprecipitated using anti-phospho-tyrosine antibodies followed by detection of PLC-c1 by Western blot analysis with anti-PLC-c1 antibodies (data not shown). The above data clearly show an increased in tyrosine phosphorylation of PLC-c1 with maximal phosphorylation observed approximately 0.5 min after addition of PGF2a . To rule out the possibility of variation in the loaded amounts of protein, immunoprecipitates were
Fig. 3. Dose dependence of PGF2a -stimulated tyrosine phosphorylation of PLC-c1. CISM cells were starved and incubated with indicated concentrations of PGF2a for 0.5 min and harvested with lysis buffer as described in Materials and methods. (A) Tyrosine phosphorylation of PLC-c1 in immunoprecipitates, (B) Western blot analysis of PLC-c1 as described in legend to Fig. 2. Results given are from one experiment that is a representative of five separate experiments (n ¼ 5).
Dose-dependence of PGF2a on tyrosine phosphorylation of PLC-c1 CISM cells were treated with indicated concentrations of PGF2a and PLC-c1 was immunoprecipitated from homogenates as described in Fig. 2. As shown in Fig. 3A, PGF2a stimulates tyrosine phosphorylation of PLC-c1 in a dose-dependent manner. The maximum increase in tyrosine phosphorylation of PLC-c1 was observed at 1 lM of PGF2a . All the samples contained comparable amounts of immunoprecipitated PLC-c1 (Fig. 3B). Effects of tyrosine kinase inhibitors on PGF2a -induced phosphorylation of PLC-c1 To investigate the role of protein tyrosine kinase in PGF2a -induced PLC-c1 phosphorylation, we used tyrosine kinase inhibitors, genistein, and tyrphostin A-25. At 1 lM, both compound blocked PGF2a -induced tyrosine phosphorylation of PLC-c1 completely (Fig. 4A). These findings suggest that a tyrosine kinase mediates PGF2a -stimulated phosphorylation of the PLC-c1 in CISM cells. In all the samples a comparable amounts of PLC-c1 was immunoprecipitated (Fig. 4B). Effects of PGF2a on p42/p44 MAP kinase activation MAP kinases are one of the most important signaling components involved in the regulation of cytoslic
Fig. 4. Effects of protein tyrosine kinase inhibitors on PGF2a -stimulated tyrosine phosphorylation of PLC-c1. CISM cells were incubated in serum-free DMEM with 1lM PGF2a in the absence or presence of 1 lM genistein or tyrphostin A-25. Inhibitors were added 15 min prior to the addition of PGF2a . (A) Tyrosine phosphorylation of PLC-c1 in immunoprecipitates, (B) Western blot analysis of PLC-c1 as described in legend to Fig. 2. Results given are from one experiment that is a representative of five separate experiments (n ¼ 5).
S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107
phospholipase A2 , arachidonic acid (AA) release, and matrix metalloproteinases (MMPs) secretion in various tissues. Several lines of evidence suggest that MMPs are involved in the regulation of intraocular pressure (IOP) in normal and glaucoma human eyes. Since, PGF2a and its analogs are widely used to lower IOP, it is important to understand PGF2a -induced signaling mechanisms. Recently, we have shown that PGF2a activates p42/p44 MAP kinase through a PKC-dependent pathway in CISM cells [4]. However, their up-stream regulation remained unclear in these cells. To answer these questions, CISM cells were stimulated with 1 lM PGF2a for various times of incubation and p42/p44 MAP kinase activity was measured by in-gel renaturation kinase assay as described in Materials and methods. As shown in Fig. 5A PGF2a stimulates p42/p44 MAP kinase activity in a time-dependent manner. There is a significant increase in the activity of p42/p44 MAP kinase after 0.5 min of PGF2a treatment, which was sustained upto 5 min. To establish a correlation between PLC-c1 and p42/p44 MAP kinase, cells were stimulated with PGF2a in the absence or presence of protein tyrosine kinase inhibitors and MAP kinase activity was measured. As shown in Fig. 6A, PGF2a -induced p42/p44 MAP kinase activation was significantly inhibited in the presence of 1 lM of genistein, herbimycin A, and tyrphostin A-25. Furthermore, PGF2a -induced p42/p44 MAP kinase activation was completely inhibited in the presence of 10 lM of these compounds (data not shown). A specific inhibitor of p42/p44 MAP kinase, PD-98059, also inhibited PGF2a -induced p42/p44 MAP kinase activation. To rule out the possibility of variation in the amounts of loaded protein samples on the gels, protein were transferred to nitrocellulose membranes and immunoblotted with anti-p42/p44 MAP kinase antibodies. All samples contained comparable amounts of p42/p44 MAP kinase (Figs. 5B and 6B).
Fig. 5. Dose dependence of PGF2a on p42/p44 MAP kinase activation. Starved CISM cells were incubated in the absence (control) or presence of 1 lM PGF2a for indicated time periods. (A) p42/p44 MAP kinase activity as described in Materials and methods, (B) Western blot analysis by anti-p42/p44 MAP kinase antibodies. Results given are from one experiment that is a representative of five separate experiments (n ¼ 5).
