©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Prostaglandin FStimulates Formation of p21-GTP Complex and Mitogen-activated Protein Kinase in NIH-3T3 Cells via G-protein-coupled Pathway (*)

Tsuyoshi Watanabe (1)(§), Iwao Waga (2), Zen-ichiro Honda (2)(¶), Kiyoshi Kurokawa (1), Takao Shimizu (2)

From the (1) Departments of Internal Medicine (Division I) and (2) Biochemistry, Faculty of Medicine, The University of Tokyo, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Prostaglandin (PG) Factivated mitogen-activated protein (MAP) kinase and MAP kinase kinase in NIH-3T3 cells by a mechanism that was completely inhibited by protein kinase inhibitors, staurosporine (20 n M) or H-7 (20 µ M), but was insensitive to pretreatment with islet-activating protein (100 ng/ml; 24 h) or 12- O-tetradecanoylphorbol 13-acetate (2.5 µ M; 24 h). PGFstimulation also led to a significant increase in RasGTP complex. Transfection of a cDNA encoding a constitutively active mutant of G-subunit (Q209L) mimicked PGF-induced MAP kinase activation, increase in RasGTP complex, and DNA synthesis in these cells, suggesting that activation of Gmediates the PGF-activation of Ras-MAP kinase pathway and mitogenesis in NIH-3T3 cells.

These data provide a new insight into regulatory mechanisms of Ras-MAP kinase pathway through heterotrimeric G-protein-mediated pathways.


INTRODUCTION

Prostaglandin (PG)() Fstimulates cell proliferation in NIH-3T3 cells (1, 2) . Activation of phospholipase C is to date the only known biochemical signal via the G-coupled PGFreceptor (2) . Such being the case, this G-coupled pathway is likely to be linked to the mitogenic response, and NIH-3T3 cells may be a useful model system to examine G-protein-mediated intracellular mechanisms linked to mitogenic responses. Possible intracellular mechanisms that may explain PGFeffects are the activation of specific serine/threonine and/or tyrosine protein kinases (PKs). Activation of phospholipase C leads to elevation of intracellular Ca([Ca]) and/or formation of diacylglycerol, which in turn leads to activation of [Ca]/calmodulin-dependent PKs and/or PKC, a family of multipotent serine/threonine kinases that elicits cellular responses, including mitogenesis (3) , respectively. We recently found that PGFreceptor-mediated, [Ca]-dependent tyrosine phosphorylation of cellular components including p125correlated well with PGF-induced mitogenesis (4) . However, the entire spectra of intracellular PK cascades and their target cellular proteins remained to be determined.

Mitogen-activated protein (MAP) kinases (MAPKs) are activated during differentiation and cell cycle transition triggered by a variety of stimuli (5) , thereby playing a key role in the kinase cascade originating from receptor activation (6) . MAPK seems to transmit mitogenic signals by phosphorylating downstream components such as transcription factors (c- myc (7) , c-jun (8) , and p62(9) ). A pathway leading from the tyrosine kinase receptor to MAP kinase activation has been elucidated; ligand-receptor interaction causes formation of the RasGTP complex, which in turn activates a kinase cascade comprising p74 (10) , MAP kinase kinase (MAPKK), and MAPK. However, another protein with MAP kinase kinase kinase activity (MEKK) has been cloned (11) . As overexpressed MEKK can activate MAPKK without activating p74, p74 and MEKK may converge on MAPKK. Recent studies revealed that MAPK is also activated through heterotrimeric G-protein-mediated mechanisms (12, 13) . It was suggested that receptor tyrosine kinase may activate MAPKK via p21 and p72, while G-protein coupled receptors may be linked to MEKK (11) . This hypothesis is supported by our recent observation that transfected platelet-activating factor receptor cDNA into Chinese hamster ovary cells mediates platelet-activating factor-induced activation of MAPK and MAPKK without detectable increase in GTP form of Ras (14) . However, lysophosphatidic acid (15, 16, 17) , thrombin (16, 18) , 2 adrenergic (19) , and M2 muscarinic (20) agonists stimulate formation of the GTP form of Ras and MAPK activity via an islet-activating protein (IAP)-sensitive pathway. Moreover, MAPK activation by lysophosphatidic acid can be blocked by dominant negative p21 or p74 (15) . Therefore, MAPK can be activated by an IAP-sensitive G-protein-coupled pathway that requires both p21 and p74. Even though MAPK activation induced by endothelin via an IAP-insensitive G-protein has been reported (21, 22) , much less is known of the involvement of p21 in MAPK activation dependent upon an IAP-insensitive G-protein.

Several subtypes of -subunit of IAP-insensitive G-proteins have shown to link to phospholipase C-; these include -subunit of G(G) family (G, G11, G14, and G16) (23, 24, 25, 26) . Subtypes of phospholipase C; phospholipase C-1 and phospholipase C-2, are known to be stimulated by specific types of G family. Phospholipase C-2 is also activated by dimers of G-protein (26) . The activity of G is regulated by the exchange of GDP and GTP and by intrinsic GTPase activity. Agonist binding to cell surface receptors stimulate G by enhancing GDP-GTP exchange, and the intrinsic GTPase activity reverses this state to inactivate G. It was reported that substitution of arginine 183 or glutamine 209 of G with cysteine or leucine, respectively, constitutively activates G by inhibiting intrinsic GTP hydrolysis (27, 28) . It was also reported that NIH-3T3 cells stably expressing G with a mutation of conserved glutamine residue or overexpressing the wild-type of G exhibited transformation of the cells (29, 30) . These recent advances have led us to direct examination of the interrelationship between activation of G and PGF-induced cellular responses in NIH-3T3 cells.

