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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