New insights into the regulation of protein kinase C and novel phorbol ester receptors.
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Protein kinase C (PKC), a family of related serine-threonine kinases, is a key player in the cellular responses mediated by the second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. The traditional view of PKCs as DAG/phospholipid-regulated proteins has expanded in the last few years by three seminal discoveries. First, PKC activity and maturation is controlled by autophosphorylation and transphosphorylation mechanisms, which includes phosphorylation of PKC isozymes by phosphoinositide-dependent protein kinases (PDKs) and tyrosine kinases. Second, PKC activity and localization are regulated by direct interaction with different types of interacting proteins. Protein-protein interactions are now recognized as important mechanisms that target individual PKCs to different intracellular compartments and confer selectivity by associating individual isozymes with specific substrates. Last, the discovery of novel phorbol ester receptors lacking kinase activity allows us to speculate that some of the biological responses elicited by phorbol esters or by activation of receptors coupled to elevation in DAG levels could be mediated by PKC-independent pathways. (+info)
Sema3A-induced growth-cone collapse is mediated by Rac1 amino acids 17-32.
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BACKGROUND: Neurons project their axons along specific pathways in order to establish appropriate connections with their target cells. The rate and direction of axonal growth is determined by interactions between the highly motile growth cone and environmental cues that can act in either an attractive or a repulsive manner. Locomotion is ultimately dependent upon the reorganisation of the actin cytoskeleton and an established role for the Rho family of small GTPases in regulating this process in non-neuronal cells identifies them as candidate signalling molecules in growth cones. An inactive form of Rac1 has recently been shown to inhibit the 'growth-cone collapse' response induced by chick Sema3A, a protein that has recently been established as an important guidance cue. The molecular basis for this inhibition remains unclear. RESULTS: We have made a series of overlapping peptides from the amino-terminal region of Rac1 and rendered them cell permeable by synthesis in tandem with an established internalisation vector. We report here that a peptide encompassing Rac1 amino acids 17-32 binds directly to the established Rac1-interacting molecules PAK, WASP, 3BP-1 and p85beta(P13K), but not to p67(Phox). Furthermore, the peptide can compete with activated Rac1 for target binding, and inhibits Sema3A-induced growth-cone collapse. We also synthesised cell-permeable peptides that correspond to the Cdc42/Rac1-binding (CRIB) motifs present in PAK and N-WASP. Our results show that a CRIB-containing peptide from PAK, but not that from N-WASP, inhibits growth-cone collapse and that the inhibitory activity correlates with binding to Rac1 and not to Cdc42. CONCLUSIONS: Our results suggest that Sema3A-induced growth-cone collapse is mediated by Rac1 amino acids 17-32, and demonstrate the feasibility of designing new cell-permeable inhibitors of small GTPases. (+info)
Distinct effects of Rac1 on differentiation of primary avian myoblasts.
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Rho family GTPases have been implicated in the regulation of the actin cytoskeleton in response to extracellular cues and in the transduction of signals from the membrane to the nucleus. Their role in development and cell differentiation, however, is little understood. Here we show that the transient expression of constitutively active Rac1 and Cdc42 in unestablished avian myoblasts is sufficient to cause inhibition of myogenin expression and block of the transition to the myocyte compartment, whereas activated RhoA affects myogenic differentiation only marginally. Activation of c-Jun N-terminal kinase (JNK) appears not to be essential for block of differentiation because, although Rac1 and Cdc42 GTPases modestly activate JNK in quail myoblasts, a Rac1 mutant defective for JNK activation can still inhibit myogenic differentiation. Stable expression of active Rac1, attained by infection with a recombinant retrovirus, is permissive for terminal differentiation, but the resulting myotubes accumulate severely reduced levels of muscle-specific proteins. This inhibition is the consequence of posttranscriptional events and suggests the presence of a novel level of regulation of myogenesis. We also show that myotubes expressing constitutively active Rac1 fail to assemble ordered sarcomeres. Conversely, a dominant-negative Rac1 variant accelerates sarcomere maturation and inhibits v-Src-induced selective disassembly of I-Z-I complexes. Collectively, our findings provide a role for Rac1 during skeletal muscle differentiation and strongly suggest that Rac1 is required downstream of v-Src in the signaling pathways responsible for the dismantling of tissue-specific supramolecular structures. (+info)
Rab5 induces Rac-independent lamellipodia formation and cell migration.
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Rab5 is a regulatory GTPase of vesicle docking and fusion that is involved in receptor-mediated endocytosis and pinocytosis. Introduction of active Rab5 in cells stimulates the rate of endocytosis and vesicle fusion, resulting in the formation of large endocytic vesicles, whereas dominant negative Rab5 inhibits vesicle fusion. Here we show that introduction of active Rab5 in fibroblasts also induced reorganization of the actin cytoskeleton but not of microtubule filaments, resulting in prominent lamellipodia formation. The Rab5-induced lamellipodia formation did not require activation of PI3-K or the GTPases Ras, Rac, Cdc42, or Rho, which are all strongly implicated in cytoskeletal reorganization. Furthermore, lamellipodia formation by insulin, Ras, or Rac was not affected by expression of dominant negative Rab5. In addition, cells expressing active Rab5 displayed a dramatic stimulation of cell migration, with the lamellipodia serving as the leading edge. Both lamellipodia formation and cell migration were dependent on actin polymerization but not on microtubules. These results demonstrate that Rab5 induces lamellipodia formation and cell migration and that the Rab5-induced lamellipodia formation occurs by a novel mechanism independent of, and distinct from, PI3-K, Ras, or Rho-family GTPases. Thus, Rab5 can control not only endocytosis but also actin cytoskeleton reorganization and cell migration, which provides strong support for an intricate relationship between these processes. (+info)
Biochemical analysis of SopE from Salmonella typhimurium, a highly efficient guanosine nucleotide exchange factor for RhoGTPases.