We report here that in NIH-3T3 cells, PGFstimulates MAPKK and MAPK by a mechanism that is inhibited by staurosporine and H-7 but is independent of classical TPA-sensitive PKC or Ca/calmodulin-dependent PKs and that G and Ras may mediate this MAPK activation.


EXPERIMENTAL PROCEDURES

Materials

Materials were obtained from the following sources: [-P]ATP (specific activity, >5,000 Ci/mmol) from Amersham Corp.; [H]thymidine (specific activity, 20.1 Ci/mmol) and myo-[H]inositol (specific activity, 45.1 Ci/mmol) from DuPont NEN; P-labeled carrier-free Pfrom ICN; myelin basic protein (MBP), 12- O-tetradecanoylphorbol 13-acetate (TPA), epidermal growth factor (EGF), insulin, forskolin, staurosporine, H-7, W-7, and KN-62 from Sigma; IAP from Funakoshi Biochemicals (Tokyo); 1,2-bis(2-aminophenoxy)ethane- N, N, N`, N`-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) from Dojin (Kumamoto, Japan); protein G-Sepharose and Q-Sepharose Fast Flow from Pharmacia LKB Biotechnology Inc; anti-v-H-ras monoclonal antibody Y13-259 from Oncogene Science; anti-MAPK (erk2) monoclonal antibody against murine recombinant p42, anti-rat MAPK R2 (erk1-CT), and anti-phosphotyrosine-conjugated Sepharose from Upstate Biotechnology (Lake Placid, NY); Pansorbin from Calbiochem; Transfectam from Sepracor (Marlborough, MA); P-81 phosphocellulose papers from Whatman; polyethyleneimine-cellulose plates from Merck. PGF, PGE, and PGDwere donated by Ono Pharmaceuticals (Osaka, Japan). A cDNA encoding guinea pig G and that encoding a GTPase deficient mutant, in which glutamine 209 was replaced with leucine (27, 28) (M6 mutant), were subcloned into an expression vector (pCDNA-1: Invitrogen Corp., San Diego, CA). All other chemicals were of analytical grade. Reagents for cell culture were from Nissui (Tokyo) and Life Technologies, Inc.

Cells and Transfection

NIH-3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS), under the conditions described elsewhere (2) . The cells were washed 3 times with FCS-free DMEM and cultured for 24 h before the assays of MAPKK and MAPK, analysis of GTP-bound Ras, or Western blot analysis.

Each plasmid DNA was transfected into NIH-3T3 cells by DEAE-dextran method, as described (31) or using Transfectam, as described in the manual provided by the supplier (Sepracor, Marlborough, MA). Typically, 2 µg of plasmid DNA was transfected into 5 10cells (counted 1 day before transfection)/60-mmdish. One day after transfection, cells from one 60-mmdish were seeded onto two wells on a six-well coaster for the measurement of [H]thymidine incorporation and phosphoinositide breakdown. Three days after transfection, the cells were washed 3 times with DMEM without FCS and cultured in FCS-free DMEM for 24 h prior to the measurement of [H]thymidine incorporation, phosphoinositide breakdown, MAPK assay, and analysis of Ras-bound GTP and GDP.

MAPK Assay

Quiescent Cells were washed twice with Tyrode buffer containing 20 m M HEPES pH 7.4 and 1 m M CaCl(HEPES-Tyrode) and then stimulated with agonists in HEPES-Tyrode for the indicated times. After a wash with ice-cold phosphate-buffered saline, the cells were lysed in ice-cold lysis buffer (20 m M Tris-HCl, pH 7.5, 25 m M -glycerophosphate, 2 m M EGTA, 1 m M phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 2 m M dithiothereitol, and 1 m M sodium orthovanadate) and centrifuged at 10,000 g for 10 min. The supernatant was used as the source of MAPK and MAPKK.

The immune complex MAPK assay was carried out essentially as described (32) . MAPK was immunoprecipitated with anti-MAPK (erk2) monoclonal antibody, with the aid of Pansorbin, washed with lysis buffer, and resuspended in the same buffer.

In the transfection experiments, MAPK was partially purified by batch treatment with Q-Sepharose; the cell lysate was mixed with 0.5 volume of Q-Sepharose beads equilibrated with lysis buffer containing 0.12 M NaCl for 30 min at 4 °C and then briefly centrifuged (3,000 g for 5 min). The resultant pellet was washed twice with the same buffer and incubated with the original volume of lysis buffer containing 0.3 M NaCl for 30 min at 4 °C. MAPK was recovered in the supernatant by centrifugation.

The sample for MAPK assay was incubated with MBP (1 mg/ml) in 25 µl of kinase buffer (20 m M Tris-HCl, pH 7.5, 10 m M MgCl, 1 m M MnCl, and 40 m M ATP) containing 1 µCi of [-P]ATP for 25 min at 25 °C. A 12-µl aliquot was spotted onto P-81 phosphocellulose paper and extensively washed with 0.5% phosphoric acid. The paper was dried, and P incorporation into MBP was measured by Cerenkov counting (14) .

In the kinase detection assay in the MBP-containing gel (gel kinase assay), the supernatant of the cell lysate was electrophoresed onto an SDS-polyacylamide gel containing 1 mg/ml MBP. Proteins were denatured in 6 M guanidine-HCl and renatured as described previously (33) . Phosphorylation of MBP was carried out in 5 ml of kinase buffer containing 25 µCi of [-P]ATP, and the gel was extensively washed with 7% acetic acid. The dried gel was subjected to an image analyzing system using FUJI BAS 2000.