1105
Fig. 6. Effects of protein tyrosine kinase inhibitors on PGF2a -stimulated p42/p44 MAP kinase activation. Starved CISM cells were incubated with 1 lM PGF2a in the absence or presence of 1 lM genistein, tyrphostin A-25, herbimycin A, or PD-98059. Inhibitors were added 15 min prior to the addition of PGF2a . (A) p42/p44 MAP kinase activity as described in Materials and methods, (B) Western blot analysis by anti-p42/p44 MAP kinase antibodies. Results given are from one experiment that is a representative of five separate experiments (n ¼ 5).
Discussion Glaucoma is a group of human disorders characterized by progressive loss of retinal ganglion cells with an associated loss of vision. Derivatives of prostaglandin F2a (i.e., activate the FP-prostanoid receptors) are the most potent and effective topical ocular hypotensive agents currently known. Physiological studies in animals, normal subjects [11,12], and glaucoma patients [13] indicate a remarkable reduction in intraocular pressure (IOP) in the presence of PGF2a and its analog latanoprost (PhXA41). The underlying molecular mechanisms of action of anti-glaucoma drugs and causes of glaucoma are still unknown. It is important to investigate signaling mechanisms induced by anti-glaucoma drugs such as PGF2a in ocular smooth muscle, which could shed more light on the molecular mechanisms underlying the IOP-lowering effects of PGF2a in glaucoma patients. The major finding of this study is that PGF2a stimulates tyrosine phosphorylation of PLC-c1 (Figs. 2 and 3). This is the first study to show a relationship between PGF2a -stimulated tyrosine phosphorylation of PLC-c1 and p42/p44 MAP kinase in CISM cells. Both PLC-c1 phosphorylation and p42/p44 MAP kinase activation were inhibited in the presence of protein tyrosine kinase inhibitors (Figs. 4 and 6). In addition, data also suggest that G-protein-coupled receptors such as the FP-receptors, which have been thought to activate only PLC-b [14], can also stimulate PLC-c1 in CISM cells. Several
1106
S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107
receptors that stimulate PLC are thought to act via G-proteins [15], because their activation of PLC is modulated by bacterial toxins, aluminum fluoride, and analogs of GTP, which are known to modify the actions of G-proteins. At present, the biochemical link between the FP-receptors and tyrosine phosphorylation of PLC-c1 is not known in CISM cells. The tyrosine phosphorylation has been reported for growth factor receptors that are themselves kinases such as PDGF and epidermal growth factors [16,17]. However, tyrosine phosphorylation has also been reported for receptors that lack kinase activity [18–20]. Previous studies have shown that the actions of PGF2a on inositol-1,4,5-trisphosphate (IP3 ) production and Ca2þ -mobilization, and contraction were inhibited by protein tyrosine kinase inhibitors in cat iris sphincter smooth muscle [14]. Angiotensin II stimulated PLC-c1 tyrosine phosphorylation by 4.5-fold at 0.5 min in rat aortic vascular smooth muscle cells [21]. Palmier et al. [22] reported that orthovanadate, a potent phosphatase inhibitor, mediated an increased generation of inositol phosphates and contraction in rat myometrium through phosphorylation and activation of PLC-c1. Gould et al. [23] reported that genistein inhibited both contraction and Ca2þ -mobilization induced by activation of receptors for histamine in porcine carotid arterial smooth muscle. In conclusion, the data obtained suggest that PGF2a induced transient tyrosine phosphorylation of PLC-c1. Furthermore, data of protein tyrosine kinase inhibitors suggest an important role of protein tyrosine kinase in PGF2a -induced phosphorylation and activation of PLCc1. One or more intracellular tyrosine kinases must be stimulated by the PGF2a –FP-receptor interaction. Which initiates a signaling cascade and leads to the activation of protein kinases including p42/p44 MAP kinase. The understanding of the signaling mechanisms of action of PGF2a in the smooth muscle of iris-ciliary body could lead to the development of rational and more effective anti-glaucoma drugs.
Acknowledgment A Combined Intramural Research Grant awarded to S. Husain from Medical College of Georgia supported this work.
References [1] R.A. Coleman, W.L. Smith, S. Narumiya, International union of pharmacology classification of prostanoid receptors: Properties, distribution, and structure of the receptors and their subtypes, Pharmacol. Rev. 46 (1994) 205–229. [2] L.Z. Bito, J. Stjernschantz, B. Resul, O.C. Miranda, S. Basu, The ocular effects of prostaglandins and the therapeutic potential of a new PGF2a analog, PhXA41 (latanoprost), for glaucoma management, J. Lipid Mediators 6 (1993) 535–543.
