rho-type guanine exchange factor


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. 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, has been purified and cloned. 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. These results suggest a role for PIX in Cdc42-to-Rac1 signaling, involving the PIX/PAK complex (Manser, 1998).

Proteins of the p21-activated kinase (Pak) family have been implicated in the regulation of gene expression, cytoskeletal architecture, and apoptosis. Although the ability of Cdc42 and Rac GTPases to activate Pak is well established, relatively little else is known about Pak regulation or the identity of Pak cellular targets. Two closely related Pak3-binding proteins, possibly arising from alternative splicing, designated p50 and p85(Cool-1) (cloned out of library), have been identified. Both isoforms of Cool contain a Src homology 3 domain that directly mediates interaction with Pak3 and tandem Dbl homology and pleckstrin homology domains. Despite the presence of the Dbl homology-pleckstrin homology motif, a characteristic of Rho family activators, activation of Cdc42 or Rac by Cool is not detectable. Instead binding of p50(Cool-1), but not p85(Cool-1), to Pak3 represses its activation by upstream activators such as the Dbl oncoprotein, indicating a novel mechanism of regulation of Pak signaling (Bagrodia, 1998).

Activation of p21-activated kinases (Paks) is achieved through binding of the GTPases Rac or Cdc42 to a conserved domain in the N-terminal regulatory region of Pak. Additional signaling components are also likely to be important in regulating Pak activation. Recently, a family of Pak-interacting guanine nucleotide exchange factors (Pix) have been identified and which are good candidates for regulating Pak activity. Using an active, truncated form of alphaPix (amino acids 155-545), stimulation of Pak1 kinase activity is observed when alphaPix155-545 is co-expressed with Cdc42 and wild-type Pak1 in COS-1 cells. This activation does not occur when a Pak1 mutant unable to bind alphaPix is coexpressed. The activation of wild-type Pak1 by alphaPix155-545 also requires that alphaPix155-545 retain functional exchange factor activity. However, the Pak1(H83,86L) mutant that does not bind Rac or Cdc42 is activated in the absence of GTPase by alphaPix155-545 and by a mutant of alphaPix155-545 that no longer has exchange factor activity. Pak1 activity was stimulated in vitro using GTPgammaS-loaded Cdc42 is also enhanced by recombinant alphaPix155-545 in a binding-dependent manner. These data suggest that Pak activity can be modulated by physical interaction with alphaPix and that this specific effect involves both exchange factor-dependent and -independent mechanisms (Daniels, 1999).

p21-activated kinase (PAK) is a common effector protein of the small GTPases Cdc42 and Rac, leading to the activation of downstream mitogen activated protein kinases. PAK also mediates polarized cytoskeletal changes induced by these GTPases. The recently identified PAK-interacting exchange factor (PIX) acts as a guanine nucleotide exchange factor on Rac, and colocalizes with PAK in a focal complex, but little is known about the associated signaling cascades, including upstream activators of PIX. One of the isoforms of PIX, alphaPIX, is activated by signaling cascades from the platelet-derived growth factor (PDGF) receptor and EphB2 receptor, and from integrin-induced signaling through phosphatidylinositol 3-kinase (PI3-kinase). alphaPIX is activated by forming a complex with these receptors either via association with PAK and Nck, or direct association with the p85 regulatory subunit of PI3-kinase. Synthetic phosphoinositide and membrane targeted PI3-kinase augments the alphaPIX activity in vivo. In Xenopus, aggregates of mesodermal cells derived from embryos microinjected with alphaPIX significantly increase the peripheral spreading on fibronectin substrate in response to PDGF through PI3-kinase. These results indicate that alphaPIX is activated by PI3-kinase, and is involved in the receptor mediated signaling leading to the activation of the kinase activity of PAK, and the migration of mesodermal cells on extracellular matrix (Yoshii, 1999).

The Cool/Pix (for PAK-interactive exchange factor) proteins directly bind to members of the PAK family of serine/threonine kinases and regulate their activity. Three members of the Cool/Pix family have shown distinct regulatory activities; p50Cool-1 inhibits Cdc42/Rac-stimulated PAK activity; p85Cool-1/beta-Pix has a permissive effect on Cdc42/Rac-stimulated activity, whereas p90Cool-2/alpha-Pix strongly activates PAK. It was initially suspected that these different functional effects were due to a binding interaction that occurs at the carboxyl terminal ends of the larger Cool/Pix proteins, thus enabling them to stimulate (or at least permit) rather than inhibit PAK activity. This led to the identification of the Cat proteins (for Cool-associated tyrosine phosphosubstrates). The Cat proteins bind to the carboxyl terminal ends of p85Cool-1 (residues 523-546) and Cool-2 (residues 647-670), and the binding of Cat to Cool-2 in fact is not necessary for the Cool-2-mediated activation of PAK. Rather, an 18 amino acid region, designated T1, that is present in the Cool-1 proteins, but missing in Cool-2, is essential for controlling the regulation of PAK activity by Cool-1/beta-Pix in vivo. Deletion of T1 yields a p85Cool-1 molecule that mimics the Cool-2 protein and is capable of strongly stimulating PAK activity. However, when T1 is added to Cool-2, the ability of Cool-2 to directly activate PAK is lost. It is concluded that T1 represents a novel regulatory domain that accounts for the specific functional effects on PAK activity exhibited by the different members of the Cool/Pix family (Feng, 2001).

