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Cdc42Hs, but Not Rac1, Inhibits Serum-stimulated Cell Cycle Progression at G1/S through a Mechanism Requiring p38/RK

Cdc42Hs, but Not Rac1, Inhibits Serum-stimulated Cell Cycle Progression at G1/S through a... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 20, Issue of May 16, pp. 13229 –13235, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Cdc42Hs, but Not Rac1, Inhibits Serum-stimulated Cell Cycle /S through a Mechanism Requiring p38/RK* Progression at G (Received for publication, November 14, 1996, and in revised form, February 10, 1997) Arpa ´ d Molna ´ r, Anne M. Theodoras‡, Leonard I. Zon§, and John M. Kyriakis¶ From the Diabetes Research Laboratory, Massachusetts General Hospital East, Charlestown, Massachusetts 02129, ‡Mitotix, Inc., Cambridge, Massachusetts 02139, and the §Howard Hughes Medical Institute, Division of Hematology/Oncology, Children’s Hospital, Boston, Massachusetts 02115 Antimitogenic stimuli such as environmental or geno- MAPK-kinase-kinases (MAPKKKs) activate MAPK/ERK ki- toxic stress, transforming growth factor-b, and the in- nases (MEKs), which, in turn, activate ERKs (1–3). In simple flammatory cytokines tumor necrosis factor and inter- eukaryotes, these pathways are activated by a variety of leukin-1 activate two extracellular signal-regulated stresses, including nutrient withdrawal and osmotic shock (3). kinase (ERK)-based signaling pathways: the stress-acti- In mammals and other multicellular eukaryotes, ERK/MAPK vated protein kinase (SAPK/JNK) pathway and the p38 pathways are activated by both environmental stresses and pathway. Activated p38 phosphorylates transcription more complex physiologic stimuli such as mitogens, develop- factors important in the regulation of cell growth and mental cues, neurotransmitters, vasoactive peptides, and in- apoptosis, including activating transcription factor 2 flammatory cytokines (1, 2, 4). (ATF2), Max, cAMP response element-binding protein- The stress-activated protein kinases (SAPKs, also referred to homologous protein/growth arrest DNA damage 153 as JNKs) and p38 are the ERK components in two mammalian (CHDP/GADD153). In turn, p38 lies downstream of the signaling pathways activated by a broad array of environmen- Rho family GTPases Cdc42Hs and Rac1, as well as at tal stresses as well as by inflammatory cytokines (1, 2). Once least three mitogen-activated protein kinase (MAPK)/ activated, the SAPKs and p38 are responsible for the phospho- ERK-kinases (MEKs): MAPK kinases-3, -6, and SAPK/ rylation and activation of transcription factors necessary for ERK-kinase-1. Although many of the stimuli that acti- the stress response including c-Jun, ATF2, Max, and CHOP vate p38 can also inhibit cell cycle progression, a clear- (1, 2, 5). cut role for the p38 pathway in cell cycle regulation has The signaling components upstream of the SAPKs and p38 not been established. Using a quantitative microinjec- which have been identified thus far suggest a complex cell- and tion approach, we show here that Cdc42Hs, but not Rac1 stimulus-dependent regulation consistent with the diversity of or RhoA, can inhibit cell cycle progression at G /S through a mechanism requiring activation of p38. These extracellular stimuli that activate these pathways. Several results suggest a novel role for Cdc42Hs in cell cycle chromatographically distinct MEKs, including SEK1/MKK4, inhibition. Furthermore, these results suggest that al- SAPK-kinase (SAPKK)4, and SAPKK5 can activate the SAPKs though both Cdc42Hs and Rac1 can activate p38 in situ, in situ and in vitro. (6 – 8). SAPKK4 and SAPKK5 are specific the effects of Cdc42Hs and Rac1 on cell cycle progres- for the SAPKs, whereas SEK1 can also activate p38. By con- sion are, in fact, quite distinct. trast, MKK3 and MKK6 are specific p38 activators (8 –10). Four families of MAPKKKs have also been implicated in SAPK and p38 regulation. MEK kinases (MEKKs) 1 and 2, as Protein kinase signal transduction pathways that employ well as the mixed lineage kinase SH3 domain-containing, pro- members of the extracellular signal-regulated kinase (ERK) / line-rich kinase (SPRK), can activate the SAPK pathway via mitogen-activated protein kinase (MAPK) family have been direct activation of SEK1 (11–13). MEKK2 can also activate the remarkably conserved in evolution (1–3). Typically these path- mitogenic MAPK/ERK1 and 2 pathway by directly phosphoryl- ways consist of a three-tiered core of protein kinases wherein ating the ERK1/2 upstream activator MEK1 (11). Tpl-2 the rat homolog of the human cot proto-oncogene product, like MEKK2, can activate both SEK1 and MEK1 (14). MEKK3 can * This work was supported by United States Public Health Service activate both the SAPK and MAPK pathways, although SEK1 Grants GM53697 and DK41513 and by Grant DAMD17-94-J-4397 from is not a MEKK3 substrate (11). In addition, transforming the United States Army Breast Cancer Research Program (to J. M. K.). growth factor-b-activated kinase-1 (TAK1) can activate p38 in The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked situ and has been implicated as a transforming growth factor- “advertisement” in accordance with 18 U.S.C. Section 1734 solely to b-activated MAPKKK upstream of SEK1, MKK3, and MKK6 indicate this fact. (15, 16). Finally, the p21-activated kinases (PAKs) and germi- To whom correspondence should be addressed: Diabetes Research nal center kinase (GCK), mammalian homologs of Saccharo- Laboratory, Massachusetts General Hospital East, 149 13th St., myces cerevisiae STE20 and SPS1, respectively, can activate Charlestown, MA 02129. Tel.: 617-726-6909; Fax: 617-726-5649; E- mail: [email protected]. the SAPK and p38 pathways, although their mechanisms of The abbreviations used are: ERK, extracellular signal-regulated action are unclear (17–20). kinase; BrdUrd, bromodeoxyuridine; CHOP, cAMP response element Genetic epistasis studies in yeast as well as biochemical and binding protein-homologous protein; GADD, growth arrest DNA dam- transfection experiments employing mammalian cells have age; GCK, germinal center kinase; HA, hemagglutinin; MAPK, mito- gen-activated protein kinase; MAPKKK, MAPK-kinase-kinase; MEK, identified members of the Ras superfamily as upstream ele- MAPK/ERK kinase; MEKK, MEK-kinase; MKK, MAPK-kinase (equiv- ments regulating the core protein kinases in ERK signaling alent to MEK); PAK, p21-activated kinase; SAPK, stress-activated pro- cascades (1, 2, 4, 19, 21–23). Thus, Ras is a critical upstream tein kinase; SAPKK, SAPK-kinase; SEK, SAPK/ERK kinase; SPRK, regulator of the Raf1 kinase, a MAPKKK in the MAPK path- SH3 domain-containing, proline-rich kinase; TAK, transforming growth factor-b-activated kinase; tpl, tumor progression locus; wt, wild-type. way (4). Similarly, in mammalian cells, the Rho family This paper is available on line at http://www-jbc.stanford.edu/jbc/ 13229 This is an Open Access article under the CC BY license. 13230 G Arrest Mediated by Cdc42Hs and p38 GTPases Rac1 and Cdc42Hs can activate the p38 and SAPK pathways. This activation may involve the direct binding of Rac1 and Cdc42Hs to the PAKs. This binding results in PAK activation (18 –20, 22–24). A complete picture of the biological functions of these stress- activated pathways is only beginning to emerge. Recent data have implicated the SAPK and p38 pathways in the induction of apoptosis in response to nerve growth factor withdrawal in PC-12 cells, as well as heat shock, genotoxic chemotherapeutics (cis-platinum), and signaling pathways, which stimulate the generation of ceramide from membrane sphingomyelin (25– 27). However, induction of apoptosis may not be the only bio- FIG.1. Representative photomicrographs of cells microin- logical consequence of activation of the SAPKs and p38. More- jected with the empty pMT3 expression vector together with an over, the specific biological roles of the SAPK and p38 pathways inert rabbit IgG (panels A and F), or with an expression vector (pMT3) harboring HA-tagged p38 (panels B and G), SAPK-p46b1 in signaling mediated by Rac1 and Cdc42Hs have not been (panels C and H), p44-MAPK (panels D and I), or p70 S6 kinase elucidated. (panels E and J) cDNAs. The expressed proteins are shown in red, In this study, we used quantitative microinjection to begin to nuclei of BrdUrd-positive cells in S phase are stained green (panels characterize the biological consequences of Cdc42Hs and Rac1 A–E). Nuclei of BrdUrd-positive