Rho, a member of the Ras superfamily of GTP-binding proteins, regulates actin polymerization resulting in the formation of stress fibers and the assembly of focal adhesions. In Swiss 3T3 cells, heterotrimeric G proteincoupled receptors for lysophosphatidic acid and gastrin releasing peptide stimulate Rho-dependent stress fiber and focal adhesion formation. The specific heterotrimeric G protein subunits mediating Rho-dependent stress fiber and focal adhesion formation have not been defined previously. We have expressed GTPase-deficient, constitutively activated G protein ␣ subunits and mixtures of ␤ and ␥ subunits in Swiss 3T3 cells. Measurement of actin polymerization and focal adhesion formation indicated that GTPase-deficient ␣ 12 and ␣ 13 , but not the activated forms of ␣ i2 or ␣ q stimulated stress fiber and focal adhesion assembly. Combinations of ␤ and ␥ subunits were unable to stimulate stress fiber or focal adhesion formation. G␣ 12 -and ␣ 13 -mediated stress fiber and focal adhesion assembly was inhibited by botulinum C3 exoenzyme, which ADP-ribosylates and inactivates Rho, indicating that ␣ 12 and ␣ 13 , but not other G protein ␣ subunits or ␤␥ complexes, regulate Rhodependent responses. The results define the integration of G 12 and G 13 with the regulation of the actin cytoskeleton.

Signal transduction pathways regulated by G12 and G13 heterotrimeric G proteins are largely unknown. Expression of activated, GTPase-deficient mutants of alpha 12 and alpha 13 alter physiological responses such as Na+/H+ exchanger activity, but the effector pathways controlling these responses have not been defined. We have found that the expression of GTPase-deficient mutants of alpha 12 (alpha 12Q229L) or alpha 13 (alpha 13Q226L) leads to robust activation of the Jun kinase/stress-activated protein kinase (JNK/SAPK) pathway. Inducible alpha 12Q229L and alpha 13Q226L expression vectors stably transfected in NIH 3T3 cells demonstrated JNK/SAPK activation but not extracellular response/mitogen-activated protein kinase activation. Transient transfection of alpha 12Q229L and alpha 13Q226L also activated the JNK/SAPK pathway in COS-1 cells. Expression of the GTPase-deficient mutant of alpha q (alpha qQ209L) but not alpha i (alpha iQ205L) or alpha s (alpha sQ227L) was also able to activate the JNK/SAPK pathway. Functional Ras signaling was required for alpha 12Q229L and alpha 13Q226L activation of the JNK/SAPK pathway; expression of competitive inhibitory N17Ras inhibited JNK/SAPK activation in response to both alpha 12Q229L and alpha 13Q226L. The results describe for the first time a Ras-dependent signal transduction pathway involving JNK/SAPK regulated by alpha 12 and alpha 13.

Dual speci®city kinases that phosphorylate the Thr-and Tyr-residues within the TXY motif of MAP-kinases of play a central role in the regulation of various processes of cell growth. These dual speci®city kinases also known as MAP kinase kinases are constituents of the sequential kinase signaling modules. Seven distinct mammalian MAP kinases kinases have been identi®ed. Some of the unique signaling properties of these kinases are discussed here.

T he mitogen-activated protein kinase (MAPK) family consists of a group of kinases responsive to a variety of environmental stimuli. MAPKs can be subdivided into three groups: extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38MAPK (1-8). Although ERK has been shown to be activated primarily by proliferative signals, JNK and p38MAPK are activated by genotoxic as well as cytotoxic stress signals (7-10). The structural organization of these kinases into specific signaling modules appears to be facilitated by scaffolding proteins such as STE5 in yeast (11)(12)(13)(14) and JNK͞stress-activated protein kinase-associated protein (JSAP) and JNK interacting proteins 1-3 in mammalian cells (15)(16)(17)(18)(19)(20). These scaffolding proteins tether different MAPK kinase kinases (MEKKs), MAPK kinases (MKKs), and MAPKs into close proximity so that the successive phosphorylation events can occur efficiently, thus conferring specificity to a particular combination of kinases for activation. Although it is well documented that these phosphorylation cascades lead to the activation of transcription factors such as Fos, Jun, and Myc, the precise mechanisms through which the specific transcription factors are recruited is not known (1, 4). In our search for proteins that associate with transcription factors such as Max and Myc, we identified a scaffolding protein, which we termed JLP for JNK-associated leucine zipper protein. Here we show that JLP brings together Max and c-Myc along with JNK and p38MAPK, as well as their upstream kinases MKK4 and MEKK3. Thus, JLP defines a family of scaffolding proteins that bring MAPKs and their target transcription factors together for the execution of specific signaling pathways. Materials and MethodsCloning of JLP. Human Max was expressed as bacterial recombinant protein, which was 32 P-labeled in vitro with heart muscle kinase (Sigma) by using [␥-32 P]ATP. The Max probe was added in the hybridization buffer Hyb75 (20 mM Hepes-KOH, pH 7.7͞75 mM KCl͞0.1 mM EDTA͞2.5 mM MgCl 2 ͞0.05% Nonidet P-40͞1% nonfat milk͞10 mM DTT) and used to screen the gt11 expression library derived from 32Dcl3 cells (21) as described (22). Four overlapping cDNAs were used to generate a cDNA sequence encoding for the full-length protein of JLP.S Tagging and Mutation of JLP. PCR was used to generate a fragment of JLP sequence (3,250-4,083 bp) whose 3Ј end contained the coding sequence of S tag (KETAAAKFERQH-MDS) followed by a stop codon. Substitution of all leucine residues [amino acids 117, 124, 131, 145, 152, and 159 in leucine zipper I (LZI) and amino acids 413 and 420 in leucine zipper II (LZII)] of JLP with alanine residues was carried out by sitedirected mutagenesis and fusion PCR by using M2 cDNA as the template as described (23). All mutations and deletion constructs were verified by sequence analysis. JLP-S 3Ј deletion mutants were created by digestion of the WT JLP-S cDNA EcoRI͞MluI fragment with SmlI, MslI, PvuII, BssSI, SpeI, and Bsu36I. JLP-S domain constructs were generate...