MAPKK Assay

MAPKK activity was assayed by measuring P incorporation into a kinase-negative mutant of a recombinant Xenopus MAPK (rMAPK) (34, 35) , which was kindly provided by Drs. Y. Gotoh and E. Nishida of Kyoto University. MAPKK was partially purified by batch treatment with Q-Sepharose; the cell lysate was mixed with 0.5 volume of Q-Sepharose beads equilibrated with lysis buffer containing 0.12 M NaCl and briefly centrifuged. Under these conditions, MAPKK activity was recovered in the supernatant. The supernatant was incubated with rMAPK (final concentration, 50 µg/ml) in 12.5 µl of the kinase buffer containing 1 µCi of [-P]ATP for 20 min at 25 °C. The sample was subjected to SDS-polyacrylamide gel electrophoresis, and P incorporation into the rMAPK band was measured using a Fuji image analyzer (FUJI BAS 2000).

Immunoprecipitation and Western blot Analysis- Quiescent cultures of NIH-3T3 cells on a 60-mmculture dish (3 10) were washed twice with HEPES-Tyrode, treated with each agonist in HEPES-Tyrode at 37 °C for 3 min and then frozen in liquid nitrogen. The cells were lysed on ice in 1 ml of solution containing 10 m M Tris-HCl, pH 7.6, 5 m M EDTA, 50 m M NaCl, 30 m M sodium pyrophosphate, 50 m M NaF, 100 µ M sodium orthovanadate, and 1% Triton X-100 (immunoprecipitaion buffer). Cell lysates were centrifuged at 15,000 rpm for 10 min. Resultant supernatants were precleared by incubation with agarose at 4 °C for 1 h. After removal of agarose by centrifugation at 15,000 rpm for 5 min, the supernatants were incubated with 100 µl of anti-phosphotyrosine conjugated to agarose at 4 °C for 4 h and then cleared by centrifugation at 15,000 rpm for 5 min. Immunoprecipitates were washed 3 times with 1 ml of immunoprecipitaion buffer and then treated with 30 µl of Laemmli's sample buffer. After proteins were separated by 8% SDS-polyacrylamide gel electrophoresis, Western blot analysis was performed using 2 µg/ml of anti-Rat MAPK (erk1-CT) as the first antibody and goat anti-rabbit IgG conjugated to horseradish peroxidase (200-fold dilution) as the second antibody as described (36) .

Analysis of Ras-bound GTP and GDP

Quiescent cells were labeled with 0.1 mCi/ml P-labeled carrier-free Pin phosphate-free DMEM medium for 4 h, and then stimulated with agonists for 3 min, unless otherwise stated. Next, the cells were lysed in Triton X-114 buffer (50 m M HEPES-NaOH, pH 7.4, 1% Triton X-114, 100 m M NaCl, 5 m M MgCl, 1 mg/ml bovine serum albumin, 1 m M phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 100 µ M GTP, 100 µ M GDP, and 1 m M ATP) supplemented with phosphatase inhibitors (1 m M sodium pyrophosphate and 1 m M sodium orthovanadate). Membrane-bound Ras was recovered by detergent phase splitting, as described (37) and immunoprecipitated with a monoclonal antibody Y13-259 with the aid of protein G-Sepharose (38) . The immune complex was extensively washed with washing buffer (50 m M HEPES-NaOH, pH 7.4, 0.1% Triton X-100, 0.05 M NaCl, 5 m M MgCl, and 1 mg/ml bovine serum albumin) (37) supplemented with phosphatase inhibitors (1 m M sodium pyrophosphate and 1 m M sodium orthovanadate). Guanine nucleotides bound to Ras were eluted and analyzed by thin-layer chromatography on a polyethyleneimine-cellulose plate. The GTP/(GTP + GDP) ratio was measured using an image analyzer (FUJI BAS 2000).

Assessment of Phosphoinositide Breakdown

Formation of inositol phosphates (IPs) for 1 min in the presence or absence of PGFwas examined as described (2) using the methods of Bijsterbosch et al. (39) .

Assay for DNA Synthesis

[H]Thymidine incorporation into DNA was measured by the method of Nakamura et al. (40) , with slight modifications. Quiescent cells (6-well coaster) were washed twice with DMEM at 37 °C and then incubated for 24 h in 1 ml of DMEM in the presence or absence of PGF. One µCi of [H]thymidine was added to each dish 6 h before harvest. [H]Thymidine incorporation into trichloroacetic acid-insoluble materials was determined.

Miscellaneous

Protein concentration was measured by Bio-Rad protein assay kits using bovine serum albumin as the standard. Statistical analyses were made by the procedure of analysis of variance.