[3] B.W. Griffin, P.E. Magnino, I.-H. Pang, N.A. Sharif, Pharmacological characterization of an FP prostaglandin receptor on rat vascular smooth muscle cells (A7r5) coupled to phosphoinositide turnover and intracellular calcium mobilization, J. Pharmacol. Exp. Ther. 286 (1998) 411–418. [4] S. Husain, A.A. Abdel-Latif, Effects of PGF2a and carbachol on MAP kinases, cytosolic phospholipase A2 and arachidonic acid release in cat iris sphincter smooth muscle cells, Exp. Eye Res. 72 (2001) 581–590. [5] H.R. Ansari, S. Husain, A.A. Abdel-Latif, Role of p42/p44 MAP kinases and calcium-dependent calmodulin kinases II in PGF2a , ionomycin, and thapsigargin-induced muscle contraction in cat iris sphincter smooth muscle, J. Pharmacol. Exp. Ther. 299 (2001) 178–186. [6] S.G. Rhee, K.D. Choi, Regulation of inositol phospholipidspecific phospholipase C isozymes, J. Biol. Chem. 267 (1992) 12393–12396. [7] A.V. Smarka, J.R. Hepler, K.O. Brown, P.C. Sternweis, Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq, Science 251 (1991) 804–807. [8] S.J. Taylor, H.Z. Chae, S.G. Rhee, J.H. Exton, Activation of the b1 isozyme of phospholipase C by alpha subunits of the Gq class of G-proteins, Nature 350 (1991) 516–518. [9] H.K. Kim, J.W. Kim, A. Zilberstein, B. Margolis, C.K. Kim, J. Schlesinger, S.G. Rhee, PDGF stimulation of inositol phospholipid hydrolysis requires PLC-c 1 phosphorylation on tyrosine residues 783 and 1254, Cell 65 (1991) 435–441. [10] S. Husain, A.A. Abdel-Latif, Role of protein kinase C-a in endothelin-1 stimulation of phospholipase A2 and arachidonic acid release in cultured cat iris sphincter smooth muscle cells, Biochim. Biophys. Acta 1392 (1998) 127–144. [11] J.R. Kerstetter, R.F. Brubaker, S.E. Wilson, L.J. Kullerstrand, Prostaglandin F2a -1-isopropylester lowers intraocular pressure without decreasing aqueous flow, Am. J. Ophthalmol. 105 (1988) 30–34. [12] P.Y. Lee, H. Shao, L. Xu, C.-K. Qu, The effect of prostaglandin F2a on intraocular pressure in normotensive human subjects, Invest. Ophthalmol. Vis. Sci. 29 (1988) 1474–1477. [13] J. Stjernschantz, G. Selen, B. Sjoquist, B. Resul, Preclinical pharmacology of latanoprost, a phenyl-substituted PGF2a analog. Ad. in prostaglandin, Thrombox. Leukotr. Res. 23 (1995) 513– 518. [14] S.Y.K. Yousufzai, A.A. Abdel-Latif, Tyrosine kinase inhibitors suppress prostaglandin F2a -induced phosphoinositide hydrolysis, Ca2þ elevation and contraction in iris sphincter smooth muscle, Eur. J. Pharmacol. 360 (1998) 185–193. [15] S.G. Rhee, K.D. Choi, Multiple forms of phospholipase C isozymes and their activation mechanisms, Adv. Second Messenger-phosphoprotein Res. 26 (1992) 35–61. [16] B. Margolis, S.G. Rhee, S. Felder, M. Mervie, R. Lyall, A. levitzki, A. Ullrich, A. Zilberstein, J. Schlessinger, EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling, Cell 57 (1989) 1101– 1107. [17] D.A. Kumjian, A. Barnstein, S.G. Rhee, T.O. Daniel, Phospholipase C-c complexes with ligand-activated plateletderived growth factor receptors. An intermediate implicated in phospholipase activation, J. Biol. Chem. 266 (1991) 3973– 3980. [18] T. Tsuda, Y. Kawahara, K. Shii, M. Koide, Y. Ishida, M. Yokoyama, Vasoconstrictor-induced protein-tyrosine phosphorylation in cultured vascular smooth muscle cells, FEBS Lett. 285 (1991) 44–48. [19] D.J. Park, H.W. Rho, S.G. Rhee, CD3 stimulation causes phosphorylation of phospholipase C-c1 on serine and tyrosine residues in a human T-cell line, Proc. Natl. Acad. Sci. USA 88 (1991) 5453–5456.
S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107 [20] R.H. Carter, D.J. Park, S.G. Rhee, D.T. Fearon, Tyrosine phosphorylation of phospholipase C induced by membrane immunoglobulin in B lymphocytes, Proc. Natl. Acad. Sci. USA 88 (1991) 2745–2749. [21] M.B. Marrero, W.G. Paxton, J.L. Duff, B.C. Berk, K.E. Bernstein, Angiotensin-II stimulates tyrosine phosphorylation of PLCc1 in vascular smooth muscle cells, J. Biol. Chem. 269 (1994) 10935–10939.
1107
[22] B. Palmier, D. Leiber, S. Harbon, Pervanadate mediated an increased generation of inositol phosphates and tension in rat myometrium. Activation and phophorylation of PLC-c1, Biol. Reprod. 54 (1996) 1383–1389. [23] E.M. Gould, C.M. Rembold, R.A. Murphy, Genistein a tyrosine kinase inhibitor reduces Ca2þ -mobilization in swine carotid artery, Am. J. Physiol. 268 (1995) 1425–1429.