cells in S phase concomitantly express- ing the injected plasmids appear yellow because of the usage of a double activation of p38. Here we report that Cdc42Hs is a potent filter and the superimposition of the red and green colors. Nuclei were inhibitor of cell cycle progression, arresting cells at the G /S also counterstained with Hoechst 33258 to identify all nuclei (panels transition point. In addition, we demonstrate that this inhibi- F–J). tion is mediated by elements of the p38 pathway. Rac1 and the RESULTS SAPKs, although able to induce characteristic cellular and biochemical responses, do not inhibit cell cycle progression Effects of ERK Family Kinases on Cell Cycle Progression— significantly. Murine NIH-3T3 fibroblasts were rendered quiescent by serum withdrawal and synchronized in G by serum readdition. Cells EXPERIMENTAL PROCEDURES were microinjected in early G with expression vectors encod- Microinjection—NIH-3T3 cells were grown on glass coverslips and ing HA-tagged p38, SAPK-p46bI, p44-MAPK, p70 S6 kinase, or synchronized in G by serum withdrawal for 24 h. After this period, less empty vector. Entry into S phase was monitored by incorpora- than 1% of the cells incorporated bromodeoxyuridine (BrdUrd) during tion of BrdUrd into DNA. After 24 h, expression of the relevant an additional 24 h of incubation. The arrested cells were released from constructs was verified by immunostaining, and the expressing serum starvation by readdition of 10% calf serum and were microin- cells were scored blindly for S phase entry. Cells injected with jected in early G with expression vectors harboring the HA-tagged or the empty vector were identified by staining for coinjected FLAG-tagged cDNAs, using a Zeiss automated microinjection system rabbit IgG. Cells were also counterstained with bisbenzimide (28). As a control, cells were microinjected with the empty vector to- gether with an inert rabbit antibody marker to identify the injected (Hoechst 33258) to visualize all nuclei and assess any abnormal cells. S phase entry was monitored by BrdUrd (100 mM) incorporation nuclear morphology. Fig. 1 shows representative photomicro- into cellular DNA (28, 29). Cells were fixed at the indicated time after graphs of the stained cells. release from serum starvation as described. Cells stained for phospho- Expression of p38 resulted in a striking 77.5% inhibition of S p38 or phospho-c-Jun were fixed with 4% paraformaldehyde according phase entry compared with cells injected with empty plasmid to the manufacturer’s suggestions. Injected DNA concentrations were: (Fig. 2a). Similar results were obtained using mink lung 100 ng/ml for HA-p38, HA-SAPK, HA-p44MAPK, HA-p70S6 kinase, Mv1Lu cells (data not shown). The inhibition of S phase entry HA-MKK3, HA-MKK3-KR, HA-SEK1, HA-SEK1-KR, HA-MKK6, M2- FLAG-GCK, and empty pMT3; 240 ng/ml for empty pMT3, HA-MKK3- by p38 is likely due to the presence of increased amounts of KR, or HA-SEK1-KR in the dominant negative experiments shown in active p38 in the microinjected cells. Staining of the injected Figs. 1, 3, and 4; and 80 ng/ml for Rho family GTPases. cells with an antibody specific for the active, phosphorylated Immunocytochemistry—Fixed coverslips were stained with anti-HA form of p38 (1, 2) revealed that the p38-expressing cells con- mouse monoclonal antibody or anti-M2-FLAG mouse monoclonal anti- tained greater amounts of phosphorylated p38 than did the body, followed by biotinylated anti-mouse antibody and streptavidin- uninjected cells (Fig. 2b). Texas Red staining as described previously (28, 29). The coinjected The ability of p38 to arrest cells in G was specific, with other rabbit IgG was detected with a biotinylated anti-rabbit antibody, fol- signaling kinases having little or no effect on cell cycle progres- lowed by streptavidin-Texas-Red staining. The incorporated BrdUrd was detected by anti-BrdUrd rat monoclonal antibody and anti-rat sion. p44-MAPK is an integral component of the mitogenic fluorescein isothiocyanate antibody. Nuclei were counterstained with pathway recruited by Ras, and constitutive activation of this Hoechst 33258 dye (1 mg/ml). To quantitate cells in S phase, BrdUrd- pathway is sufficient to transform NIH-3T3 cells (4). Not sur- positive cells were scored blindly as a percent of the number of injected prisingly, expression of p44-MAPK modestly enhanced G /S cells expressing the tagged cDNAs. To stain for p38 following anti-HA progression (91% of p44-MAPK-expressing cells in S phase staining, rabbit anti-p38 antibody was used followed by a 9-amino-6- versus 69% of empty plasmid-injected cells, Fig. 2a). p70 S6 chloro-2-methoxyacridine conjugated anti-rabbit antibody staining. The kinase is also activated by mitogens through mechanisms in- phosphorylated portion of the expressed HA-p38, the endogenous p38 volving phosphatidylinositol 3-kinase and FRAP/RAFT/TOR protein, or c-Jun protein was detected by anti-phospho-p38 or anti- phospho-c-Jun antibodies, followed by biotinylated anti-rabbit antibody (32, 33). Inhibition of p70 S6 kinase blocks G /S transition (34). and streptavidin-9-amino-6-chloro-2-methoxyacridine or streptavidin- Expression of p70 S6 kinase from microinjected plasmid had no fluorescein isothiocyanate staining. Actin stress fibers were stained significant effect on G /S transition (Fig. 2a). SAPKs are pref- with fluorescein phalloidin as described (30). Membrane ruffling was erentially activated by the same antimitogenic stimuli as is p38 detected by staining as described (31). (1, 2). To our surprise, however, expression of injected SAPK, Cloning—MKK3 and MKK6 were cloned by polymerase chain reac- which increased intracellular SAPK activity and, consequently, tion from human B cell (RAMOS) cDNA. p38, SAPK, MKK3, and SEK1 substantially elevated the level of phosphorylated c-Jun (a cDNAs were expressed from the pMT3 (HA-tagged) vector. GCK, SAPK substrate) in the injected cells (Fig. 2b), resulted in only MKK6, Cdc42Hs, Rac1 and RhoA plasmids were expressed from the pCMV5-M2-FLAG vector. modest inhibition of S phase entry (55% in S phase; Fig. 2a). In G Arrest Mediated by Cdc42Hs and p38 13231 FIG.3. Strong activation of SAPK does not promote G arrest. GCK inhibits cell cycle progression at G /S via a p38-dependent mech- anism. Cells were injected with the indicated plasmids and assayed for cell cycle progression. For each injection set, the ratios of S phase cells/total expressing cells were: empty plasmid: 53/85, 67/96, 80/117, 53/73: GCK: 16/59, 9/44, 15/33, 6/29; GCK plus KR-MKK3: 37/62, 47/78, 19/40, 9/17; GCK plus SAPK plus KR-MKK3: 21/42, 25/43, 11/21, 12/25. lie upstream of p38 in stress signaling cascades could block S phase entry. MKK3, MKK6, and SEK1 are three mammalian MEKs that can activate p38 in situ and in vitro (9, 10). Al- though MKK3 and MKK6 are apparently specific for p38, SEK1 is also a strong SAPK activator (6, 9, 10). Expression of HA-tagged, wild-type SEK1, MKK3, or MKK6 from microin- jected plasmids strongly inhibited S phase entry (14% in S phase for SEK1, 17% in S phase for MKK3, 15% in S phase for MKK6, 69% in S phase for empty plasmid). By contrast, ex- pression of kinase-inactive mutant SEK1 or MKK3 plasmids, wherein the lysine residue critical for ATP binding has been mutated to arginine (KR mutants), was without significant effect (Fig. 4a). FIG.2. Inhibition of G /S transition by p38. Part a, quantitation Despite the lack of intrinsic activity, however, kinase-inac- of cell cycle progression. Cells were microinjected with expression plas- mids encoding HA-tagged constructs indicated. Data are expressed as tive SEK1 and MKK3 mutants can act as dominant inhibitors percent of expressing cells in S phase (calculated as the ratio of S phase of coexpressed p38 activation and biological activity and can expressing cells 3 total expressing cells ). Shown are mean 6 S.D. For thus be used to assess the requirement for p38 activation for a each injection set the calculated ratios (S phase expressing cells/total particular biological function (6, 9, 10, 25–27). Accordingly, expressing cells) were: empty plasmid: 36/51, 28/47, 27/45, 52/66, 76/90; p38: 2/19, 4/28, 12/74, 6/28, 3/27; SAPK: 12/21, 9/20, 16/29, 12/21, to test whether activation of p38 was necessary for p38-medi- 84/129; p44-MAPK: 23/23, 18/22, 57/61, 184/205; p70 S6 kinase: 36/51, ated cell cycle arrest, we coinjected p38 with KR-MKK3 or 19/27, 13/20, 24/31. Part b, expression of microinjected p38 elevates the