RESULTS

The addition of 1 µ M PGFcaused a transient activation of MAPK and MAPKK with a peak at around 3 min, while 1 µ M TPA induced a time-dependent increase of MAPK activity up to 30 min and EGF/insulin activated MAPK with a peak value at around 5 min and a sustained phase within 60 min (Fig. 1). Dose dependence of PGFon MAPK and MAPKK activation with EDof around 10 M was similar to that for elevation of [Ca], formation of IPs, and [H]thymidine incorporation, as described previously (2) (Fig. 2). Pretreatment of the cells with IAP did not affect the dose-dependence of PGFon MAPKK activation, even under conditions that are assumed to ADP ribosylate almost all of the IAP substrate, as described (2) (Fig. 2). A gel MAPK assay of the supernatant of cell lysate (see ``Experimental Procedures'') (Fig. 3 A) and that of anti-42-kDa MAPK immunoprecipitates (data not shown) showed that a 42-kDa MAPK was the main MAPK activated by PGs in the NIH-3T3 cells. We also found that MAPK and MAPKK were activated by PGs (1 µ M); PGF> PGE, PGD, which correlates with the order of potencies of these PGs to activate the elevation of [Ca], formation of IPs, and [H]thymidine incorporation, as described previously (2) (Fig. 3, A and B). These two kinases (MAPK and MAPKK) were also activated by EGF (100 ng/ml)/insulin (1 µ M) TPA (1 µ M) > ionomycin (100 n M), but not by forskolin (10 µ M) (Fig. 3, A and B). Western blot analysis with anti-rat MAPK (erk1-CT) of immunoprecipitates with antiphosphotyrosine-conjugated Sepharose of cellular lysates demonstrated that a MAPK with a molecular mass around 42-44 kDa was tyrosine phosphorylated (Fig. 4). The PGF-induced activation of MAPK was almost completely inhibited by staurosporine (20 n M) and by H-7 (20 µ M) at concentrations that inhibit PKC (41, 42) , but not by Ca/calmodulin kinase inhibitors; W-7 or KN-62, even at concentrations that inhibit calmodulin-dependent kinases (20 µ M) (43, 44) ; nor by removing extracellular Ca. The [Ca]chelator, BAPTA, only partially attenuated PGF-induced activation of MAPK activity, even under conditions that almost completely prevented the PGF-induced elevation of [Ca](50 µ M, 15 min) (4) (Table I). Pretreatment of the cells with TPA (2.5 µ M, 24 h), which is assumed to down-regulate classical PKC, did not affect the PGF-induced activation of MAPK ().


Figure 2: Dose-response of PGFon MAPK and MAPKK activities. Quiescent NIH-3T3 cells (3 10/60-mm diameter culture dish) in HEPES-Tyrode stimulated with the indicated concentrations of PGFat 37 °C for 3 min, and then immersed in liquid nitrogen. MAPK activity was measured by immune complex assay () and MAPKK assay in control cells () or in cells pretreated with IAP (100 ng/ml; 18 h) () were performed as described under ``Experimental Procedures.'' Each symbol represents the mean of duplicate samples.




Figure 3: Effects of various agents on MAPK ( A) and MAPKK ( B) activities. Quiescent NIH-3T3 cells (3 10/60-mm diameter culture dish) in HEPES-Tyrode were stimulated with various agonists at 37 °C for 3 min. MAPK activity of the supernatant of cell lysate measured by gel kinase assay, and MAPKK assays were performed as described under ``Experimental Procedures.'' One µ M of PGF, PGD, PGE, U46619, iloprost or TPA, a combined use of 100 ng/ml EGF and 1 µ M insulin ( EGF/Ins.), 100 n M ionomycin ( Iono.), or 10 µ M forskolin ( Forsk.) was used as an agonist. MAPKK assay was done in the presence (+rMAPK) or absence (-rMAPK) of rMAPK, as described under ``Experimental Procedures.'' Results typical of experiments repeated at least three times are depicted.




Figure 4: Western blot analysis with anti-rat MAPK monoclonal antibody (erk-1 CT) of anti-phosphotyrosine immunoprecipitates of NIH-3T3 cell lysates. Quiescent NIH-3T3 cells (3 10/60-mm diameter culture dish) in HEPES-Tyrode buffer were stimulated with 1 µ M PGFor the combined use of 100 ng/ml EGF and 1 µ M insulin at 37 °C for 3 min. Immunoprecipitation of cell lysates with Sepharose-conjugated anti-phosphotyrosine (100 µl gel/dish) and Western blot analysis with anti-rat MAPK (erk1-CT) (2 µg/ml) were done as described under ``Experimental Procedures.''



The ratio of GTP/GTP + GDP bound to p21 was also significantly increased by PGFor PGEstimulation by about 30% compared with the basal level, while that ratio was increased by 78% with the combined use of EGF and insulin (Fig. 5).


Figure 5: Effects of PGs and EGF/insulin on GTP form of Ras in NIH-3T3 cells. Quiescent NIH-3T3 cells (3 10/60-mm diameter culture dish) labeled with P carrier-free Pwere stimulated with vehicle ( control), 1 µ M PGF, 1 µ M PGE, or the combined use of 100 ng/ml EGF and 1 µ M insulin ( EGF/Ins.) at 37 °C for 3 min. Labeling of the cells and analysis of Ras-bound GDP and GTP were done as described under ``Experimental Procedures.'' A, a representative thin layer chromatogram from five independent experiments, visualized using a Fuji image analyzer (FUJI BAS 2000). B, the ratio of radioactive GTP/GTP + GDP bound to p21 measured using an image analyzer system (FUJI BAS 2000). Each column and bar represents the mean ± S.E. ( n = 6). Statistical analyses for differences between agonist-stimulated groups versus control were made using the procedure of analysis of variance. *, p < 0.05; **, p < 0.01.