Prostaglandin F2a stimulates tyrosine phosphorylation of phospholipase C-c1 Shahid Husaina,* and Farahdiba Jafrib a
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA b Department of Clinical Pharmacy, Medical College of Georgia, Augusta, GA 30912, USA Received 4 September 2002
Abstract In this study, we investigated the ability of prostaglandin F2a (PGF2a ) to induce tyrosine phosphorylation of phospholipase C-c1 (PLC-c1) in cat iris sphincter smooth muscle (CISM) cells. PGF2a ð1 lMÞ-stimulated PLC-c1 tyrosine phosphorylation in a time- and dose-dependent manner with a maximum increase of 3-fold at 0.5 min. The protein tyrosine kinase inhibitors, genistein, and tyrphostin A-25, blocked the stimulatory effects of PGF2a , suggesting involvement of protein tyrosine kinase activity in the physiological actions of the PGF2a . Furthermore, PGF2a -induced p42/p44 MAP kinase activation was also completely blocked by protein tyrosine kinase inhibitors. In summary, these findings show that PGF2a stimulates tyrosine phosphorylation of PLC-c1 in CISM cells and indicate that PGF2a -stimulated tyrosine phosphorylation is responsible for an early signal transduction event. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Phospholipase C-c1; Tyrosine phosphorylation; Prostaglandin F2a ; Non-vascular smooth muscle
Prostaglandins (PG) exert a broad range of physiological and pharmacological actions in a wide variety of tissues through interaction with specific cell-surface receptors. All PG receptors identified to date are seventransmembrane proteins that couple to specific Gproteins mediating the formation of cAMP or inositol phosphate/diacylglycerol second messengers [1]. In the eye, prostaglandin F2a ðPGF2a Þ and its analog latanoprost (PhXA41) mediate, through prostaglandin F2a (FP) receptors, a broad range of biological effects including smooth muscle contraction and reduction of intraocular pressure (IOP) in glaucoma patients [2]. FP receptors activation in a number of cell types has been shown to result in the generation of inositol phosphates via phospholipase C (PLC) activation with a subsequent mobilization of intracellular Ca2þ [3] and activation of a number of protein kinases such as Ca2þ -calmodulin-dependent protein kinase II, myosin light chain kinase, protein kinase C, and mitogen-activated protein (MAP) kinase [4,5].
*
Corresponding author. Fax: 1-706-721-6608. E-mail address: [email protected] (S. Husain).
Three families of mammalian PLC isozymes designated b, c, and d have been described based on their molecular structure and mechanisms of regulation [6]. The PLC-b group has been shown to be regulated by the Gq-class of heterotrimeric GTP-binding proteins [7,8]. The PLC-c family of isozymes appears to be regulated by tyrosine phosphorylation [9]. The regulatory mechanisms of PLC-d has not yet been elucidated although they do not appear to be stimulated by G-proteins or tyrosine phosphorylation [6]. Little information is available concerning PGF2a -induced PLC-c1 phosphorylation and down-stream signaling pathways in ocular smooth muscle. Therefore, the purpose of the present study was to determine the effects of PGF2a on tyrosine phosphorylation of PLC-c1 and identification of down-stream signaling targets in cat iris sphincter smooth muscle (CISM) cells. We show that PGF2a induces transient phosphorylation of PLC-c1, which leads to the activation of mitogen-activated protein (MAP) kinases in CISM cells. Furthermore, PGF2a -induced phosphorylation of PLC-c1 and p42/p44 MAP kinase were completely inhibited in the presence of protein tyrosine kinase inhibitors.
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 2 3 4 7 - 1
S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107
Materials and methods Materials Anti-PLC-c1 and anti-p42/p44 MAP kinase antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). [c-32 P]ATP (specific activity, 3000 Ci/mmol) was obtained from Amersham Life Science (Arlington Height, IL). PGF2a , genistein, and herbimycin A, were obtained from Calbiochem (La Jolla, CA). Mylein basic protein, protein A–Sepharose, and tyrphostin A-25 were purchased from Sigma Chemical, (St. Louis, MO). Fetal bovine serum was obtained from Hyclone (Logan, UT) and all other cell culture supplies were obtained from Cell gro (Herndon, VA). Methods Cell culture. Iris sphincter smooth muscle cells were isolated from 4- to 6-months-old cats as described previously [10]. Briefly, the eyes were enucleated immediately after the death of the animal and sphincter muscle was dissected out, further cleaned and cut into 1– 2 mm2 pieces. The explants were placed in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mg ml1 collagenase type IA, 10% fetal bovine serum (FBS) and 50 lg ml1 gentamicin and then incubated for 1–2 h at 37 °C with occasional gentle shaking. The cell suspension was centrifuged at 200g and resuspended in DMEM/F-12 supplemented with 10% (v/v) FBS and 100 U ml1 penicillin, 100 lg ml1 streptomycin, and 0.25 lg ml1 amphotericin B in 5% CO2 humidified atmosphere. The contaminating fibroblasts were removed as previously described [10]. After 3 days, one third of the culture medium was replaced with fresh medium. The smooth muscle cells were subcultured at a split ratio of 1:3 using 0.05% trypsin and 0.02% EDTA. The cells of 3–7 passages were used in this study. Immunoprecipitation of PLC-c1 and Western blotting. CISM cells were starved in serum-free DMEM for 24 h and treated with PGF2a for indicated time periods. The inhibitors were added 15 min prior to the addition of the PGF2a . After treatment, cells were lysed in 50 mM Tris– HCl buffer, pH 8.0, containing 100 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, 50 mM NaF, 1 mM Na3 VO4 , 5 mM PMSF, 10 lg ml1 leupeptin, and 50 lg ml1 aprotinin for 20 min on ice. Cell lysates were centrifuged at 2500g for 10 min and the supernatant was used either for immunoprecipitation of PLC-c1 or for immuno-detection of p42/p44 MAP kinase. PLC-c1 was isolated by immunoprecipitation using rabbit anti-PLC-c1 polyclonal antibodies for 2 h followed by an incubation for 12 h with protein A–Sepharose at 4 °C. The immunoprecipitates were washed and extracted with Laemmli buffer as described earlier [10]. The proteins were resolved on 10% SDS–polyacrylamide gels and then transferred to nitrocellulose membranes. The membranes were probed with anti-phospho-tyrosine, anti-PLC-c1, or anti-p42/p44 MAP kinase antibodies followed by incubation with secondary antibodies (HRP-conjugated goat anti-rabbit IgG at 1:3000 dilution) for 1 h at 20 °C as previously described [4,10]. For chemiluminescent detection, the membranes were treated with ECL reagent for 1 min and subsequently exposed to ECL hyperfilm for 1–2 min. Measurement of p42/p44 MAP kinase activity in CISM cells. MAP kinase activity was characterized by the in situ myelin basic protein (MBP) phosphorylation assay as described earlier [4,10]. Briefly, quiescent cells were stimulated with 1 lM PGF2a and scraped into ice-cold extraction buffer (20 mM b-glycero-phosphate, 20 mM NaF, 2 mM EDTA, 0.2 mM sodium vanadate, 1 mM PMSF, 25 lg ml1 leupeptin, 10 lg ml1 aprotinin, and 0.3% (v/v) b-mercaptoethanol, pH 7.5). The cell extracts were centrifuged at 10,000g for 10 min at 4 °C, and the supernatant was resolved on a 10% SDS–polyacrylamide gel co-polymerized with 0.5 mg ml1 MBP. After electrophoresis, the gels were washed with 50 mM Tris–HCl buffer, pH 8.0, containing 20% (v/v) propanol to remove SDS, then washed with denaturing buffer (50 mM Tris–HCl, pH 8.0, containing 6 M guanidine hydrochloride and 5 mM
1103
b-mercaptoethanol). The enzymes on the gel were then renatured by washing with 50 mM Tris–HCl buffer, pH 8.0, containing 0.04% (v/v) Tween 40 and 5 mM b-mercaptoethanol at 4 °C for 21 h. The gel was then preincubated with assay buffer containing 40 mM Hepes, pH 8.0, 10 mM MgCl2 , 2 mM dithiothreitol, and 0.1 mM EGTA at 30 °C for 30 min. The MAP kinase activity was determined by incubating the gel with 20 ml of the assay buffer, which contained 20 lM ATP and 100 lCi [c-32 P]ATP, at 30 °C for 1 h. After extensive washing in 5% (w/v) trichloroacetic acid containing 10 mM sodium pyrophosphate, the gel was dried and autoradiographed at )70 °C.
Results and discussion Results Immunochemical identification of PLC-c1 We examined the presence of PLC-c1 in cat iris sphincter smooth muscle (CISM) cells by polyclonal antibodies specific for PLC-c1 isoform. The anti-PLCc1 antibodies reacted with a 145-kDa protein. As shown in Fig. 1, PLC-c1 is predominantly present in cytosolic fraction. Cytosolic and membrane fractions were obtained by centrifugation at 100,000g. The specificity of PLC-c1 band was confirmed by loss of the immunoreactive band upon incubation with the appropriate blocking peptide (i.e., an antigenic peptide that was used to produce PLC-c1 antibodies). PLC-c1 band was completely disappeared when 1:1 ratio of antibodies and blocking peptides were used, suggesting that 145-kDa band correspond to PLC-c1 isoform. Time course study of PGF2a -induced tyrosine phosphorylation of PLC-c1 To investigate the effects of PGF2a on PLC-c1 phosphorylation, PLC-c1 was immunoprecipitated from crude homogenates of PGF2a treated and untreated (control) CISM cells with polyclonal antibodies specific to PLC-c1. PGF2a -stimulated tyrosine phosphorylation of PLC-c1 in CISM cells was analyzed by two different protocols. First, anti-PLC-c1 antibodies were used to immunoprecipitate the PLC-c1 from PGF2a treated and
Fig. 1. Immunochemical indentification of PLC-c1 in CISM cells. Western blot analysis of PLC-c1 in cytosol (3), membrane (2), and total homogenate (1) of CISM cells. Appropriate amounts (10–15 lg protein) were subjected to 10% SDS–PAGE. Proteins were transferred to nitrocellulose membranes and immunoblotted with antibodies specific to PLC-c1 as described in Materials and methods. Results given are from a representative experiment of three separate experiments.
1104
S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107
probed with anti-PLC-c1 antibodies. These antibodies selectively recognized one band of 145 kDa and all the samples showed a comparable amounts of immunoprecipitated PLC-c1 in these cells (Fig. 2B).