PIX is a Rho-family guanine nucleotide exchange factor that binds PAK. Two isoforms of PIX have been identified that differ in their N termini. Here, the identification of a new splice variant of betaPIX, designated beta2PIX, is reported that is the dominant species in brain and that lacks the region of approximately 120 residues with predicted coiled-coil structure at the C terminus of beta1PIX. Instead, beta2PIX contains a serine-rich C terminus. To determine whether these splice variants differ in their cellular function, the effect of expressing these proteins in HeLa cells was examined. The coiled-coil region plays a key role in the localization of beta1PIX to the cell periphery and is also responsible for PIX dimerization. Overexpression of beta1, but not beta2PIX, drives formation of membrane ruffles and microvillus-like structures (via activation of Rac1 and Cdc42, respectively), indicating that its function requires localized activation of these GTPases. Thus, beta1PIX, like other RhoGEFs, exerts specific morphological functions that are dependent on its intracellular location and are mediated by its C-terminal dimerization domain (Koh, 2001).

betaPix (PAK-interacting exchange factor) is a recently identified guanine nucleotide exchange factor for Rho family small G protein Cdc42/Rac. The protein interacts with p21-activated protein kinase (PAK) through its SH3 domain. The effect of betaPix on MAP kinase signaling and cytoskeletal rearrangement has been studied in NIH3T3 fibroblast cells. Overexpression of betaPix enhances the activation of p38 in the absence of other stimuli and also induces translocation of p38 to the nucleus. This betaPix-induced p38 activation is blocked by coexpression of dominant-negative Cdc42/Rac or kinase-inactive PAK, indicating that the effect of betaPix on p38 is exerted through the Cdc42/Rac-PAK pathway and requires PAK kinase activity. The essential role of betaPix in growth factor-stimulated p38 activation is evidenced by the blocking of platelet-derived growth factor-induced p38 activation in the cells expressing betaPix SH3m (W43K) and betaPix DHm (L238R,L239R). In addition, SB203580, a p38 inhibitor, and kinase-inactive p38 (T180A,Y182F) block membrane ruffling induced by betaPix, suggesting that p38 might be involved in mediating betaPix-induced membrane ruffling. The results in this study suggest that betaPix might have a role in nuclear signaling, as well as in actin cytoskeleton regulation, and that some part of these cellular functions is possibly mediated by p38 MAP kinase (Lee, 2001).

Pix, a Pak-interacting exchange factor, is known to be involved in the regulation of Cdc42/Rac GTPases and Pak kinase activity. The cDNAs encoding two betaPix isoforms have been cloned from a mouse brain cDNA library. Both of the cloned genes, designated betaPix-b and betaPix-c (GenBank Accession Nos. AF247654 and AF247655, respectively), have a novel insert region consisting of 59 amino acid residues. In betaPix-c, 75 amino acid residues are deleted in the proline-rich region at the carboxyl-terminus of betaPix. In situ hybridization studies with insert region-specific probes in rat embryo show that insert region-containing isoforms are expressed mainly in the central nervous system. Moreover, the temporal expression pattern of isoforms is correlated with the active neurogenesis period in the cerebral cortex and cerebellum. These results strongly suggest that betaPix isoforms may play important roles in the cellular events required for brain development such as neuronal migration (Kim, 2000).

BetaPix, a Pak-interacting guanine nucleotide exchange factor is known to be involved in the regulation of Cdc42/Rac GTPases and Pak kinase activity. Currently, three 1Pix isoforms, betaPix-a, -b, and -c have been reported. In this study, the cDNA of a novel Pix splice variant was isolated from a mouse brain cDNA library. The cloned betaPix isoform, named betaPix-d, lacks a leucine zipper domain that is present in other Pix isoforms, and has an 11 amino acid addition at carboxyl terminus and a distinct 3'-UTR. Analysis of the tissue distribution of betaPix-d using RT-PCR has revealed that its message is present mainly in brain and testis but at lower levels in heart, spleen, lung, liver, skeletal muscle and kidney. In situ hybridization studies with the 13Pix-d specific probes in the rat embryo show that betaPix-d isoform is expressed mainly in the central nervous system. Moreover, temporal expression pattern of the isoform is correlated with the active neurogenesis period in the cerebral cortex and cerebellum during rat brain development. These findings suggest that betaPix-d isoform may be developmentally regulated (T. Kim, 2001).