In order to test the involvement of G activation, we performed transfection study using the M6 mutant cDNA, wild-type G cDNA, and the vector pCDNA-1. [H]Thymidine incorporation, IPs formation, MAPK activity, and the ratio of GTP/GDP + GTP bound to p21 at the basal levels ( open columns in Fig. 6, A-D, respectively) were significantly higher in transfectants of the M6 mutant cDNA than in those of the vector control (172, 154, 122, and 155% versus the control, respectively). PGF-induced stimulation of [H]thymidine incorporation, IPs formation, and RasGTP complex were not significant in transfectants of the M6 mutant, and PGF-stimulation of MAPK in those transfectants was less significant compared to the control cells (stimulation by 21% ( p < 0.05) versus 42% ( p < 0.01)) (Fig. 6 B). There were no significant differences in IPs formation, DNA synthesis, and MAPK activity between transfectants of the wild-type G cDNA and the vector control.


Figure 6: [H]Thymidine incorporation ( A), the formation of IPs ( B), MAPK activity (C), and the ratio of GTP/GDP+GTP bound to p21 ( D) in NIH-3T3 cells transfected with the wild-type G cDNA (G), those with the constitutively active G mutant cDNA (M6) and those with the vector: pCDNA-1 ( control). Transfectants were treated with 1 µ M PGF( closed columns) or vehicle ( open columns) in FCS-free DMEM ( A), HEPES-Tyrode containing 10 m M LiCl ( B), HEPES-Tyrode ( C), or FCS- and phosphate-free DMEM ( D), and each assay was done as described under ``Experimental Procedures.'' MAPK activity was partially purified by batch treatment with Q-Sepharose ( C). The sample numbers are three for each group in A and B, six for each group in C, and eight for each group in D. Each column and bar represent the mean ± S.E. Statistical differences between the two groups were determined by the procedure of analysis of variance. *, p < 0.05; **, p < 0.01; ***, p < 0.001. N.S., not significant.




DISCUSSION

In NIH-3T3 cells, PGF, PGD, and PGEwere reported to activate phospholipase C via a G-coupled receptor(s), which is assumed to be responsible for cellular responses evoked by these PGs, such as mitogenesis (2) . One mechanism that may participate in intracellular signaling leading to mitogenesis is activation of the PK cascade comprising specific tyrosine and/or serine/threonine PKs, an event that occurs following receptor tyrosine kinase activation (5) , even though the precise intracellular signaling mechanisms triggering by phospholipase C activation leading to mitogenesis remain to be elucidated. The activation of phospholipase C may lead to PKC activation by liberation of diacylglycerol from cleaved phosphatidylinositol. Concomitantly, the creation of inositol 1,4,5-trisphosphates may elevate [Ca]and subsequently activate Ca/calmodulin-dependent PKs. Tyrosine phosphorylation of cellular components was also reported to be induced by phospholipase C activating agonists such as vasoactive peptides or by neuropeptides (45) . We have recently demonstrated that PG-evoked [Ca]-dependent tyrosine phosphorylation of cellular components via a G-coupled receptor pathway correlated with PG-induced DNA synthesis (4) . Thus, tyrosine phosphorylation of cellular components may participate in signaling pathways to mitogenesis. However, the identification of kinases and their target proteins has not been made, except that p125and/or p130 were seen to function as substrates for tyrosine phosphorylated by bombesin, vasopressin (45) , or PGF(4) in mouse 3T3 cells. MAPKs are considered to play key roles in kinase cascade triggered by stimulation of receptor tyrosine kinases leading to cell cycle transition and to differentiation (5, 6, 22, 46, 47) . MAPKs are also activated through heterotrimeric G-protein-mediated mechanisms (15, 16, 17, 18, 19, 20) . However, the effect of PG stimulation on MAPK cascade in the NIH-3T3 cell system has apparently not been reported.

In the present study, we found that PGF, PGD, and PGEactivate MAPK and the direct upstream activator, MAPKK, in NIH-3T3 cells. Potencies of these PGs to activate MAPK and MAPKK correlate with those that stimulate phospholipase C in NIH-3T3 cells (2) (Fig. 3, A and B). Dose dependence and sensitivity to IAP of PGFon MAPKK and MAPK activation are much the same as those for phospholipase C activation (Fig. 2), supporting our conclusion that this activation of MAPK is mediated via a G-coupled receptor (see below).

MAPKs are a family comprising enzymes with molecular mass between 40 and 58 kDa (48) and are activated by phosphorylation of their intrinsic tyrosine and threonine residues (34, 49) . Based on the results from a gel MAPK assay of the supernatant of cell lysate (Fig. 3 A) and that of anti-42-kDa MAPK immunoprecipitates (data not shown), a 42-kDa MAPK seems to be the main MAPK activated by PGs in NIH-3T3 cells. We also noted tyrosine phosphorylation of a MAPK with a molecular mass around 42 kDa by Western blots with anti-42-, -43-, and -44-kDa MAPKs monoclonal antibody of immunoprecipitates of cell lysates with anti-phosphotyrosine Sepharose (Fig. 4).