KR-SEK1. Consistent with a requirement for active p38, both level of active phosphorylated p38; injection of SAPK elevates the level KR-SEK1 and KR-MKK3 completely inhibited the ability of of c-Jun phosphorylated at Ser-63 and Ser-73. Anti-HA staining of the expressed p38 (panel A) and SAPK (panel C). Anti-phospho-p38 stain- p38 to block S phase entry (Fig. 4b) without preventing p38 ing (panel B), anti-phospho-c-Jun staining (panel D). expression (Fig. 4c). Effects of Rho Family GTPases on Cell Cycle Progression— view of this result, we explored the effects on cell cycle progres- Cdc42Hs, a member of the Rho subfamily of small GTP-binding sion of strong activation of the SAPKs. proteins, is a human homolog of S. cerevisiae CDC42 (36). GCK is a mammalian homolog of S. cerevisiae SPS1 which, Cdc42p activates Ste20p as part of the yeast mating pheromone when expressed transiently, is constitutively active (1, 2, 17, signaling pathway. This pathway activates two ERKs, Fus3p 35). Coexpression of GCK and SAPK results in potent SAPK and Kss1p, and culminates in G cell cycle arrest (3, 21). activation in the absence of external stimuli. At low levels of Likewise, GTP-charged Cdc42Hs or the related GTPase Rac1 expression, GCK activation of SAPK is specific, with no p38 or can activate the mammalian STE20 homologs PAK1 or PAK3 ERK1 activation seen (17). At high levels of expression, how- in situ and in vitro; and recent evidence indicates that Cdc42Hs ever, GCK can activate p38 modestly. Injection of GCK into and Rac1, but not RhoA, can activate p38 (and the SAPKs) in NIH-3T3 cells resulted in inhibition of G /S transition to a situ via a mechanism requiring PAKs (18, 19, 22–24, 37). To degree commensurate with that obtained upon injection of p38 investigate the potential effects of Rho family GTPases on cell alone (25% in S phase versus 67% in S phase for cells injected cycle arrest, we microinjected expression plasmids encoding with empty vector, Fig. 3). If KR-MKK3, a kinase-dead domi- wild-type and mutant forms of Cdc42Hs, Rac1 or RhoA into nant inhibitory mutant of MKK3, which specifically blocks p38, synchronized NIH-3T3 cells. Expression of wt-Cdc42Hs re- but not SAPK activation (9 and see Fig. 4) is coinjected with sulted in a striking arrest of the cell cycle at G /S, exceeding GCK, this G arrest is abolished completely whether or not that seen for p38. Of the cells injected with empty plasmid, SAPK is coinjected with the GCK (Fig. 3). Thus, SAPK activa- entry into S phase was apparent within 20 h after release from tion likely does not contribute to GCK-mediated cell cycle serum starvation; and 75% of the cells injected with empty arrest. plasmid had transited to S phase within 26 h. By contrast, less Effects of MEKs Upstream of p38 on Cell Cycle Progres- than 7% of the cells expressing wt-Cdc42Hs entered S phase sion—To investigate further the role of activation of p38 in within 30 h after serum readdition (Fig. 5a). This represents a inhibition of G /S transition, we asked if components known to dramatic 90.7% decrease in S phase entry (Fig. 5, a and b). Expression of wt-Cdc42Hs from injected plasmid also elevated T. Yuasa and J. M. Kyriakis, manuscript in preparation. the level of activated, endogenous p38 phosphorylated at the 13232 G Arrest Mediated by Cdc42Hs and p38 although having no effect on Cdc42Hs expression (Fig. 5d), resulted in a partial reversal of the cell cycle inhibitory effects of Cdc42Hs with 37% of the cells progressing to S phase, 44% of control (Fig. 5b). These results suggest that p38 activation is a necessary component in the mechanism by which Cdc42Hs inhibits cell cycle progression. To investigate further the role of Cdc42Hs in G arrest, we next sought to determine the effects of constitutively active and dominant interfering mutants of Cdc42Hs on cell cycle progres- sion. V12-Cdc42Hs is a GTPase-defective mutant of Cdc42 which is constitutively active. Microinjection of V12-Cdc42Hs, although still strongly able to arrest cells in G , is less effective than wild-type (Fig. 5b). We attribute this to the possible non- specific activation of mitogenic pathways by the activated Cdc42Hs mutant. N17-Cdc42Hs is a dominant interfering mu- tant of Cdc42Hs which is thought to act by sequestering nucle- otide exchangers for Cdc42Hs and other Rho family GTPases (38). Thus N17-Cdc42Hs can act either by blocking Cdc42Hs, Rac1 or RhoA activation (38). Consistent with this, microinjec- tion of N17-Cdc42Hs does not activate endogenous p38, as detected by staining with antibodies to phospho-p38 (Fig. 5c). However, to our surprise, N17-Cdc42Hs inhibited G /S progres- sion nearly one-third as effectively as wt-Cdc42Hs (Fig. 5b). This result supports the contention that the N17 mutant pre- vents activation of other Rho family GTPases, such as Rac1, which are required for cell growth. It is conceivable that injection of wt-Cdc42Hs could nonspe- cifically recruit other Rho family GTPases that inhibit cell cycle progression. To investigate the specificity of cell cycle inhibi- tion by Cdc42Hs, we assayed for cell cycle inhibition mediated by other members of the Rho family. To our surprise, injection of either a wild-type or constitutively active allele of Rac1 (V12-Rac1), either of which can activate p38 in situ (19), was far less effective at inhibition of G /S transition than was wt-Cdc42Hs (50% in S phase for both Rac1 constructs versus 67% in S phase for cells injected with empty vector; Fig. 6a), even though injection of either wt- or V12-Rac1 was sufficient to activate p38 to an extent commensurate with that induced by Cdc42Hs, as judged by staining for activated endogenous p38 with anti-phospho-p38 antibodies (Fig. 6b). Moreover, in- jection of V12-Rac1 stimulated membrane ruffling, a charac- teristic response of the cell to activation of Rac1 (31) (Fig. 6c). This result is consistent with earlier observations indicating that Rac1 is in fact a necessary component for Ras transforma- tion (39). Thus, although Rac and Cdc42Hs can both activate p38 in situ (18, 19, 23), each may also activate additional pathways that together result in distinct biological effects. FIG.4. Effect of expression of p38-activating MEKs on cell RhoA has not been shown to lie upstream of p38 or the SAPKs cycle progression: requirement for active p38 for G arrest. Part (22, 23). Injection of a constitutively active (V14) RhoA allele, a, quantitation of S phase entry of cells injected with HA-tagged SEK1, although stimulating the characteristic formation of actin MKK3, MKK6, KR-SEK1, or KR-MKK3. Data shown are mean 6 S.D. stress fibers (30) (Fig. 6c), yielded cell cycle results similar to For each injection set the ratios of S phase expressing cells/total ex- pressing cells were: MKK3: 6/55, 11/55, 3/22, 9/40, 12/67; SEK1: 8/50, those obtained upon injection of Rac1 (49% in S phase, Fig. 6a). 2/22, 5/36, 4/24; MKK6: 7/47, 6/62, 5/33; KR-MKK3: 17/27, 9/16, 10/15, Thus, among Rho family GTPases, cell cycle inhibition appears 26/49, 47/78; KR-SEK1: 45/85, 12/23, 12/20, 14/33; empty vector: 28/47, to be specific to Cdc42Hs. 27/45, 52/66, 76/90, 46/67. Part b, activation of p38 is necessary for p38-mediated inhibition of cell cycle progression. KR-MKK3 and KR- DISCUSSION SEK1 block p38 inhibition of G /S transition. Shown are mean 6 S.D. For each injection set the ratios of S phase cells/expressing cells were: p38 and the SAPKs are activated by an array of ligands that empty plasmid: 80/113, 33/48, 64/101; p38: 6/27; 8/30, 6/32; p38 plus are known to be either antimitogenic or proapoptotic (1, 2). In KR-MKK3: 18/30, 19/32, 18/27; p38 plus KR-SEK1: 14/20, 18/26, 16/28. contrast to the mitogenic pathways, which are regulated Part C, immunostaining of cells coinjected with the HA tagged p38 and largely by polypeptide ligands coupled to tyrosine kinases, the KR-MKK3 cDNAs. Expression of both proteins is shown in panel A. That the S phase transition was due to the inhibitory effect of KR- stimuli that activate the stress pathways are remarkably di- MKK3 and not a lack of expression of p38 was verified by directly verse and include ionizing radiation, heat shock, chemical DNA staining the expressed p38 protein (panel B). damage, oxidative stress, reperfusion injury, and the inflam- matory cytokines tumor necrosis factor-a and interleukin-1. sites critical for activation, as determined by staining with an Thus, the upstream molecular components that feed into the antibody specific for the activated