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RhoGTPases are key regulators of eukaryotic cell physiology. The bacterial enteropathogen Salmonella typhimurium modulates host cell physiology by translocating specific toxins into the cytoplasm of host cells that induce responses such as apoptotic cell death in macrophages, the production of proinflammatory cytokines, the rearrangement of the host cell actin cytoskeleton (membrane ruffling), and bacterial entry into host cells. One of the translocated toxins is SopE, which has been shown to bind to RhoGTPases of the host cell and to activate RhoGTPase signaling. SopE is sufficient to induce profuse membrane ruffling in Cos cells and to facilitate efficient bacterial internalization. We show here that SopE belongs to a novel class of bacterial toxins that modulate RhoGTPase function by transient interaction. Surface plasmon resonance measurements revealed that the kinetics of formation and dissociation of the SopE.CDC42 complex are in the same order of magnitude as those described for complex formation of GTPases of the Ras superfamily with their cognate guanine nucleotide exchange factors (GEFs). In the presence of excess GDP, dissociation of the SopE.CDC42 complex was accelerated more than 1000-fold. SopE-mediated guanine nucleotide exchange was very efficient (e.g. exchange rates almost 10(5)-fold above the level of the uncatalyzed reaction; substrate affinity), and the kinetic constants were similar to those described for guanine nucleotide exchange mediated by CDC25 or RCC1. Far-UV CD spectroscopy revealed that SopE has a high content of alpha-helical structure, a feature also found in Dbl homology domains, Sec7-like domains, and the Ras-GEF domain of Sos. Despite the lack of any obvious sequence similarity, our data suggest that SopE may closely mimic eukaryotic GEFs. (+info)
Requirement for Ras/Rac1-mediated p38 and c-Jun N-terminal kinase signaling in Stat3 transcriptional activity induced by the Src oncoprotein.
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Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors. However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis. In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation. Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3. We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis. Expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK). Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts. Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation. In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine. Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation. Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein. These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis. (+info)
Reciprocal signaling between heterotrimeric G proteins and the p21-stimulated protein kinase.
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p21-activated protein kinase (PAK)-1 phosphorylated Galpha(z), a member of the Galpha(i) family that is found in the brain, platelets, and adrenal medulla. Phosphorylation approached 1 mol of phosphate/mol of Galpha(z) in vitro. In transfected cells, Galpha(z) was phosphorylated both by wild-type PAK1 when stimulated by the GTP-binding protein Rac1 and by constitutively active PAK1 mutants. In vitro, phosphorylation occurred only at Ser(16), one of two Ser residues that are the major substrate sites for protein kinase C (PKC). PAK1 did not phosphorylate other Galpha subunits (i1, i2, i3, o, s, or q). PAK1-phosphorylated Galpha(z) was resistant both to RGSZ1, a G(z)-selective GTPase-activating protein (GAP), and to RGS4, a relatively nonselective GAP for the G(i) and G(q) families of G proteins. Phosphorylation of Ser(27) by PKC did not alter sensitivity to either GAP. The previously described inhibition of G(z) GAPs by PKC is therefore mediated by phosphorylation of Ser(16). Phosphorylation of either Ser(16) by PAK1 or Ser(27) by PKC decreased the affinity of Galpha(z) for Gbetagamma; phosphorylation of both residues by PKC caused no further effect. PAK1 thus regulates Galpha(z) function by attenuating the inhibitory effects of both GAPs and Gbetagamma. In this context, the kinase activity of PAK1 toward several protein substrates was directly inhibited by Gbetagamma, suggesting that PAK1 acts as a Gbetagamma-regulated effector protein. This inhibition of mammalian PAK1 by Gbetagamma contrasts with the stimulation of the PAK homolog Ste20p in Saccharomyces cerevisiae by the Gbetagamma homolog Ste4p/Ste18p. (+info)
Activation of activator protein 1 and stress response kinases in epithelial cells colonized by Helicobacter pylori encoding the cag pathogenicity island.
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Helicobacter pylori interacts with the apical membrane of the gastric epithelium and induces a number of proinflammatory cytokines/chemokines. The subsequent infiltration of macrophages and granulocytes into the mucosa leads to gastric inflammation accompanied by epithelial degeneration. Gastric diseases, e.g. peptic ulcer or gastric adenocarcinoma, are more common among people infected with H. pylori strains producing VacA (vacuolating cytotoxin A) and possessing a cag (cytotoxin-associated antigen A) pathogenicity island. For the induction of the cytokine/chemokine genes in response to H. pylori, we studied the signaling leading to the nuclear activation of the early response transcription factor activator protein 1 (AP-1). We found that H. pylori strains carrying the pathogenicity island induce activation of AP-1 and nuclear factor kappaB. In contrast to the wild type or an isogenic strain without the vacA gene, isogenic H. pylori strains with mutations in certain cag genes revealed only weak AP-1 and nuclear factor kappaB activation. In respect to the molecular components that direct AP-1 activity, our results indicate a cascade of the cellular stress response kinases c-Jun N-terminal kinase, MAP kinase kinase 4, and p21-activated kinase, and small Rho-GTPases including Rac1 and Cdc42, which contributes to the activation of proinflammatory cytokines/chemokines induced by H. pylori encoding the cag pathogenicity island. (+info)