Fig. 2. Time course study for PGF2a -stimulated tyrosine phosphorylation of PLC-c1. Starved CISM cells were treated for the indicated times periods with 1 lM PGF2a and harvested with lysis buffer as described in Materials and methods. In panel A, proteins were immunoprecipitated with an anti-PLC-c1 antibodies and developed with anti-phospho-tyrosine antibodies. Panel B shows Western blot analysis of immunoprecipitates that were probed with anti-PLC-c1 antibodies. Results given are from one experiment that is a representative of five separate experiments (n ¼ 5).
untreated homogenates. Western blot analysis of PLCc1 immunoprecipitates with anti-phospho-tyrosine antibodies showed an increase in tyrosine phosphorylation of PLC-c1 by 3-fold relative to control levels (Fig. 2A). The peak response was seen at 0.5 min and returned to near control levels by 5 min. Similar results were seen when tyrosine-phosphorylated protein were immunoprecipitated using anti-phospho-tyrosine antibodies followed by detection of PLC-c1 by Western blot analysis with anti-PLC-c1 antibodies (data not shown). The above data clearly show an increased in tyrosine phosphorylation of PLC-c1 with maximal phosphorylation observed approximately 0.5 min after addition of PGF2a . To rule out the possibility of variation in the loaded amounts of protein, immunoprecipitates were
Fig. 3. Dose dependence of PGF2a -stimulated tyrosine phosphorylation of PLC-c1. CISM cells were starved and incubated with indicated concentrations of PGF2a for 0.5 min and harvested with lysis buffer as described in Materials and methods. (A) Tyrosine phosphorylation of PLC-c1 in immunoprecipitates, (B) Western blot analysis of PLC-c1 as described in legend to Fig. 2. Results given are from one experiment that is a representative of five separate experiments (n ¼ 5).
Dose-dependence of PGF2a on tyrosine phosphorylation of PLC-c1 CISM cells were treated with indicated concentrations of PGF2a and PLC-c1 was immunoprecipitated from homogenates as described in Fig. 2. As shown in Fig. 3A, PGF2a stimulates tyrosine phosphorylation of PLC-c1 in a dose-dependent manner. The maximum increase in tyrosine phosphorylation of PLC-c1 was observed at 1 lM of PGF2a . All the samples contained comparable amounts of immunoprecipitated PLC-c1 (Fig. 3B). Effects of tyrosine kinase inhibitors on PGF2a -induced phosphorylation of PLC-c1 To investigate the role of protein tyrosine kinase in PGF2a -induced PLC-c1 phosphorylation, we used tyrosine kinase inhibitors, genistein, and tyrphostin A-25. At 1 lM, both compound blocked PGF2a -induced tyrosine phosphorylation of PLC-c1 completely (Fig. 4A). These findings suggest that a tyrosine kinase mediates PGF2a -stimulated phosphorylation of the PLC-c1 in CISM cells. In all the samples a comparable amounts of PLC-c1 was immunoprecipitated (Fig. 4B). Effects of PGF2a on p42/p44 MAP kinase activation MAP kinases are one of the most important signaling components involved in the regulation of cytoslic
Fig. 4. Effects of protein tyrosine kinase inhibitors on PGF2a -stimulated tyrosine phosphorylation of PLC-c1. CISM cells were incubated in serum-free DMEM with 1lM PGF2a in the absence or presence of 1 lM genistein or tyrphostin A-25. Inhibitors were added 15 min prior to the addition of PGF2a . (A) Tyrosine phosphorylation of PLC-c1 in immunoprecipitates, (B) Western blot analysis of PLC-c1 as described in legend to Fig. 2. Results given are from one experiment that is a representative of five separate experiments (n ¼ 5).
S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107
phospholipase A2 , arachidonic acid (AA) release, and matrix metalloproteinases (MMPs) secretion in various tissues. Several lines of evidence suggest that MMPs are involved in the regulation of intraocular pressure (IOP) in normal and glaucoma human eyes. Since, PGF2a and its analogs are widely used to lower IOP, it is important to understand PGF2a -induced signaling mechanisms. Recently, we have shown that PGF2a activates p42/p44 MAP kinase through a PKC-dependent pathway in CISM cells [4]. However, their up-stream regulation remained unclear in these cells. To answer these questions, CISM cells were stimulated with 1 lM PGF2a for various times of incubation and p42/p44 MAP kinase activity was measured by in-gel renaturation kinase assay as described in Materials and methods. As shown in Fig. 5A PGF2a stimulates p42/p44 MAP kinase activity in a time-dependent manner. There is a significant increase in the activity of p42/p44 MAP kinase after 0.5 min of PGF2a treatment, which was sustained upto 5 min. To establish a correlation between PLC-c1 and p42/p44 MAP kinase, cells were stimulated with PGF2a in the absence or presence of protein tyrosine kinase inhibitors and MAP kinase activity was measured. As shown in Fig. 6A, PGF2a -induced p42/p44 MAP kinase activation was significantly inhibited in the presence of 1 lM of genistein, herbimycin A, and tyrphostin A-25. Furthermore, PGF2a -induced p42/p44 MAP kinase activation was completely inhibited in the presence of 10 lM of these compounds (data not shown). A specific inhibitor of p42/p44 MAP kinase, PD-98059, also inhibited PGF2a -induced p42/p44 MAP kinase activation. To rule out the possibility of variation in the amounts of loaded protein samples on the gels, protein were transferred to nitrocellulose membranes and immunoblotted with anti-p42/p44 MAP kinase antibodies. All samples contained comparable amounts of p42/p44 MAP kinase (Figs. 5B and 6B).