form of p38 (Fig. 5c). p38 and SAPK pathways are accordingly complex and diverse. Coexpression of dominant interfering (KR) SEK1 and MKK3, The Rho family GTPases Cdc42Hs and Rac1 represent two G Arrest Mediated by Cdc42Hs and p38 13233 distinct mechanisms of p38 and SAPK activation with different biological functions. We have demonstrated herein that activation of Cdc42Hs inhibits cell cycle progression at G /S. Insofar as Cdc42Hs (and p38) inhibition of cell cycle progression can be at least partially reversed upon expression of kinase-dead MKK3 and SEK1, we conclude that recruitment of p38 is a critical component of Cdc42Hs-mediated cell cycle arrest. In addition, we have shown that p38 or elements immediately upstream of p38 (SEK1, MKK3, or MKK6) can also arrest cells in G . By con- trast, Rac1, which can also activate p38 (19, 23), fails to inhibit strongly cell cycle progression. Indeed, Rac1 has been impli- cated in Ras transformation (39) and may recruit additional signaling pathways that prevent inhibition of the cell cycle or promote cell cycle progression. Thus, although activation of p38 alone is sufficient to arrest cells in G , we cannot rule out that p38, which can be activated weakly by mitogenic signals, could, in conjunction with other signaling pathways, lead to responses distinct from G arrest. These ideas are summarized in the model shown in Fig. 7. Our results are in contrast with data implicating Rho family GTPases, notably Cdc42Hs, in cell growth and transformation (40). It is conceivable that the concomitant recruitment of Ras and several Rho family GTPase signaling pathways could re- sult in cell growth, whereas activation of Cdc42Hs alone or in conjunction with other antimitogenic pathways results in cell cycle arrest. In this regard, care must be taken in the interpre- tation of results concerning Cdc42Hs mutants. Our results presented in Fig. 5 suggest that wt-Cdc42Hs is the most potent inhibitor of cell cycle progression, whereas a constitutively active mutant (V12-Cdc42Hs), although still growth inhibitory, is less so than wild-type. This result may reflect the ability of the overexpressed, constitutively active Cdc42Hs mutant to recruit nonspecifically mitogenic signaling mechanisms, possi- bly including those normally regulated by other Rho family GTPases, more effectively than the overexpressed wild-type Cdc42Hs. By the same token, overexpression of dominant neg- ative Cdc42Hs, although unable to activate endogenous p38, was growth inhibitory, possibly because of nonspecific seques- tering of the activation machinery for other Rho family GTPases. For example, Ost, a proto-oncogene, is a guanine nucleotide exchanger for Rho, Cdc42Hs, and Rac1 (38). We do not believe that Cdc42Hs inhibition of G /S transition is the result of nonspecific recruitment of p38 inasmuch as wild-type p38 itself, as well as MKK3, MKK6, and SEK1, all p38 activators, are also able to arrest NIH-3T3 cells at G /S. Moreover, recent evidence indicates that p38 overexpression inhibits mitogen induction of G cyclins (41). Thus it is not surprising that p38 activation, by a Cdc42Hs-dependent mech- anism, could result in growth arrest. Moreover, precedent for Cdc42Hs as a G inhibitor has been observed in lower eu- karyotes. Thus, S. cerevisiae Cdc42p is an essential component of the mating pheromone response pathway, which also results in G arrest (3, 21). Inasmuch as the experiments implicating Cdc42Hs in cell growth have employed Swiss 3T3 cells (40), our FIG.5. Cdc42Hs, an upstream activator of p38, inhibits G /S transition, whereas coexpression of Cdc42Hs with KR-MKK3, KR-SEK1 blocks Cdc42Hs-mediated cell cycle inhibition. Part a, staining (panels A and C, respectively). Staining for phospho-p38 is time course of cell cycle progression in cells injected with the expression shown in panels B and D. Arrows indicate nuclear phospho-p38 staining plasmids indicated. Part b, quantitation of S phase cells 26 h after (panel B) or mark the nuclei of the injected cells (panel D). Part d, release from serum starvation. Shown are the mean 6 S.D. For each expression of KR-SEK and KR-MKK3 has no effect on expression of injection set the ratios of S phase expressing cells/total expressing cells coinjected Cdc42Hs. The expressed proteins (in red) were stained with were: empty vector: 29/37, 98/157, 130/172, 108/143, 150/197; wt- anti-FLAG (panels A and B), and the rabbit IgG coinjected with the Cdc42Hs: 9/96, 2/25; 3/62, 1/45, 15/129, 14/145; wt-Cdc42Hs plus KR- empty vectors were stained with anti-rabbit antibody (panel C). Nuclei MKK3 and KR-SEK1: 45/102, 24/68, 12/35, 26/80; V12-Cdc42Hs: 11/42, of cells in S phase are stained with anti-BrdUrd and shown in green 18/50, 11/49; N17-Cdc42Hs: 7/34, 6/25, 10/35, 8/34. Part c, expression of (panels A–C). All nuclei were stained with Hoechst 33258 (panels D–F). wt-Cdc42Hs increases endogenous phosphorylation of p38 at the regu- Injected cDNAs were: wt-Cdc42Hs (panels A and D), wt-Cdc42Hs plus latory sites, whereas expression of N17-Cdc42Hs does not. Expression KR-MKK3 and KR-SEK1 (panels B and F), empty vectors plus rabbit of the injected wt- and N17-Cdc42 cDNAs was verified with anti-FLAG IgG (panels C and F). 13234 G Arrest Mediated by Cdc42Hs and p38 FIG.7. Model for cell cycle regulation by Rho family GTPases and the p38 pathway. Bold arrows indicate strong signaling inputs. Dashed arrows indicate weaker inputs. The reasons for G arrest in response to stress signals have not been clearly defined; however, it is plausible to propose that cell cycle arrest would be followed by repair of stress-related damage. Alternatively, the cell could arrest in G and await restoration of a normal cellular milieu conducive to continued growth. The targets of Cdc42Hs and p38 which mediate cell cycle arrest are not known. However, inhibition of induction of G cyclins (41) is a logical candidate. Moreover, CHOP, a p38 substrate, promotes G arrest as part of the response to geno- toxic stress (5, 42). Finally, several recent studies indicate that recruitment of the SAPKs or p38 results in apoptosis. Withdrawal of nerve growth factor from PC-12 cells activates both the SAPKs and p38 and promotes apoptosis. Expression of KR-MKK3 or KR- SEK1 blocks this effect, and constitutively active mutants of MKK3 or SEK1 promote apoptosis in the presence of nerve growth factor (25). Likewise, thermotolerant fibroblasts dis- play defective SAPK activation and cell death in response to heat shock. Expression of SEK1 restores heat sensitivity to these cells (26). Finally, treatment of macrophage cell lines with agonists that stimulate sphingomyelin hydrolysis (tumor necrosis factor, UV radiation, x-irradiation, oxidant stress) re- sults in apoptosis, a response that can be reversed upon ex- pression of dominant inhibitory SEK1 (27). Despite these find- ings, in none of the experiments shown in Figs. 1– 6 did we see evidence of apoptosis. Thus, cycle arrest at G /S, rather than apoptosis, appears to be the characteristic response of NIH-3T3 FIG.6. Rac1 and RhoA do not strongly block cell cycle progres- cells to activation of the Cdc42Hs/p38 pathway, indicating that sion. Part a, cells were microinjected with the indicated plasmids and different cells respond distinctly to p38 activation. quantitated for cell cycle progression. For each injection set the ratios of S phase expressing cells/total expressing cells were: empty plasmid: Acknowledgments—We thank J. Settleman for Rac, Rho, and 53/85, 67/96, 80/117, 53/73; wt-Rac1: 24/53, 28/55, 58/113; V12-Rac1: Cdc42Hs cDNAs; J. H. Kehrl for GCK cDNA; A. Nebreda for anti-p38 35/59, 30/64, 23/50; V14-RhoA: 19/35, 16/39, 39/86, 49/101, 31/59. Part antiserum; J. Avruch for p70 S6 kinase cDNA and for a critical reading b, injection of wt- or V12-Rac1 activates p38 to the same degree as does of the manuscript; D. Ron for CHOP cDNA; M. Pagano, R. Bruns, and wt-Cdc42Hs. Cells were injected with wt- (panels A and C) or V12-Rac1 S. Brill for advice; the Massachusetts General Hospital Molecular On- (panels B and D) and stained for expression of the injected construct cology Laboratory for use of their microinjection apparatus; and the (panels A and B) or endogenous phospho-p38 (panels C and D). 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Cdc42Hs, but Not Rac1, Inhibits Serum-stimulated Cell Cycle Progression at G1/S through a Mechanism Requiring p38/RK

Molnár, Árpád; Theodoras, Anne M.; Zon, Leonard I.; Kyriakis, John M.
