The PAK family of kinases are regulated through interaction with the small GTPases Cdc42 and Rac1, but little is known of the signaling components immediately upstream or downstream of these proteins. We have purified and cloned a new class of Rho-p21 guanine nucleotide exchange factor binding tightly through its N-terminal SH3 domain to a conserved proline-rich PAK sequence with a Kd of 24 nM. This PAK-interacting exchange factor (PIX), which is widely expressed and enriched in Cdc42- and Rac1-driven focal complexes, is required for PAK recruitment to these sites. PIX can induce membrane ruffling, with an associated activation of Rac1. Our results suggest a role for PIX in Cdc42-to-Rac1 signaling, involving the PIX/PAK complex.

The GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, motility, and cytokinesis. We recently reported on a p150 serine/threonine kinase (termed ROK␣) binding RhoA only in its active GTP-bound state and on its cDNA; introduction of RhoA into HeLa cells resulted in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROK␣ being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK␣ contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROK␤) with 90% identity to ROK␣ in the kinase domain. Both ROK␣ and ROK␤, which had a molecular mass of 160 kDa, contained a highly conserved cysteine/histidine-rich domain located within a putative pleckstrin homology domain. The kinases bound RhoA, RhoB, and RhoC but not Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain caused partial or complete loss of Rho binding. The morphological effects of ROK␣ were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK␣. Full-length ROK␣ promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK␣ truncated at the C terminus promoted this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibited protein kinase activity which was required for stress fiber formation; the kinase-dead ROK␣K112A and N-terminally truncated mutants showed no such promotion. The latter mutant instead induced disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects were mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROK␣ appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes.In mammals, the Ras-related Rho subfamily includes RhoA, -B, and -C, Rac1 and -2, and Cdc42, which play pivotal roles in cytoskeletal control and cell morphology. RhoA has been implicated in stress fiber formation (33, 34), whereas Rac1 (36) and Cdc42 (15,29) are involved in lamellipodial and filopodial formation, respectively. Fibroblasts injected with these GTPases display a set of distinctive morphological changes, suggestive of probable hierarchy in the order Cdc42, Rac, and Rho. These changes demand a high level of flexibility in the dynamic reorganization of actin microfilaments in cells. RhoA, -B, and -C have about 85% identity and appear to have different cellular localizations (1). Although their exact roles in cells have not been clearly defined, these GTPases have been implicated in a variety of cellular activities either directly by overexpression of wil...

The activation dynamics of the transcription factor NF-kappaB exhibit damped oscillatory behavior when cells are stimulated by tumor necrosis factor-alpha (TNFalpha) but stable behavior when stimulated by lipopolysaccharide (LPS). LPS binding to Toll-like receptor 4 (TLR4) causes activation of NF-kappaB that requires two downstream pathways, each of which when isolated exhibits damped oscillatory behavior. Computational modeling of the two TLR4-dependent signaling pathways suggests that one pathway requires a time delay to establish early anti-phase activation of NF-kappaB by the two pathways. The MyD88-independent pathway required Inferon regulatory factor 3-dependent expression of TNFalpha to activate NF-kappaB, and the time required for TNFalpha synthesis established the delay.

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The transcription factor NF-kappaB regulates a wide variety of genes involved in multiple processes. Although the apparent consensus sequence of DNA binding sites for NF-kappaB (kappaB sites) is very broad, the sites active in any one gene show remarkable evolutionary stability. Using a lentivirus-based methodology for implantation of gene regulatory sequences we show that for genes with two kappaB sites, both are required for activity. Swapping sites between kappaB-dependent genes altered NF-kappaB dimer specificity of the promoters and revealed that two kappaB sites can function together as a module to regulate gene activation. Further, although the sequence of the kappaB site is important for determining kappaB family member specificity, rather than determining the ability of a particular dimer to bind effectively, the sequence affects which coactivators will form productive interactions with the bound NF-kappaB dimer. This suggests that binding sites may impart a specific configuration to bound transcription factors.

The Rho GTPases play distinctive roles in cytoskeletal reorganization associated with growth and differentiation. The Cdc42/Rac-binding p21-activated kinase (PAK) and Rho-binding kinase (ROK) act as morphological effectors for these GTPases. We have isolated two related novel brain kinases whose p21-binding domains resemble that of PAK whereas the kinase domains resemble that of myotonic dystrophy kinase-related ROK. These ϳ190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, which is known to be crucial for activating actin-myosin contractility. The p21-binding domain binds GTP-Cdc42 but not GDP-Cdc42. The multidomain structure includes a cysteine-rich motif resembling those of protein kinase C and n-chimaerin and a putative pleckstrin homology domain. MRCK␣ and Cdc42

Hepatocellular carcinoma (HCC) is a major malignancy in many parts of the world, especially in Asia and Africa. Loss of heterozygosity (LOH) on the long arm of chromosome 13 has been reported in HCC. In search of tumor suppressor genes in this region, here we have identified DLC2 (for deleted in liver cancer 2) at 13q12.3 encoding a novel Rho family GTPase-activating protein (GAP). DLC2 mRNA is ubiquitously expressed in normal tissues but was significantly underexpressed in 18% (8/ 45) of human HCCs. DLC2 is homologous to DLC1, a previously identified tumor suppressor gene at 8p22-p21.3 frequently deleted in HCC. DLC2 encodes a novel protein with a RhoGAP domain, a SAM (sterile ␣ motif) domain related to p73/p63, and a lipid-binding StARrelated lipid transfer (START) domain. Biochemical analysis indicates that DLC2 protein has GAP activity specific for small GTPases RhoA and Cdc42. Expression of the GAP domain of DLC2 sufficiently inhibits the Rho-mediated formation of actin stress fibers. Introduction of human DLC2 into mouse fibroblasts suppresses Ras signaling and Ras-induced cellular transformation in a GAP-dependent manner. Taken together, our findings suggest a role for DLC2 in growth suppression and hepatocarcinogenesis.

Dramatic transient changes resulting in a stellate morphology are induced in many cell types on treatment with agents that enhance intracellular cAMP levels. Thrombin fully protects cells from this inductive effect of cAMP through the thrombin receptor. The protective effect of thrombin was shown to be Rho-dependent. Clostridium botulinum C3 exoenzyme, which inactivates RhoA functions, abolished the ability of thrombin to protect cells from responding to increased cAMP levels. A constitutively activated RhoA V14 mutant protein also prevented cells from responding to cAMP. RhoA can be specifically phosphorylated at Ser-188 by the cAMPactivated protein kinase A (PKA). We demonstrate that RhoA V14A188 , which cannot be phosphorylated by PKA in vitro, is more effective than RhoA V14 in preventing cells from responding to cAMP and in inducing actin stress fiber formation. This suggests that PKA phosphorylation of RhoA impairs its biological activity in vivo. ROK␣, a RhoA-associated serine/threonine kinase can also prevent cells from responding to cAMP with shape changes. Phosphorylation of RhoA by PKA in vitro decreases the binding of RhoA to ROK␣. These results indicate that RhoA and cAMP have antagonistic roles in regulating cellular morphology and suggest that cAMPmediated down-regulation of RhoA binding to its effector ROK␣ may be involved in this antagonism.