Fig. 5. Dose dependence of PGF2a on p42/p44 MAP kinase activation. Starved CISM cells were incubated in the absence (control) or presence of 1 lM PGF2a for indicated time periods. (A) p42/p44 MAP kinase activity as described in Materials and methods, (B) Western blot analysis by anti-p42/p44 MAP kinase antibodies. Results given are from one experiment that is a representative of five separate experiments (n ¼ 5).
1105
Fig. 6. Effects of protein tyrosine kinase inhibitors on PGF2a -stimulated p42/p44 MAP kinase activation. Starved CISM cells were incubated with 1 lM PGF2a in the absence or presence of 1 lM genistein, tyrphostin A-25, herbimycin A, or PD-98059. Inhibitors were added 15 min prior to the addition of PGF2a . (A) p42/p44 MAP kinase activity as described in Materials and methods, (B) Western blot analysis by anti-p42/p44 MAP kinase antibodies. Results given are from one experiment that is a representative of five separate experiments (n ¼ 5).
Discussion Glaucoma is a group of human disorders characterized by progressive loss of retinal ganglion cells with an associated loss of vision. Derivatives of prostaglandin F2a (i.e., activate the FP-prostanoid receptors) are the most potent and effective topical ocular hypotensive agents currently known. Physiological studies in animals, normal subjects [11,12], and glaucoma patients [13] indicate a remarkable reduction in intraocular pressure (IOP) in the presence of PGF2a and its analog latanoprost (PhXA41). The underlying molecular mechanisms of action of anti-glaucoma drugs and causes of glaucoma are still unknown. It is important to investigate signaling mechanisms induced by anti-glaucoma drugs such as PGF2a in ocular smooth muscle, which could shed more light on the molecular mechanisms underlying the IOP-lowering effects of PGF2a in glaucoma patients. The major finding of this study is that PGF2a stimulates tyrosine phosphorylation of PLC-c1 (Figs. 2 and 3). This is the first study to show a relationship between PGF2a -stimulated tyrosine phosphorylation of PLC-c1 and p42/p44 MAP kinase in CISM cells. Both PLC-c1 phosphorylation and p42/p44 MAP kinase activation were inhibited in the presence of protein tyrosine kinase inhibitors (Figs. 4 and 6). In addition, data also suggest that G-protein-coupled receptors such as the FP-receptors, which have been thought to activate only PLC-b [14], can also stimulate PLC-c1 in CISM cells. Several
1106
S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107
receptors that stimulate PLC are thought to act via G-proteins [15], because their activation of PLC is modulated by bacterial toxins, aluminum fluoride, and analogs of GTP, which are known to modify the actions of G-proteins. At present, the biochemical link between the FP-receptors and tyrosine phosphorylation of PLC-c1 is not known in CISM cells. The tyrosine phosphorylation has been reported for growth factor receptors that are themselves kinases such as PDGF and epidermal growth factors [16,17]. However, tyrosine phosphorylation has also been reported for receptors that lack kinase activity [18–20]. Previous studies have shown that the actions of PGF2a on inositol-1,4,5-trisphosphate (IP3 ) production and Ca2þ -mobilization, and contraction were inhibited by protein tyrosine kinase inhibitors in cat iris sphincter smooth muscle [14]. Angiotensin II stimulated PLC-c1 tyrosine phosphorylation by 4.5-fold at 0.5 min in rat aortic vascular smooth muscle cells [21]. Palmier et al. [22] reported that orthovanadate, a potent phosphatase inhibitor, mediated an increased generation of inositol phosphates and contraction in rat myometrium through phosphorylation and activation of PLC-c1. Gould et al. [23] reported that genistein inhibited both contraction and Ca2þ -mobilization induced by activation of receptors for histamine in porcine carotid arterial smooth muscle. In conclusion, the data obtained suggest that PGF2a induced transient tyrosine phosphorylation of PLC-c1. Furthermore, data of protein tyrosine kinase inhibitors suggest an important role of protein tyrosine kinase in PGF2a -induced phosphorylation and activation of PLCc1. One or more intracellular tyrosine kinases must be stimulated by the PGF2a –FP-receptor interaction. Which initiates a signaling cascade and leads to the activation of protein kinases including p42/p44 MAP kinase. The understanding of the signaling mechanisms of action of PGF2a in the smooth muscle of iris-ciliary body could lead to the development of rational and more effective anti-glaucoma drugs.
Acknowledgment A Combined Intramural Research Grant awarded to S. Husain from Medical College of Georgia supported this work.
References [1] R.A. Coleman, W.L. Smith, S. Narumiya, International union of pharmacology classification of prostanoid receptors: Properties, distribution, and structure of the receptors and their subtypes, Pharmacol. Rev. 46 (1994) 205–229. [2] L.Z. Bito, J. Stjernschantz, B. Resul, O.C. Miranda, S. Basu, The ocular effects of prostaglandins and the therapeutic potential of a new PGF2a analog, PhXA41 (latanoprost), for glaucoma management, J. Lipid Mediators 6 (1993) 535–543.