Journal of Biological Chemistry – May 1, 1997
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 20, Issue of May 16, pp. 13229 –13235, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Cdc42Hs, but Not Rac1, Inhibits Serum-stimulated Cell Cycle /S through a Mechanism Requiring p38/RK* Progression at G (Received for publication, November 14, 1996, and in revised form, February 10, 1997) Arpa ´ d Molna ´ r, Anne M. Theodoras‡, Leonard I. Zon§, and John M. Kyriakis¶ From the Diabetes Research Laboratory, Massachusetts General Hospital East, Charlestown, Massachusetts 02129, ‡Mitotix, Inc., Cambridge, Massachusetts 02139, and the §Howard Hughes Medical Institute, Division of Hematology/Oncology, Children’s Hospital, Boston, Massachusetts 02115 Antimitogenic stimuli such as environmental or geno- MAPK-kinase-kinases (MAPKKKs) activate MAPK/ERK ki- toxic stress, transforming growth factor-b, and the in- nases (MEKs), which, in turn, activate ERKs (1–3). In simple flammatory cytokines tumor necrosis factor and inter- eukaryotes, these pathways are activated by a variety of leukin-1 activate two extracellular signal-regulated stresses, including nutrient withdrawal and osmotic shock (3). kinase (ERK)-based signaling pathways: the stress-acti- In mammals and other multicellular eukaryotes, ERK/MAPK vated protein kinase (SAPK/JNK) pathway and the p38 pathways are activated by both environmental stresses and pathway. Activated p38 phosphorylates transcription more complex physiologic stimuli such as mitogens, develop- factors important in the regulation of cell growth and mental cues, neurotransmitters, vasoactive peptides, and in- apoptosis, including activating transcription factor 2 flammatory cytokines (1, 2, 4). (ATF2), Max, cAMP response element-binding protein- The stress-activated protein kinases (SAPKs, also referred to homologous protein/growth arrest DNA damage 153 as JNKs) and p38 are the ERK components in two mammalian (CHDP/GADD153). In turn, p38 lies downstream of the signaling pathways activated by a broad array of environmen- Rho family GTPases Cdc42Hs and Rac1, as well as at tal stresses as well as by inflammatory cytokines (1, 2). Once least three mitogen-activated protein kinase (MAPK)/ activated, the SAPKs and p38 are responsible for the phospho- ERK-kinases (MEKs): MAPK kinases-3, -6, and SAPK/ rylation and activation of transcription factors necessary for ERK-kinase-1. Although many of the stimuli that acti- the stress response including c-Jun, ATF2, Max, and CHOP vate p38 can also inhibit cell cycle progression, a clear- (1, 2, 5). cut role for the p38 pathway in cell cycle regulation has The signaling components upstream of the SAPKs and p38 not been established. Using a quantitative microinjec- which have been identified thus far suggest a complex cell- and tion approach, we show here that Cdc42Hs, but not Rac1 stimulus-dependent regulation consistent with the diversity of or RhoA, can inhibit cell cycle progression at G /S through a mechanism requiring activation of p38. These extracellular stimuli that activate these pathways. Several results suggest a novel role for Cdc42Hs in cell cycle chromatographically distinct MEKs, including SEK1/MKK4, inhibition. Furthermore, these results suggest that al- SAPK-kinase (SAPKK)4, and SAPKK5 can activate the SAPKs though both Cdc42Hs and Rac1 can activate p38 in situ, in situ and in vitro. (6 – 8). SAPKK4 and SAPKK5 are specific the effects of Cdc42Hs and Rac1 on cell cycle progres- for the SAPKs, whereas SEK1 can also activate p38. By con- sion are, in fact, quite distinct. trast, MKK3 and MKK6 are specific p38 activators (8 –10). Four families of MAPKKKs have also been implicated in SAPK and p38 regulation. MEK kinases (MEKKs) 1 and 2, as Protein kinase signal transduction pathways that employ well as the mixed lineage kinase SH3 domain-containing, pro- members of the extracellular signal-regulated kinase (ERK) / line-rich kinase (SPRK), can activate the SAPK pathway via mitogen-activated protein kinase (MAPK) family have been direct activation of SEK1 (11–13). MEKK2 can also activate the remarkably conserved in evolution (1–3). Typically these path- mitogenic MAPK/ERK1 and 2 pathway by directly phosphoryl- ways consist of a three-tiered core of protein kinases wherein ating the ERK1/2 upstream activator MEK1 (11). Tpl-2 the rat homolog of the human cot proto-oncogene product, like MEKK2, can activate both SEK1 and MEK1 (14). MEKK3 can * This work was supported by United States Public Health Service activate both the SAPK and MAPK pathways, although SEK1 Grants GM53697 and DK41513 and by Grant DAMD17-94-J-4397 from is not a MEKK3 substrate (11). In addition, transforming the United States Army Breast Cancer Research Program (to J. M. K.). growth factor-b-activated kinase-1 (TAK1) can activate p38 in The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked situ and has been implicated as a transforming growth factor- “advertisement” in accordance with 18 U.S.C. Section 1734 solely to b-activated MAPKKK upstream of SEK1, MKK3, and MKK6 indicate this fact. (15, 16). Finally, the p21-activated kinases (PAKs) and germi- To whom correspondence should be addressed: Diabetes Research nal center kinase (GCK), mammalian homologs of Saccharo- Laboratory, Massachusetts General Hospital East, 149 13th St., myces cerevisiae STE20 and SPS1, respectively, can activate Charlestown, MA 02129. Tel.: 617-726-6909; Fax: 617-726-5649; E- mail: [email protected]. the SAPK and p38 pathways, although their mechanisms of The abbreviations used are: ERK, extracellular signal-regulated action are unclear (17–20). kinase; BrdUrd, bromodeoxyuridine; CHOP, cAMP response element Genetic epistasis studies in yeast as well as biochemical and binding protein-homologous protein; GADD, growth arrest DNA dam- transfection experiments employing mammalian cells have age; GCK, germinal center kinase; HA, hemagglutinin; MAPK, mito- gen-activated protein kinase; MAPKKK, MAPK-kinase-kinase; MEK, identified members of the Ras superfamily as upstream ele- MAPK/ERK kinase; MEKK, MEK-kinase; MKK, MAPK-kinase (equiv- ments regulating the core protein kinases in ERK signaling alent to MEK); PAK, p21-activated kinase; SAPK, stress-activated pro- cascades (1, 2, 4, 19, 21–23). Thus, Ras is a critical upstream tein kinase; SAPKK, SAPK-kinase; SEK, SAPK/ERK kinase; SPRK, regulator of the Raf1 kinase, a MAPKKK in the MAPK path- SH3 domain-containing, proline-rich kinase; TAK, transforming growth factor-b-activated kinase; tpl, tumor progression locus; wt, wild-type. way (4). Similarly, in mammalian cells, the Rho family This paper is available on line at http://www-jbc.stanford.edu/jbc/ 13229 This is an Open Access article under the CC BY license. 13230 G Arrest Mediated by Cdc42Hs and p38 GTPases Rac1 and Cdc42Hs can activate the p38 and SAPK pathways. This activation may involve the direct binding of Rac1 and Cdc42Hs to the PAKs. This binding results in PAK activation (18 –20, 22–24). A complete picture of the biological functions of these stress- activated pathways is only beginning to emerge. Recent data have implicated the SAPK and p38 pathways in the induction of apoptosis in response to nerve growth factor withdrawal in PC-12 cells, as well as heat shock, genotoxic chemotherapeutics (cis-platinum), and signaling pathways, which stimulate the generation of ceramide from membrane sphingomyelin (25– 27). However, induction of apoptosis may not be the only bio- FIG.1. Representative photomicrographs of cells microin- logical consequence of activation of the SAPKs and p38. More- jected with the empty pMT3 expression vector together with an over, the specific biological roles of the SAPK and p38 pathways inert rabbit IgG (panels A and F), or with an expression vector (pMT3) harboring HA-tagged p38 (panels B and G), SAPK-p46b1 in signaling mediated by Rac1 and Cdc42Hs have not been (panels C and H), p44-MAPK (panels D and I), or p70 S6 kinase elucidated. (panels E and J) cDNAs. The expressed proteins are shown in red, In this study, we used quantitative microinjection to begin to nuclei of BrdUrd-positive cells in S phase are stained green (panels characterize the biological consequences of Cdc42Hs and Rac1 A–E). Nuclei of BrdUrd-positive cells in S phase concomitantly express- ing the injected plasmids appear yellow because of the usage of a double activation of p38. Here