Two pathways have been proposed for MAPK activation: via MEKK activation and via Ras-Raf activation. The pathway via MEKK and that via Ras-Raf may be linked to tyrosine kinase receptors and G-protein-coupled receptors, respectively (11) . However, activation of MAPK by various agonists via p21 and p74 in an IAP-sensitive fashion was reported (15, 16, 17, 18, 19, 20) . In the present study, we noted a small, but significant Ras activation induced by PGFand PGE. The potencies of PGFand EGF/insulin to increase the GTP form of Ras (30 and 78% versus the basal level, respectively) are comparative to those that activate MAPKK activity (2.56- and 7.55-fold, respectively). 1) Because PGFdid not inhibit forskolin-induced cAMP formation,() neglecting a possibility that the PGFreceptor is linked to Gin NIH-3T3 cells and 2) because IAP-pretreatment of the cells did not affect PGF-induced activation of MAPK activation, even under conditions that all of the 41 kDa IAP-substrate (G) is assumed to be ADP-ribosylated (100 ng/ml; 24 h) (2) , this PGF-induced activation of Ras-MAPK pathway may be mediated by an IAP-insensitive G-protein, G, instead of G. This hypothesis was further supported by transfection experiments; transfection of cDNA encoding an active mutant of G (27, 28) , but not that of the wild G, into NIH-3T3 cells led to constitutional activation of MAPK activity and increase in GTP form of Ras, thereby providing direct evidence of the G-mediated activation of the Ras-MAPK pathway. However, while no further increase in IPs formation, RasGTP complex and DNA synthesis were observed with PGFin the M6 transfectants, the MAPK response appears to show a PGF-dependent increase in the M6 transfectants. Thus, it is still possible that other G-proteins may be partly involved in this response. Neverthless, to our knowledge, this is the first direct demonstration of Ras and MAPK activation by an IAP-insensitive G-protein-dependent pathway.

With regard to the regulatory mechanisms of MAPK activity, the activation of MAPK was independent of extracellular Caand Ca/calmodulin kinases, but sensitive to PK inhibitors staurosporine and H-7 (). These characteristics are similar to those of the tyrosine phosphorylation of cellular components including p125and [H]thymidine incorporation, both induced by PGFand PGE(4) . However, the PGF-induced tyrosine phosphorylation is completely blocked by BAPTA under the same condition as we used in the present study (50 µ M, 15 min) and is mimicked by ionomycin (0.1 µ M) (4) , while the PGF-induced MAPK activation was only partially dependent upon [Ca]. These results suggest that the tyrosine phosphorylation of major cellular proteins and the activation of MAPK both induced by PGFstimulation are regulated by independent mechanisms. PGF-induced MAPK activation was not affected by down-regulation of classical PKC with TPA-pretreatment of the cells but completely inhibited by staurosporine and H-7 at concentrations that inhibit PKC (41, 42) . In relation to these results, an isozyme of PKC, which was stimulated by acidic phospholipids and phosphatidylinositol 3,4,5-trisphosphate, but not by Ca, TPA, or diacylglycerol, was purified (PKC) (50) . In addition, a staurosporine-sensitive PK was proposed for lysophosphatidic acid-induced Ras activation through an IAP-sensitive G-protein in Rat-1 fibroblasts (17) . Thus, it is possible that PKC or an unidentified staurosporine-sensitive PK may participate in PGF-induced activation of MAPK in NIH-3T3 cells. This PK will need to be identified if intracellular mechanisms of G-protein-mediated Ras-MAPK activation are to be understood.

It is important to question the role of PGF-MAPK cascade on PGF-induced cell proliferation. In the present work, we found that the PGF-induced MAPK activation has characteristics similar to the PGF-induced DNA synthesis as the PGF-induced tyrosine phosphorylation of major cellular proteins including p125(4) . At present, we cannot completely answer what pathway is the main one leading to mitogenesis. In relation to this question, however, transient transfection of a cDNA encoding an active mutant of G (27, 28) leads to a constitutional activation of phospholipase C, Ras-MAPK pathway, and [H]thymidine incorporation, and attenuates further enhancement of these parameters by PGF(Fig. 6). Therefore, the PGF-induced activation of Ras-MAPK pathway may, at least in part, play some role in PGF-induced cellular responses such as cell proliferation. Recent reports demonstrated that expression of a mutant G or over-expression of the wild G in NIH-3T3 cells induced transformation in the presence of FCS (29, 30) . We used NIH-3T3 cells in quiescent culture under FCS-free conditions to assess PGFeffects on mitogenesis of NIH-3T3 cells. It is, therefore, suggested that the over-expression of the wild G per se has no apparent effect on DNA synthesis in the absence of agonists (Fig. 6 A), but it did have transforming effects with continuous exposure to mitogens. We also compared the contents of phosphotyrosyl cellular proteins in transfectants of the M6 mutant cDNA and those of the vector, but we found no significant differences between these two types of transfectants, regardless of whether the cells were stimulated with PGF(data not shown). Therefore, PGF-induced [Ca]-dependent tyrosine phosphorylation may not be mediated through G activation, and may be independent of cell proliferation. However, it is also possible that PGF-induced tyrosine phosphorylation may be mediated through the action of free dimers, as shown in case of phospholipase C-2 activation (51) , and may lead to DNA synthesis in this cell line. Other studies using stable transformants of the M6 mutant, those of a dominant inhibitory mutant of G, or those of specific types of and subunits (51) need to be tested.

Our results, taken together, suggest 1) that PGFactivates MAPKK and MAPK by a mechanism that depends partially on [Ca]and totally on a PK(s) sensitive to staurosporine and to H-7, but not on classical PKC nor calmodulin-dependent PKs, 2) that Gand Ras may be involved in this PGF-induced MAPK activation, and 3) that this PGFreceptor-Ras-MAPK cascade may be responsible for the effects of PGFin NIH-3T3 cells.

  
Table: Effects of various inhibitors on PGF-induced activation of MAPK

A typical result from three independent experiments with similar results. Each datum is the mean of duplicate samples. The radioactivity in the no addition control sample (100%) was 1707 cpm.


  
Table: Effects of PKC down-regulation on PGF-induced activation of MAPK

Each datum is the mean ± S.D. ( n = 3).