[3] B.W. Griffin, P.E. Magnino, I.-H. Pang, N.A. Sharif, Pharmacological characterization of an FP prostaglandin receptor on rat vascular smooth muscle cells (A7r5) coupled to phosphoinositide turnover and intracellular calcium mobilization, J. Pharmacol. Exp. Ther. 286 (1998) 411–418. [4] S. Husain, A.A. Abdel-Latif, Effects of PGF2a and carbachol on MAP kinases, cytosolic phospholipase A2 and arachidonic acid release in cat iris sphincter smooth muscle cells, Exp. Eye Res. 72 (2001) 581–590. [5] H.R. Ansari, S. Husain, A.A. Abdel-Latif, Role of p42/p44 MAP kinases and calcium-dependent calmodulin kinases II in PGF2a , ionomycin, and thapsigargin-induced muscle contraction in cat iris sphincter smooth muscle, J. Pharmacol. Exp. Ther. 299 (2001) 178–186. [6] S.G. Rhee, K.D. Choi, Regulation of inositol phospholipidspecific phospholipase C isozymes, J. Biol. Chem. 267 (1992) 12393–12396. [7] A.V. Smarka, J.R. Hepler, K.O. Brown, P.C. Sternweis, Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq, Science 251 (1991) 804–807. [8] S.J. Taylor, H.Z. Chae, S.G. Rhee, J.H. Exton, Activation of the b1 isozyme of phospholipase C by alpha subunits of the Gq class of G-proteins, Nature 350 (1991) 516–518. [9] H.K. Kim, J.W. Kim, A. Zilberstein, B. Margolis, C.K. Kim, J. Schlesinger, S.G. Rhee, PDGF stimulation of inositol phospholipid hydrolysis requires PLC-c 1 phosphorylation on tyrosine residues 783 and 1254, Cell 65 (1991) 435–441. [10] S. Husain, A.A. Abdel-Latif, Role of protein kinase C-a in endothelin-1 stimulation of phospholipase A2 and arachidonic acid release in cultured cat iris sphincter smooth muscle cells, Biochim. Biophys. Acta 1392 (1998) 127–144. [11] J.R. Kerstetter, R.F. Brubaker, S.E. Wilson, L.J. Kullerstrand, Prostaglandin F2a -1-isopropylester lowers intraocular pressure without decreasing aqueous flow, Am. J. Ophthalmol. 105 (1988) 30–34. [12] P.Y. Lee, H. Shao, L. Xu, C.-K. Qu, The effect of prostaglandin F2a on intraocular pressure in normotensive human subjects, Invest. Ophthalmol. Vis. Sci. 29 (1988) 1474–1477. [13] J. Stjernschantz, G. Selen, B. Sjoquist, B. Resul, Preclinical pharmacology of latanoprost, a phenyl-substituted PGF2a analog. Ad. in prostaglandin, Thrombox. Leukotr. Res. 23 (1995) 513– 518. [14] S.Y.K. Yousufzai, A.A. Abdel-Latif, Tyrosine kinase inhibitors suppress prostaglandin F2a -induced phosphoinositide hydrolysis, Ca2þ elevation and contraction in iris sphincter smooth muscle, Eur. J. Pharmacol. 360 (1998) 185–193. [15] S.G. Rhee, K.D. Choi, Multiple forms of phospholipase C isozymes and their activation mechanisms, Adv. Second Messenger-phosphoprotein Res. 26 (1992) 35–61. [16] B. Margolis, S.G. Rhee, S. Felder, M. Mervie, R. Lyall, A. levitzki, A. Ullrich, A. Zilberstein, J. Schlessinger, EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling, Cell 57 (1989) 1101– 1107. [17] D.A. Kumjian, A. Barnstein, S.G. Rhee, T.O. Daniel, Phospholipase C-c complexes with ligand-activated plateletderived growth factor receptors. An intermediate implicated in phospholipase activation, J. Biol. Chem. 266 (1991) 3973– 3980. [18] T. Tsuda, Y. Kawahara, K. Shii, M. Koide, Y. Ishida, M. Yokoyama, Vasoconstrictor-induced protein-tyrosine phosphorylation in cultured vascular smooth muscle cells, FEBS Lett. 285 (1991) 44–48. [19] D.J. Park, H.W. Rho, S.G. Rhee, CD3 stimulation causes phosphorylation of phospholipase C-c1 on serine and tyrosine residues in a human T-cell line, Proc. Natl. Acad. Sci. USA 88 (1991) 5453–5456.
S. Husain, F. Jafri / Biochemical and Biophysical Research Communications 297 (2002) 1102–1107 [20] R.H. Carter, D.J. Park, S.G. Rhee, D.T. Fearon, Tyrosine phosphorylation of phospholipase C induced by membrane immunoglobulin in B lymphocytes, Proc. Natl. Acad. Sci. USA 88 (1991) 2745–2749. [21] M.B. Marrero, W.G. Paxton, J.L. Duff, B.C. Berk, K.E. Bernstein, Angiotensin-II stimulates tyrosine phosphorylation of PLCc1 in vascular smooth muscle cells, J. Biol. Chem. 269 (1994) 10935–10939.
1107
[22] B. Palmier, D. Leiber, S. Harbon, Pervanadate mediated an increased generation of inositol phosphates and tension in rat myometrium. Activation and phophorylation of PLC-c1, Biol. Reprod. 54 (1996) 1383–1389. [23] E.M. Gould, C.M. Rembold, R.A. Murphy, Genistein a tyrosine kinase inhibitor reduces Ca2þ -mobilization in swine carotid artery, Am. J. Physiol. 268 (1995) 1425–1429.