we report that Cdc42Hs is a potent filter and the superimposition of the red and green colors. Nuclei were inhibitor of cell cycle progression, arresting cells at the G /S also counterstained with Hoechst 33258 to identify all nuclei (panels transition point. In addition, we demonstrate that this inhibi- F–J). tion is mediated by elements of the p38 pathway. Rac1 and the RESULTS SAPKs, although able to induce characteristic cellular and biochemical responses, do not inhibit cell cycle progression Effects of ERK Family Kinases on Cell Cycle Progression— significantly. Murine NIH-3T3 fibroblasts were rendered quiescent by serum withdrawal and synchronized in G by serum readdition. Cells EXPERIMENTAL PROCEDURES were microinjected in early G with expression vectors encod- Microinjection—NIH-3T3 cells were grown on glass coverslips and ing HA-tagged p38, SAPK-p46bI, p44-MAPK, p70 S6 kinase, or synchronized in G by serum withdrawal for 24 h. After this period, less empty vector. Entry into S phase was monitored by incorpora- than 1% of the cells incorporated bromodeoxyuridine (BrdUrd) during tion of BrdUrd into DNA. After 24 h, expression of the relevant an additional 24 h of incubation. The arrested cells were released from constructs was verified by immunostaining, and the expressing serum starvation by readdition of 10% calf serum and were microin- cells were scored blindly for S phase entry. Cells injected with jected in early G with expression vectors harboring the HA-tagged or the empty vector were identified by staining for coinjected FLAG-tagged cDNAs, using a Zeiss automated microinjection system rabbit IgG. Cells were also counterstained with bisbenzimide (28). As a control, cells were microinjected with the empty vector to- gether with an inert rabbit antibody marker to identify the injected (Hoechst 33258) to visualize all nuclei and assess any abnormal cells. S phase entry was monitored by BrdUrd (100 mM) incorporation nuclear morphology. Fig. 1 shows representative photomicro- into cellular DNA (28, 29). Cells were fixed at the indicated time after graphs of the stained cells. release from serum starvation as described. Cells stained for phospho- Expression of p38 resulted in a striking 77.5% inhibition of S p38 or phospho-c-Jun were fixed with 4% paraformaldehyde according phase entry compared with cells injected with empty plasmid to the manufacturer’s suggestions. Injected DNA concentrations were: (Fig. 2a). Similar results were obtained using mink lung 100 ng/ml for HA-p38, HA-SAPK, HA-p44MAPK, HA-p70S6 kinase, Mv1Lu cells (data not shown). The inhibition of S phase entry HA-MKK3, HA-MKK3-KR, HA-SEK1, HA-SEK1-KR, HA-MKK6, M2- FLAG-GCK, and empty pMT3; 240 ng/ml for empty pMT3, HA-MKK3- by p38 is likely due to the presence of increased amounts of KR, or HA-SEK1-KR in the dominant negative experiments shown in active p38 in the microinjected cells. Staining of the injected Figs. 1, 3, and 4; and 80 ng/ml for Rho family GTPases. cells with an antibody specific for the active, phosphorylated Immunocytochemistry—Fixed coverslips were stained with anti-HA form of p38 (1, 2) revealed that the p38-expressing cells con- mouse monoclonal antibody or anti-M2-FLAG mouse monoclonal anti- tained greater amounts of phosphorylated p38 than did the body, followed by biotinylated anti-mouse antibody and streptavidin- uninjected cells (Fig. 2b). Texas Red staining as described previously (28, 29). The coinjected The ability of p38 to arrest cells in G was specific, with other rabbit IgG was detected with a biotinylated anti-rabbit antibody, fol- signaling kinases having little or no effect on cell cycle progres- lowed by streptavidin-Texas-Red staining. The incorporated BrdUrd was detected by anti-BrdUrd rat monoclonal antibody and anti-rat sion. p44-MAPK is an integral component of the mitogenic fluorescein isothiocyanate antibody. Nuclei were counterstained with pathway recruited by Ras, and constitutive activation of this Hoechst 33258 dye (1 mg/ml). To quantitate cells in S phase, BrdUrd- pathway is sufficient to transform NIH-3T3 cells (4). Not sur- positive cells were scored blindly as a percent of the number of injected prisingly, expression of p44-MAPK modestly enhanced G /S cells expressing the tagged cDNAs. To stain for p38 following anti-HA progression (91% of p44-MAPK-expressing cells in S phase staining, rabbit anti-p38 antibody was used followed by a 9-amino-6- versus 69% of empty plasmid-injected cells, Fig. 2a). p70 S6 chloro-2-methoxyacridine conjugated anti-rabbit antibody staining. The kinase is also activated by mitogens through mechanisms in- phosphorylated portion of the expressed HA-p38, the endogenous p38 volving phosphatidylinositol 3-kinase and FRAP/RAFT/TOR protein, or c-Jun protein was detected by anti-phospho-p38 or anti- phospho-c-Jun antibodies, followed by biotinylated anti-rabbit antibody (32, 33). Inhibition of p70 S6 kinase blocks G /S transition (34). and streptavidin-9-amino-6-chloro-2-methoxyacridine or streptavidin- Expression of p70 S6 kinase from microinjected plasmid had no fluorescein isothiocyanate staining. Actin stress fibers were stained significant effect on G /S transition (Fig. 2a). SAPKs are pref- with fluorescein phalloidin as described (30). Membrane ruffling was erentially activated by the same antimitogenic stimuli as is p38 detected by staining as described (31). (1, 2). To our surprise, however, expression of injected SAPK, Cloning—MKK3 and MKK6 were cloned by polymerase chain reac- which increased intracellular SAPK activity and, consequently, tion from human B cell (RAMOS) cDNA. p38, SAPK, MKK3, and SEK1 substantially elevated the level of phosphorylated c-Jun (a cDNAs were expressed from the pMT3 (HA-tagged) vector. GCK, SAPK substrate) in the injected cells (Fig. 2b), resulted in only MKK6, Cdc42Hs, Rac1 and RhoA plasmids were expressed from the pCMV5-M2-FLAG vector. modest inhibition of S phase entry (55% in S phase; Fig. 2a). In G Arrest Mediated by Cdc42Hs and p38 13231 FIG.3. Strong activation of SAPK does not promote G arrest. GCK inhibits cell cycle progression at G /S via a p38-dependent mech- anism. Cells were injected with the indicated plasmids and assayed for cell cycle progression. For each injection set, the ratios of S phase cells/total expressing cells were: empty plasmid: 53/85, 67/96, 80/117, 53/73: GCK: 16/59, 9/44, 15/33, 6/29; GCK plus KR-MKK3: 37/62, 47/78, 19/40, 9/17; GCK plus SAPK plus KR-MKK3: 21/42, 25/43, 11/21, 12/25. lie upstream of p38 in stress signaling cascades could block S phase entry. MKK3, MKK6, and SEK1 are three mammalian MEKs that can activate p38 in situ and in vitro (9, 10). Al- though MKK3 and MKK6 are apparently specific for p38, SEK1 is also a strong SAPK activator (6, 9, 10). Expression of HA-tagged, wild-type SEK1, MKK3, or MKK6 from microin- jected plasmids strongly inhibited S phase entry (14% in S phase for SEK1, 17% in S phase for MKK3, 15% in S phase for MKK6, 69% in S phase for empty plasmid). By contrast, ex- pression of kinase-inactive mutant SEK1 or MKK3 plasmids, wherein the lysine residue critical for ATP binding has been mutated to arginine (KR mutants), was without significant effect (Fig. 4a). FIG.2. Inhibition of G /S transition by p38. Part a, quantitation Despite the lack of intrinsic activity, however, kinase-inac- of cell cycle progression. Cells were microinjected with expression plas- mids encoding HA-tagged constructs indicated. Data are expressed as tive SEK1 and MKK3 mutants can act as dominant inhibitors percent of expressing cells in S phase (calculated as the ratio of S phase of coexpressed p38 activation and biological activity and can expressing cells 3 total expressing cells ). Shown are mean 6 S.D. For thus be used to assess the requirement for p38 activation for a each injection set the calculated ratios (S phase expressing cells/total particular biological function (6, 9, 10, 25–27). Accordingly, expressing cells) were: empty plasmid: 36/51, 28/47, 27/45, 52/66, 76/90; p38: 2/19, 4/28, 12/74, 6/28, 3/27; SAPK: 12/21, 9/20, 16/29, 12/21, to test whether activation of p38 was necessary for p38-medi- 84/129; p44-MAPK: 23/23, 18/22, 57/61, 184/205; p70 S6 kinase: 36/51, ated cell cycle arrest, we coinjected p38 with KR-MKK3 or 19/27, 13/20, 24/31. Part b, expression of microinjected p38 elevates the KR-SEK1. Consistent with a requirement for active p38, both