FOOTNOTES

*
This work was supported in part by Grants-in-aid for Scientific Research 04670377 and 02404036 from the Ministry of Education, Science, and Culture of Japan and by grants from the Mochida Memorial Foundation for Medical and Pharmaceutical Research, the Uehara Memorial Foundation, the Yamanouchi Foundation for Research on Metabolic Disorders, and the Sankyo Foundation of Life Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel: 81-3-3815-5411 (ext. 3002); Fax: 81-3-5802-2944.

Present address: Dept. of Internal Medicine and Physical Therapy, University of Tokyo, Tokyo 113, Japan.

The abbreviations used are: PG, prostaglandin; PK, protein kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; MAPKK, MAP kinase kinase; MEKK, a protein with MAPKK kinase activity independent of the p21-p71 pathway; MBP, myelin basic protein; TPA, 12- O-tetradecanoylphorbol 13-acetate; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane- N, N, N`, N`-tetraacetic acid tetraacetoxymethyl ester; IAP, islet-activating protein; FCS, fetal calf serum; EGF, epidermal growth factor; rMAP, a kinase-negative mutant of a recombinant Xenopus MAPK; G, -subunit of G-protein; IPs, inositol phosphates; DMEM, Dulbecco's modified Eagle's medium.

T. Watanabe, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. Y. Gotoh and E. Nishida (Institute of Virus Research, Kyoto University) for providing rMAPK, and M. Ohara for helpful comments.