Pix, a p21-activated kinase-interacting exchange factor, is known to be involved in the regulation of Cdc42/Rac GTPases. The 85-kDa betaPix-a protein contains an Src homology 3 domain, the tandem Dbl homology and Pleckstrin homology domains, a proline-rich region, and a GIT1-binding domain. In addition to those domains, betaPix-a also contains a putative leucine zipper domain at the C-terminal end. The leucine zipper domain mediates the formation of betaPix-a homodimers. Using in vitro and in vivo methodologies, it has been shown that deletion of the leucine zipper domain is sufficient to abolish betaPix-a homodimerization. In NIH3T3 fibroblast cells, expression of wild type betaPix-a induces the formation of membrane ruffles. However, cells expressing the leucine zipper domain deletion mutant can not form membrane ruffle structures. Moreover, platelet-derived growth factor-mediated cytoskeletal changes are completely blocked by the leucine zipper domain deletion mutant. The results suggest that the leucine zipper domain enables betaPix-a to homodimerize, and homodimerization is essential for betaPix-a signaling functions leading to the cytoskeletal reorganization (S. Kim, 2001).

p21-activated kinases (PAKs) are implicated in integrin signalings, and have been proposed to associate with paxillin indirectly. Paxillin can bind directly to PAK3. Several representative focal adhesion proteins were examined, and it was found that paxillin is the sole protein that associates with PAK3. PAK3 associates with the alpha and beta isoforms of paxillin, but not with gamma. Paxillin alpha associates with both the kinase-inactive and the Cdc42-activated forms of PAK3 in vivo, without affecting the activation states of the kinase. A number of different functions have been ascribed to PAKs; and PAKs can bind directly to growth factor signaling-adaptor molecule, Nck, and a guanine nucleotide exchanger, betaPIX. Paxillin alpha can compete with Nck and betaPIX in the binding of PAK3. Moreover, paxillin alpha can be phosphorylated by PAK3 at serine. Therefore, paxillin alpha, but not gamma, appears to be capable of linking both the kinase-inactive and activated forms of PAK3 to integrins independent of Nck and betaPIX, because Nck links PAK1 to growth factor receptors. These results also reveal that paxillin is involved in highly complexed protein-protein interactions in integrin signaling (Hashimoto, 2001).

X-linked forms of mental retardation (XLMR) include a variety of different disorders and may account for up to 25% of all inherited cases of mental retardation. So far, seven X-chromosomal genes mutated in nonspecific mental retardation (MRX) have been identified: FMR2, GDI1, RPS6KA3, IL1RAPL, TM4SF2, OPHN1 and PAK3. The products of the latter two have been implicated in regulation of neural plasticity by controlling the activity of small GTPases of the Rho family. A new MRX gene, ARHGEF6 (also known as alphaPIX or Cool-2), encoding a protein with homology to guanine nucleotide exchange factors for Rho GTPases (Rho GEF), has been identified. Molecular analysis of a reciprocal X/21 translocation in a male with mental retardation shows that this gene in Xq26 is disrupted by the rearrangement. Mutation screening of 119 patients with nonspecific mental retardation revealed a mutation in the first intron of ARHGEF6 (IVS1-11T-->C) in all affected males in a large Dutch family. The mutation resulted in preferential skipping of exon 2, predicting a protein lacking 28 amino acids. ARHGEF6 is the eighth MRX gene identified so far and the third such gene to encode a protein that interacts with Rho GTPases (Kutsche, 2000).

Borjeson-Forssman-Lehmann syndrome (BFLS) is a syndromic X-linked mental retardation that has been mapped by linkage to Xq26-q27. A nonsyndromic mental retardation family, MRX27, has also been localized to a region of the X chromosome overlapping Xq26-q27. The gene for ARHGEF6 (also known as alphaPIX or Cool-2), a newly identified guanine nucleotide exchange factor, was identified as a potential candidate XLMR gene, due to its location within the BFLS and MRX27 critical regions and its function in the regulation of PAK3 (a known MRX gene). The full coding sequence and genomic structure of the gene for ARHGEF6 was established in silico, based on available genomic, EST, and cDNA sequence information. Mutation analysis in BFLS- and MRX27-affected individuals was carried out. No mutations were found in two BFLS families or MRX27. Although ARHGEF6 is unlikely to be the gene responsible for either BFLS or MRX27, it remains a prime candidate for nonspecific or syndromic mental retardation linked to Xq26 (Lower, 2001).