level of active phosphorylated p38; injection of SAPK elevates the level KR-SEK1 and KR-MKK3 completely inhibited the ability of of c-Jun phosphorylated at Ser-63 and Ser-73. Anti-HA staining of the expressed p38 (panel A) and SAPK (panel C). Anti-phospho-p38 stain- p38 to block S phase entry (Fig. 4b) without preventing p38 ing (panel B), anti-phospho-c-Jun staining (panel D). expression (Fig. 4c). Effects of Rho Family GTPases on Cell Cycle Progression— view of this result, we explored the effects on cell cycle progres- Cdc42Hs, a member of the Rho subfamily of small GTP-binding sion of strong activation of the SAPKs. proteins, is a human homolog of S. cerevisiae CDC42 (36). GCK is a mammalian homolog of S. cerevisiae SPS1 which, Cdc42p activates Ste20p as part of the yeast mating pheromone when expressed transiently, is constitutively active (1, 2, 17, signaling pathway. This pathway activates two ERKs, Fus3p 35). Coexpression of GCK and SAPK results in potent SAPK and Kss1p, and culminates in G cell cycle arrest (3, 21). activation in the absence of external stimuli. At low levels of Likewise, GTP-charged Cdc42Hs or the related GTPase Rac1 expression, GCK activation of SAPK is specific, with no p38 or can activate the mammalian STE20 homologs PAK1 or PAK3 ERK1 activation seen (17). At high levels of expression, how- in situ and in vitro; and recent evidence indicates that Cdc42Hs ever, GCK can activate p38 modestly. Injection of GCK into and Rac1, but not RhoA, can activate p38 (and the SAPKs) in NIH-3T3 cells resulted in inhibition of G /S transition to a situ via a mechanism requiring PAKs (18, 19, 22–24, 37). To degree commensurate with that obtained upon injection of p38 investigate the potential effects of Rho family GTPases on cell alone (25% in S phase versus 67% in S phase for cells injected cycle arrest, we microinjected expression plasmids encoding with empty vector, Fig. 3). If KR-MKK3, a kinase-dead domi- wild-type and mutant forms of Cdc42Hs, Rac1 or RhoA into nant inhibitory mutant of MKK3, which specifically blocks p38, synchronized NIH-3T3 cells. Expression of wt-Cdc42Hs re- but not SAPK activation (9 and see Fig. 4) is coinjected with sulted in a striking arrest of the cell cycle at G /S, exceeding GCK, this G arrest is abolished completely whether or not that seen for p38. Of the cells injected with empty plasmid, SAPK is coinjected with the GCK (Fig. 3). Thus, SAPK activa- entry into S phase was apparent within 20 h after release from tion likely does not contribute to GCK-mediated cell cycle serum starvation; and 75% of the cells injected with empty arrest. plasmid had transited to S phase within 26 h. By contrast, less Effects of MEKs Upstream of p38 on Cell Cycle Progres- than 7% of the cells expressing wt-Cdc42Hs entered S phase sion—To investigate further the role of activation of p38 in within 30 h after serum readdition (Fig. 5a). This represents a inhibition of G /S transition, we asked if components known to dramatic 90.7% decrease in S phase entry (Fig. 5, a and b). Expression of wt-Cdc42Hs from injected plasmid also elevated T. Yuasa and J. M. Kyriakis, manuscript in preparation. the level of activated, endogenous p38 phosphorylated at the 13232 G Arrest Mediated by Cdc42Hs and p38 although having no effect on Cdc42Hs expression (Fig. 5d), resulted in a partial reversal of the cell cycle inhibitory effects of Cdc42Hs with 37% of the cells progressing to S phase, 44% of control (Fig. 5b). These results suggest that p38 activation is a necessary component in the mechanism by which Cdc42Hs inhibits cell cycle progression. To investigate further the role of Cdc42Hs in G arrest, we next sought to determine the effects of constitutively active and dominant interfering mutants of Cdc42Hs on cell cycle progres- sion. V12-Cdc42Hs is a GTPase-defective mutant of Cdc42 which is constitutively active. Microinjection of V12-Cdc42Hs, although still strongly able to arrest cells in G , is less effective than wild-type (Fig. 5b). We attribute this to the possible non- specific activation of mitogenic pathways by the activated Cdc42Hs mutant. N17-Cdc42Hs is a dominant interfering mu- tant of Cdc42Hs which is thought to act by sequestering nucle- otide exchangers for Cdc42Hs and other Rho family GTPases (38). Thus N17-Cdc42Hs can act either by blocking Cdc42Hs, Rac1 or RhoA activation (38). Consistent with this, microinjec- tion of N17-Cdc42Hs does not activate endogenous p38, as detected by staining with antibodies to phospho-p38 (Fig. 5c). However, to our surprise, N17-Cdc42Hs inhibited G /S progres- sion nearly one-third as effectively as wt-Cdc42Hs (Fig. 5b). This result supports the contention that the N17 mutant pre- vents activation of other Rho family GTPases, such as Rac1, which are required for cell growth. It is conceivable that injection of wt-Cdc42Hs could nonspe- cifically recruit other Rho family GTPases that inhibit cell cycle progression. To investigate the specificity of cell cycle inhibi- tion by Cdc42Hs, we assayed for cell cycle inhibition mediated by other members of the Rho family. To our surprise, injection of either a wild-type or constitutively active allele of Rac1 (V12-Rac1), either of which can activate p38 in situ (19), was far less effective at inhibition of G /S transition than was wt-Cdc42Hs (50% in S phase for both Rac1 constructs versus 67% in S phase for cells injected with empty vector; Fig. 6a), even though injection of either wt- or V12-Rac1 was sufficient to activate p38 to an extent commensurate with that induced by Cdc42Hs, as judged by staining for activated endogenous p38 with anti-phospho-p38 antibodies (Fig. 6b). Moreover, in- jection of V12-Rac1 stimulated membrane ruffling, a charac- teristic response of the cell to activation of Rac1 (31) (Fig. 6c). This result is consistent with earlier observations indicating that Rac1 is in fact a necessary component for Ras transforma- tion (39). Thus, although Rac and Cdc42Hs can both activate p38 in situ (18, 19, 23), each may also activate additional pathways that together result in distinct biological effects. FIG.4. Effect of expression of p38-activating MEKs on cell RhoA has not been shown to lie upstream of p38 or the SAPKs cycle progression: requirement for active p38 for G arrest. Part (22, 23). Injection of a constitutively active (V14) RhoA allele, a, quantitation of S phase entry of cells injected with HA-tagged SEK1, although stimulating the characteristic formation of actin MKK3, MKK6, KR-SEK1, or KR-MKK3. Data shown are mean 6 S.D. stress fibers (30) (Fig. 6c), yielded cell cycle results similar to For each injection set the ratios of S phase expressing cells/total ex- pressing cells were: MKK3: 6/55, 11/55, 3/22, 9/40, 12/67; SEK1: 8/50, those obtained upon injection of Rac1 (49% in S phase, Fig. 6a). 2/22, 5/36, 4/24; MKK6: 7/47, 6/62, 5/33; KR-MKK3: 17/27, 9/16, 10/15, Thus, among Rho family GTPases, cell cycle inhibition appears 26/49, 47/78; KR-SEK1: 45/85, 12/23, 12/20, 14/33; empty vector: 28/47, to be specific to Cdc42Hs. 27/45, 52/66, 76/90, 46/67. Part b, activation of p38 is necessary for p38-mediated inhibition of cell cycle progression. KR-MKK3 and KR- DISCUSSION SEK1 block p38 inhibition of G /S transition. Shown are mean 6 S.D. For each injection set the ratios of S phase cells/expressing cells were: p38 and the SAPKs are activated by an array of ligands that empty plasmid: 80/113, 33/48, 64/101; p38: 6/27; 8/30, 6/32; p38 plus are known to be either antimitogenic or proapoptotic (1, 2). In KR-MKK3: 18/30, 19/32, 18/27; p38 plus KR-SEK1: 14/20, 18/26, 16/28. contrast to the mitogenic pathways, which are regulated Part C, immunostaining of cells coinjected with the HA tagged p38 and largely by polypeptide ligands coupled to tyrosine kinases, the KR-MKK3 cDNAs. Expression of both proteins is shown in panel A. That the S phase transition was due to the inhibitory effect of KR- stimuli that activate the stress pathways are remarkably di- MKK3 and not a lack of expression of p38 was verified by directly verse and include ionizing radiation, heat shock, chemical DNA staining the expressed p38 protein (panel B). damage, oxidative stress, reperfusion injury, and the inflam- matory cytokines tumor necrosis factor-a and interleukin-1. sites critical for activation, as determined by staining with an Thus, the upstream molecular components that feed into the