REFERENCES
  1. De Asua, L. J., Clingan, D., and Rudland, P. S. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 2724-2728 [Abstract]
  2. Nakao, A., Watanabe, T., Taniguchi, S., Nakamura, M., Honda, Z., Shimizu, T., and Kurokawa, K. (1993) J. Cell. Physiol. 155, 257-264 [Medline] [Order article via Infotrieve]
  3. Nishizuka, Y. (1986) Science 233, 305-312 [Medline] [Order article via Infotrieve]
  4. Watanabe, T., Nakao, A., Emerling, D., Hashimoto, Y., Tsukamoto, K., Horie, Y., Kinoshita, M., and Kurokawa, K. (1994) J. Biol. Chem. 269, 17619-17625 [Abstract/Free Full Text]
  5. Ray, L. B., and Sturgill, T. W. (1987) Proc. Natl. Acad. Sci. U. S. A.. 84, 1502-1506 [Abstract]
  6. Nishida, E., and Gotoh, Y. (1993) Trends Biochem. Sci. 18, 128-131 [CrossRef][Medline] [Order article via Infotrieve]
  7. Seth, A., Alvatez, E., Gupta, S., and Davis, R. J. (1991) J. Biol. Chem. 266, 23521-23524 [Abstract/Free Full Text]
  8. Baker, S. J., Kerppola, T. K., Luk, D., Vanderberg, M. T., Marshak, D. R., Curran, T., and Abate, C. (1992) Mol. Cell. Biol. 12, 4694-4705 [Abstract]
  9. Gille, H., Sharrocks, A. D., and Shaw, P. E. (1992) Nature 358, 414-417 [CrossRef][Medline] [Order article via Infotrieve]
  10. Wood, K. W., Sarnecki, C., Roberts, T. M., and Blenis, J. (1992) Cell 68, 1041-1050 [Medline] [Order article via Infotrieve]
  11. Lange-Carter, C. A., Pleoman, C. M., Gardner, A. M., Blumer, K. J., and Johnson, G. L. (1993) Science 260, 315-319 [Medline] [Order article via Infotrieve]
  12. Gupta, S. K., Gallego, C., Johnson, G. L., and Heasley, L. E. (1992) J. Biol. Chem. 267, 7987-7990 [Abstract/Free Full Text]
  13. Kahan, C., Seuwen, K., Meloche, S., and Pouyssegur, J. (1992) J. Biol. Chem. 267, 13369-13375 [Abstract/Free Full Text]
  14. Honda, Z., Takano, T., Gotoh, Y., Nishida, E., Ito, K., and Shimizu, T. (1994) J. Biol. Chem. 269, 2307-2315 [Abstract/Free Full Text]
  15. Howe, L. R., and Marshall, C. J. (1993) J. Biol. Chem. 268, 20717-20720 [Abstract/Free Full Text]
  16. van Corven, E. J., Hordijk, P. L., Bos, J. L., and Moolenaar, W. H. (1993) Proc. Natl. Acad. Sci. U. S. A.. 90, 1257-1261 [Abstract]
  17. Hordijk, P. L., Verlaan, I., van Corven, E. J., and Moolenaar, W. H. (1994) J. Biol. Chem. 269, 645-651 [Abstract/Free Full Text]
  18. LaMorte, V. J., Kennedy, E. D., Collins, L. R., Goldstein, D., Harootunian, A. T., Brown, J. H., and Feramisco, J. R. (1993) J. Biol. Chem. 268, 19411-19415 [Abstract/Free Full Text]
  19. Alblas, J., van Corven, E. J., Hordijk, P. L., Milligan, G., and Moolenaar, W. H. (1993) J. Biol. Chem. 268, 22235-22238 [Abstract/Free Full Text]
  20. Winitz, S., Russell, M., Qian, N.-X., Gardner, A., Dwyer, L., and Johnson, G. L. (1993) J. Biol. Chem. 268, 19196-19199 [Abstract/Free Full Text]
  21. Wang, Y., Simonson, M. S., Pouyssegur, J., and Dunn, M. J. (1992) Biochem. J. 287, 589-594 [Medline] [Order article via Infotrieve]
  22. Caraubon, S., Parker, P. J., Strosberg, A. D., and Couraud, P. O. (1993) Biochem. J. 293, 381-386 [Medline] [Order article via Infotrieve]
  23. Strathmann, M., and Simon, M. I. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9113-9117 [Abstract]
  24. Taylor, S. J., Chae, H. Z., Rhee, S. G., and Exton, J. H. (1991) Nature 350, 516-518 [CrossRef][Medline] [Order article via Infotrieve]
  25. Smrcka, A. V., Hepler, J. R., Brown, K. O., and Sternweis, P. C. (1991) Science 251, 804-807 [Medline] [Order article via Infotrieve]
  26. Birnbaumer, L. (1992) Cell 71, 1069-1072 [Medline] [Order article via Infotrieve]
  27. Conklin, B. R., Chabre, O., Wong, Y. H., Federman, A. D., and Bournet, H. R. (1992) J. Biol. Chem. 267, 31-34 [Abstract/Free Full Text]
  28. Wu, D., Lee, C. H., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 1811-1817 [Abstract/Free Full Text]
  29. Kalinec, G., Nazarali, A. J., Hermouet, S., Xu, N., and Gutkind, J. S. (1992) Mol. Cell. Biol. 12, 4687-4693 [Abstract]
  30. de Vivo, M., Chen, J., Codina, J., and Iyengar, R. (1992) J. Biol. Chem. 267, 18263-18266 [Abstract/Free Full Text]
  31. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, pp. 16.42-16.44, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  32. Tobe, K., Kadowaki, T., Tamemoto, H., Ueki, K., Hara, K., Koshio, O., Momomura, K., Gotoh, Y., Nishida, E., Akamuma, Y., Yazaki, Y., and Kasuga, M. (1991) J. Biol. Chem. 266, 24793-24803 [Abstract/Free Full Text]
  33. Gotoh, Y., Nishida, E., Yamashita, T., Hoshi, M., Kawasaki, M., and Sakai, H. (1990) Eur. J. Biochem. 193, 661-669 [Abstract]
  34. Kosako, H., Nishida, E., and Gotoh, Y. (1993) EMBO J. 12, 787-794 [Abstract]
  35. Matsuda, S., Gotoh, Y., and Nishida, E. (1993) J. Biol. Chem. 268, 3277-3281 [Abstract/Free Full Text]
  36. Watanabe, T., Shimizu, T., Nakao, A., Taniguchi, S., Arata, Y., Teramoto, T., Seyama, Y., Ui, M., and Kurokawa, K. (1991) Biochim. Biophys. Acta 1074, 398-405 [Medline] [Order article via Infotrieve]
  37. Burgering, B. M. T., Medema, R. H., Maassen, J. A., van de Wetering, M. L., van der Eb, A. J., McCormic, F., and Bos, J. L. (1991) EMBO J. 10, 1103-1109 [Abstract]
  38. Satoh, T., Endo, M., Nakafuku, M., and Kaziro, Y. (1990) Proc. Natl. Acad. Sci. U. S. A.. 87, 5993-5997 [Abstract]
  39. Bijsterbosch, M. K., Meade, C. J., Turner, G. A., and Klaus, G. G. (1985) Cell 41, 999-1006 [CrossRef][Medline] [Order article via Infotrieve]
  40. Nakamura, T., Tomita, Y., and Ichihara, A. (1983) J. Biochem. ( Tokyo) 94, 1029-1035 [Abstract]
  41. Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M., and Tomita, F. (1986) Biochem. Biophys. Res. Commun. 135, 397-402 [Medline] [Order article via Infotrieve]
  42. Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry 23, 5036-5041 [Medline] [Order article via Infotrieve]
  43. Kanamori, M., Naka, M., Asano, M., and Hidaka, H. (1981) J. Pharmacol. Exp. Ther. 217, 494-499 [Medline] [Order article via Infotrieve]
  44. Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mitsutani, A., Terasawa, M., and Hidaka, H. (1990) J. Biol. Chem. 265, 4315-4320 [Abstract/Free Full Text]
  45. Zachary, I., Sinett-Smith, J., and Rozengurt, E. (1992) J. Biol. Chem. 267, 19031-19034 [Abstract/Free Full Text]
  46. Gotoh, Y., Nishida, E., Matsuda, S., Shiina, N., Kosako, H., Shimokawa, K., Akiyama, T., Ohta, K., and Sakai, H. (1991) Nature 349, 251-254 [CrossRef][Medline] [Order article via Infotrieve]
  47. Gotoh, Y., Moriyama, K., Matsuda, S., Okumura, E., Kishimoto, T., Kawasaki, H., Suzuki, K., Yahara, Y., Sakai, H., and Nishida, E. (1991) EMBO J. 10, 2661-2668 [Abstract]
  48. Boulton, T. G., Nye, S. H., Robbins, D. J., Ip, N. Y., Radziejewska, E., Morgenbesser, S. D., DePinho, R. A., Panayotos, N., Cobb, M. H., and Yancopoulos, G. D. (1991) Cell 65, 663-675 [Medline] [Order article via Infotrieve]
  49. Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akiyama, T., Gotoh, Y., and Nishida, E. (1992) EMBO J. 11, 973-982 [Abstract]
  50. Nakanishi, H., Brewer, K. A., and Exton, J. H. (1993) J. Biol. Chem. 268, 13-16 [Abstract/Free Full Text]
  51. Katz, A., Wu, D., and Simon, M. I. (1992) Nature 360, 686-689 [CrossRef][Medline] [Order article via Infotrieve]

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