G␣ 13 , the ␣-subunit of the heterotrimeric G protein G13, has been shown to stimulate cell migration in addition to inducing oncogenic transformation. Cta, a Drosophila ortholog of G13, has been shown to be critical for cell migration leading to the ventral furrow formation in Drosophila embryos. Loss of G␣ 13 has been shown to disrupt cell migration associated with angiogenesis in developing mouse embryos. Whereas these observations point to the vital role of G13-orthologs in regulating cell migration, widely across the species barrier, the mechanism by which G␣ 13 couples to cytoskeleton and cell migration is largely unknown. Here we show that G␣ 13 physically interacts with Hax-1, a cytoskeleton-associated, cortactin-interacting intracellular protein, and this interaction is required for G␣ 13 -stimulated cell migration. Hax-1 interaction is specific to G␣ 13 , and this interaction is more pronounced with the mutationally or functionally activated form of G␣ 13 as compared with the wild-type G␣ 13 . Expression of Hax-1 reduces the formation of actin stress fibers and focal adhesion complexes in G␣ 13 -expressing NIH3T3 cells. Coexpression of Hax-1 also attenuates G␣ 13 -stimulated activity of Rho while potentiating G␣ 13 -stimulated activity of Rac. The presence of a quadnary complex consisting of G␣ 13 , Hax-1, Rac, and cortactin indicates the role of Hax-1 in tethering G␣ 13 to the cytoskeletal component(s) involved in cell movement. Whereas the expression of Hax-1 potentiates G␣ 13 -mediated cell movement, silencing of endogenous Hax-1 with Hax-1-specific small interfering RNAs drastically reduces G␣ 13 -mediated cell migration. These findings, along with the observation that Hax-1 is overexpressed in metastatic tumors and tumor cell lines, suggest a novel role for the association of oncogenic G␣ 13 and Hax-1 in tumor metastasis.Cell migration plays a vital role in different biological processes ranging from embryogenesis to immune response (1, 2). However, an aberrant activation of cell migration in neoplastic cells results in tumor metastasis. Cells migrate in response to different cues through the coordinated interactions of actinand/or microtubule-associated cytoskeletal proteins (3, 4). G protein-coupled receptors and their cognate G proteins play a major role in regulating cell migration and chemokinesis (5). The G12 family of G proteins, defined by ␣-subunits G␣ 12 and G␣ 13 , has been shown to activate novel signaling pathways involved in cell growth and neoplastic transformation (6). Two lines of evidence indicate that the ␣-subunit of G13, G␣ 13 , is primarily involved in the regulation of cell migration (7-9). The first is the observation that Cta, an ortholog of G␣ 13 , is critically required for cell movement during Drosophila embryogenesis (7). The second is the finding that G␣ 13 -null (G␣ 13 Ϫ/Ϫ) fibroblasts show the loss of chemokinetic response to thrombin or lysophosphatidic acid receptor-mediated cell movement (8, 9).Existing models of cell movement suggest that the initial movements i...

G Proteins provide signal transduction mechanisms to seven transmembrane receptors. Recent studies have indicated that the a-subunits as well as the bg-subunits of these proteins regulate several critical signaling pathways involved in cell proliferation, di erentiation and apoptosis. Of the 17 a-subunits that have been cloned, at least ten of them have been shown to couple mitogenic signaling in ®broblast cells. Activating mutations in Ga s , Ga i2 , and Ga 12 have been correlated with di erent types of tumors. In addition, the ability of the bg-subunits to activate mitogenic pathways in di erent cell-types has been de®ned. The present review brie¯y summarizes the diverse and novel signaling pathways regulated by the a-as well as the bg-subunits of G proteins in regulating cell proliferation.

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