antibody specific for the activated form of p38 (Fig. 5c). p38 and SAPK pathways are accordingly complex and diverse. Coexpression of dominant interfering (KR) SEK1 and MKK3, The Rho family GTPases Cdc42Hs and Rac1 represent two G Arrest Mediated by Cdc42Hs and p38 13233 distinct mechanisms of p38 and SAPK activation with different biological functions. We have demonstrated herein that activation of Cdc42Hs inhibits cell cycle progression at G /S. Insofar as Cdc42Hs (and p38) inhibition of cell cycle progression can be at least partially reversed upon expression of kinase-dead MKK3 and SEK1, we conclude that recruitment of p38 is a critical component of Cdc42Hs-mediated cell cycle arrest. In addition, we have shown that p38 or elements immediately upstream of p38 (SEK1, MKK3, or MKK6) can also arrest cells in G . By con- trast, Rac1, which can also activate p38 (19, 23), fails to inhibit strongly cell cycle progression. Indeed, Rac1 has been impli- cated in Ras transformation (39) and may recruit additional signaling pathways that prevent inhibition of the cell cycle or promote cell cycle progression. Thus, although activation of p38 alone is sufficient to arrest cells in G , we cannot rule out that p38, which can be activated weakly by mitogenic signals, could, in conjunction with other signaling pathways, lead to responses distinct from G arrest. These ideas are summarized in the model shown in Fig. 7. Our results are in contrast with data implicating Rho family GTPases, notably Cdc42Hs, in cell growth and transformation (40). It is conceivable that the concomitant recruitment of Ras and several Rho family GTPase signaling pathways could re- sult in cell growth, whereas activation of Cdc42Hs alone or in conjunction with other antimitogenic pathways results in cell cycle arrest. In this regard, care must be taken in the interpre- tation of results concerning Cdc42Hs mutants. Our results presented in Fig. 5 suggest that wt-Cdc42Hs is the most potent inhibitor of cell cycle progression, whereas a constitutively active mutant (V12-Cdc42Hs), although still growth inhibitory, is less so than wild-type. This result may reflect the ability of the overexpressed, constitutively active Cdc42Hs mutant to recruit nonspecifically mitogenic signaling mechanisms, possi- bly including those normally regulated by other Rho family GTPases, more effectively than the overexpressed wild-type Cdc42Hs. By the same token, overexpression of dominant neg- ative Cdc42Hs, although unable to activate endogenous p38, was growth inhibitory, possibly because of nonspecific seques- tering of the activation machinery for other Rho family GTPases. For example, Ost, a proto-oncogene, is a guanine nucleotide exchanger for Rho, Cdc42Hs, and Rac1 (38). We do not believe that Cdc42Hs inhibition of G /S transition is the result of nonspecific recruitment of p38 inasmuch as wild-type p38 itself, as well as MKK3, MKK6, and SEK1, all p38 activators, are also able to arrest NIH-3T3 cells at G /S. Moreover, recent evidence indicates that p38 overexpression inhibits mitogen induction of G cyclins (41). Thus it is not surprising that p38 activation, by a Cdc42Hs-dependent mech- anism, could result in growth arrest. Moreover, precedent for Cdc42Hs as a G inhibitor has been observed in lower eu- karyotes. Thus, S. cerevisiae Cdc42p is an essential component of the mating pheromone response pathway, which also results in G arrest (3, 21). Inasmuch as the experiments implicating Cdc42Hs in cell growth have employed Swiss 3T3 cells (40), our FIG.5. Cdc42Hs, an upstream activator of p38, inhibits G /S transition, whereas coexpression of Cdc42Hs with KR-MKK3, KR-SEK1 blocks Cdc42Hs-mediated cell cycle inhibition. Part a, staining (panels A and C, respectively). Staining for phospho-p38 is time course of cell cycle progression in cells injected with the expression shown in panels B and D. Arrows indicate nuclear phospho-p38 staining plasmids indicated. Part b, quantitation of S phase cells 26 h after (panel B) or mark the nuclei of the injected cells (panel D). Part d, release from serum starvation. Shown are the mean 6 S.D. For each expression of KR-SEK and KR-MKK3 has no effect on expression of injection set the ratios of S phase expressing cells/total expressing cells coinjected Cdc42Hs. The expressed proteins (in red) were stained with were: empty vector: 29/37, 98/157, 130/172, 108/143, 150/197; wt- anti-FLAG (panels A and B), and the rabbit IgG coinjected with the Cdc42Hs: 9/96, 2/25; 3/62, 1/45, 15/129, 14/145; wt-Cdc42Hs plus KR- empty vectors were stained with anti-rabbit antibody (panel C). Nuclei MKK3 and KR-SEK1: 45/102, 24/68, 12/35, 26/80; V12-Cdc42Hs: 11/42, of cells in S phase are stained with anti-BrdUrd and shown in green 18/50, 11/49; N17-Cdc42Hs: 7/34, 6/25, 10/35, 8/34. Part c, expression of (panels A–C). All nuclei were stained with Hoechst 33258 (panels D–F). wt-Cdc42Hs increases endogenous phosphorylation of p38 at the regu- Injected cDNAs were: wt-Cdc42Hs (panels A and D), wt-Cdc42Hs plus latory sites, whereas expression of N17-Cdc42Hs does not. Expression KR-MKK3 and KR-SEK1 (panels B and F), empty vectors plus rabbit of the injected wt- and N17-Cdc42 cDNAs was verified with anti-FLAG IgG (panels C and F). 13234 G Arrest Mediated by Cdc42Hs and p38 FIG.7. Model for cell cycle regulation by Rho family GTPases and the p38 pathway. Bold arrows indicate strong signaling inputs. Dashed arrows indicate weaker inputs. The reasons for G arrest in response to stress signals have not been clearly defined; however, it is plausible to propose that cell cycle arrest would be followed by repair of stress-related damage. Alternatively, the cell could arrest in G and await restoration of a normal cellular milieu conducive to continued growth. The targets of Cdc42Hs and p38 which mediate cell cycle arrest are not known. However, inhibition of induction of G cyclins (41) is a logical candidate. Moreover, CHOP, a p38 substrate, promotes G arrest as part of the response to geno- toxic stress (5, 42). Finally, several recent studies indicate that recruitment of the SAPKs or p38 results in apoptosis. Withdrawal of nerve growth factor from PC-12 cells activates both the SAPKs and p38 and promotes apoptosis. Expression of KR-MKK3 or KR- SEK1 blocks this effect, and constitutively active mutants of MKK3 or SEK1 promote apoptosis in the presence of nerve growth factor (25). Likewise, thermotolerant fibroblasts dis- play defective SAPK activation and cell death in response to heat shock. Expression of SEK1 restores heat sensitivity to these cells (26). Finally, treatment of macrophage cell lines with agonists that stimulate sphingomyelin hydrolysis (tumor necrosis factor, UV radiation, x-irradiation, oxidant stress) re- sults in apoptosis, a response that can be reversed upon ex- pression of dominant inhibitory SEK1 (27). Despite these find- ings, in none of the experiments shown in Figs. 1– 6 did we see evidence of apoptosis. Thus, cycle arrest at G /S, rather than apoptosis, appears to be the characteristic response of NIH-3T3 FIG.6. Rac1 and RhoA do not strongly block cell cycle progres- cells to activation of the Cdc42Hs/p38 pathway, indicating that sion. Part a, cells were microinjected with the indicated plasmids and different cells respond distinctly to p38 activation. quantitated for cell cycle progression. For each injection set the ratios of S phase expressing cells/total expressing cells were: empty plasmid: Acknowledgments—We thank J. Settleman for Rac, Rho, and 53/85, 67/96, 80/117, 53/73; wt-Rac1: 24/53, 28/55, 58/113; V12-Rac1: Cdc42Hs cDNAs; J. H. Kehrl for GCK cDNA; A. Nebreda for anti-p38 35/59, 30/64, 23/50; V14-RhoA: 19/35, 16/39, 39/86, 49/101, 31/59. Part antiserum; J. Avruch for p70 S6 kinase cDNA and for a critical reading b, injection of wt- or V12-Rac1 activates p38 to the same degree as does of the manuscript; D. Ron for CHOP cDNA; M. Pagano, R. Bruns, and wt-Cdc42Hs. Cells were injected with wt- (panels A and C) or V12-Rac1 S. Brill for advice; the Massachusetts General Hospital Molecular On- (panels B and D) and stained for expression of the injected construct cology Laboratory for use of their microinjection apparatus; and the (panels A and B) or endogenous phospho-p38 (panels C and D). 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Published: May 1, 1997

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