Benzo[a]pyrene [B(a)P], a potent procarcinogen found in combustion products such as diesel exhaust and cigarette smoke, has been recently shown to activate the c-Jun NH(2)-terminal kinase 1 (JNK1) and induce caspase-3-mediated apoptosis in Hepa1c1c7 cells. However, the molecules of the signaling pathway that control the mitogen-activated protein kinase cascades induced by B(a)P and the interaction between those and apoptosis by B(a)P have not been well defined. B(a)P promotes Cdc42/Rac1, p21-activated kinase 1 (PAK1), and JNK1 activities in 293T and HeLa cells. Moreover, alpha-PAK-interacting exchange factor (alpha PIX) mRNA and its protein expression are upregulated by B(a)P. While overexpression of an active mutant of alpha PIX (DeltaCH) facilitates B(a)P-induced activation of Cdc42/Rac1, PAK1, and JNK1, overexpression of mutated alphaPIX (which lacks guanine nucleotide exchange factor activity), SH3 domain-deleted alphaPIX (which lacks the ability to bind PAK, kinase-negative PAK1), and kinase-negative SEK1 inhibit B(a)P-triggered JNK1 activation. Interestingly, overexpression of alphaPIX and a catalytically active mutant PAK1 accelerates B(a)P-induced apoptosis in HeLa cells, whereas alphaPIX (Delta SH3), PAK1 (K299R), and SEK 1 (K220A, K224L) inhibit B(a)P-initiated apoptosis. Finally, a preferential caspase inhibitor, Z-Asp-CH2-DCB, strongly blocks the alphaPIX (Delta CH)-enhanced apoptosis in cells treated with B(a)P but does not block PAK1/JNK1 activation. Taken together, these results indicate that alphaPIX plays a crucial role in B(a)P-induced apoptosis through activation of the JNK1 pathway kinases (Yoshii, 2001).

Plexins are widely expressed transmembrane proteins that, in the nervous system, mediate repulsive signals of semaphorins. However, the molecular nature of plexin-mediated signal transduction remains poorly understood. Plexin-B family members associate through their C termini with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG (leukemia-associated Rho GEF). The molecular determinants of the interaction between plexin-B1 and PDZ-RhoGEF or LARG were analyzed. PDZ-RhoGEF mutants lacking the RGS domain or the DH/PH domain are still capable of interacting with plexin-B1 as effectively as wild-type, full-length PDZ-RhoGEF. In contrast, the PDZ-RhoGEF mutant lacking the PDZ domain does not interact with plexin-B1, indicating that the PDZ domain of PDZ-RhoGEF is required for its interaction with plexin-B1. Activation of plexin-B1 by semaphorin 4D regulates combined PDZ-RhoGEF and LARG activity leading to RhoA activation. In addition, a dominant-negative form of PDZ-RhoGEF blocks semaphorin 4D-induced growth cone collapse in primary hippocampal neurons. This study indicates that the interaction of mammalian plexin-B family members with the multidomain proteins PDZ-RhoGEF and LARG represents an essential molecular link between plexin-B and localized, Rho-mediated downstream signaling events that underlie various plexin-mediated cellular phenomena including axonal growth cone collapse (Swiercz, 2002).

Paracingulin regulates the activity of Rac1 and RhoA GTPases by recruiting Tiam1 and GEF-H1 to epithelial junctions

Small GTPases control key cellular events, including formation of cell-cell junctions and gene expression, and are regulated by activating and inhibiting factors. This study characterized the junctional protein paracingulin as a novel regulator of the activity of two small GTPases, Rac1 and RhoA, through the functional interaction with their respective activators, Tiam1 and GEF-H1. In confluent epithelial monolayers, paracingulin depletion leads to increased RhoA activity and increased expression of mRNA for the tight junction protein claudin-2. During tight junction assembly by the calcium-switch, Rac1 shows two transient peaks of activity, at earlier (10-20 min) and later (3-8 h) time points. Paracingulin depletion reduces such peaks of Rac1 activation in a Tiam1-dependent manner, resulting in a delay in junction formation. Paracingulin physically interacts with GEF-H1 and Tiam1 in vivo and in vitro, and it is required for their efficient recruitment to junctions, based on immunofluorescence and biochemical experiments. These results provide the first description of a junctional protein that interacts with GEFs for both Rac1 and RhoA, and identify a novel molecular mechanism whereby Rac1 is activated during junction formation (Guillemot, 2008).

rho-type guanine exchange factor : Biological Overview | Developmental Biology | Effects of Mutation | References

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