Ral-specific guanine nucleotide exchange factor activity opposes other Ras effectors in PC12 cells by inhibiting neurite outgrowth.
Ras proteins can activate at least three classes of downstream target proteins: Raf kinases, phosphatidylinositol-3 phosphate (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). In NIH 3T3 cells, activated Ral-GEFs contribute to Ras-induced cell proliferation and oncogenic transformation by complementing the activities of Raf and PI3 kinases. In PC12 cells, activated Raf and PI3 kinases mediate Ras-induced cell cycle arrest and differentiation into a neuronal phenotype. Here, we show that in PC12 cells, Ral-GEF activity acts opposite to other Ras effectors. Elevation of Ral-GEF activity induced by transfection of a mutant Ras protein that preferentially activates Ral-GEFs, or by transfection of the catalytic domain of the Ral-GEF Rgr, suppressed cell cycle arrest and neurite outgrowth induced by nerve growth factor (NGF) treatment. In addition, Rgr reduced neurite outgrowth induced by a mutant Ras protein that preferentially activates Raf kinases. Furthermore, inhibition of Ral-GEF activity by expression of a dominant negative Ral mutant accelerated cell cycle arrest and enhanced neurite outgrowth in response to NGF treatment. Ral-GEF activity may function, at least in part, through inhibition of the Rho family GTPases, CDC42 and Rac. In contrast to Ras, which was activated for hours by NGF treatment, Ral was activated for only approximately 20 min. These findings suggest that one function of Ral-GEF signaling induced by NGF is to delay the onset of cell cycle arrest and neurite outgrowth induced by other Ras effectors. They also demonstrate that Ras has the potential to promote both antidifferentiation and prodifferentiation signaling pathways through activation of distinct effector proteins. Thus, in some cell types the ratio of activities among Ras effectors and their temporal regulation may be important determinants for cell fate decisions between proliferation and differentiation. (+info
A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho.
Small GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. Here, we used a consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) to search DNA data bases, and identified a novel human GEF for Rho-related GTPases harboring structural features indicative of its possible regulatory mechanism(s). This protein contained a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF that was termed Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activated biological and biochemical pathways specific for Rho, and activation of these pathways required an intact DH and PH domain. However, the PDZ domain was dispensable for these functions, and mutants lacking the LH domain were more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain revealed a limited homology with the catalytic region of a newly identified GTPase-activating protein for heterotrimeric G proteins, RGS14. This prompted us to investigate whether PDZ-RhoGEF could interact with representative members of each G protein family. We found that PDZ-RhoGEF was able to form, in vivo, stable complexes with two members of the Galpha12 family, Galpha12 and Galpha13, and that this interaction was mediated by the LH domain. Furthermore, we obtained evidence to suggest that PDZ-RhoGEF mediates the activation of Rho by Galpha12 and Galpha13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: by stimulating the activity of members of the Galpha12 family which, in turn, activate an exchange factor acting on Rho. (+info
Regulation of p190 Rho-GAP by v-Src is linked to cytoskeletal disruption during transformation.
The v-Src oncoprotein perturbs the dynamic regulation of the cellular cytoskeletal and adhesion network by a mechanism that is poorly understood. Here, we have examined in detail the effects of a temperature-dependent v-Src protein on the regulation of p190 RhoGAP, a GTPase activating protein (GAP) that has been implicated in disruption of the organised actin cytoskeleton, and addressed the dependence of v-Src-induced stress fibre loss on inhibition of Rho activity. We found that activation of v-Src induced association of tyrosine phosphorylated p190 with p120(RasGAP) and stimulation of p120(RasGAP)-associated RhoGAP activity, although p120(RasGAP) itself was not a target for phosphorylation by v-Src in chicken embryo cells. These events required the catalytic activity of v-Src and were linked to loss of actin stress fibres during morphological transformation and not mitogenic signalling. Furthermore, these effects were rapidly reversible since switching off v-Src led to dissociation of the p190/p120(RasGAP) complex, inactivation of p120(RasGAP)-associated RhoGAP activity and re-induction of actin stress fibres. In addition, transient transfection of Val14-RhoA, a constitutively active Rho protein that is insensitive to RhoGAPs, suppressed v-Src-induced stress fibre loss and cell transformation. Thus, we show here for the first time that an activated Src kinase requires the inactivation of Rho-mediated actin stress fibre assembly to induce its effects on actin disorganisation. Moreover, our work supports p190 as a strong candidate effector of v-Src-induced cytoskeletal disruption, most likely mediated by antagonism of the cellular function of Rho. (+info
Facilitation of signal onset and termination by adenylyl cyclase.
The alpha subunit (Gsalpha) of the stimulatory heterotrimeric guanosine triphosphate binding protein (G protein) Gs activates all isoforms of mammalian adenylyl cyclase. Adenylyl cyclase (Type V) and its subdomains, which interact with Gsalpha, promoted inactivation of the G protein by increasing its guanosine triphosphatase (GTPase) activity. Adenylyl cyclase and its subdomains also augmented the receptor-mediated activation of heterotrimeric Gs and thereby facilitated the rapid onset of signaling. These findings demonstrate that adenylyl cyclase functions as a GTPase activating protein (GAP) for the monomeric Gsalpha and enhances the GTP/GDP exchange factor (GEF) activity of receptors. (+info
alphaPix stimulates p21-activated kinase activity through exchange factor-dependent and -independent mechanisms.
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), we observe stimulation of Pak1 kinase activity when alphaPix155-545 is co-expressed with Cdc42 and wild-type Pak1 in COS-1 cells. This activation does not occur when we co-express a Pak1 mutant unable to bind alphaPix. 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 stimulated in vitro using GTPgammaS-loaded Cdc42 was 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. (+info
EFA6, a sec7 domain-containing exchange factor for ARF6, coordinates membrane recycling and actin cytoskeleton organization.
We have identified a human cDNA encoding a novel protein, exchange factor for ARF6 (EFA6), which contains Sec7 and pleckstrin homology domains. EFA6 promotes efficient guanine nucleotide exchange on ARF6 and is distinct from the ARNO family of ARF1 exchange factors. The protein localizes to a dense matrix on the cytoplasmic face of plasma membrane invaginations, induced on its expression. We show that EFA6 regulates endosomal membrane recycling and promotes the redistribution of transferrin receptors to the cell surface. Furthermore, expression of EFA6 induces actin-based membrane ruffles that are inhibited by co-expression of dominant-inhibitory mutant forms of ARF6 or Rac1. Our results demonstrate that by catalyzing nucleotide exchange on ARF6 at the plasma membrane and by regulating Rac1 activation, EFA6 coordinates endocytosis with cytoskeletal rearrangements. (+info
Conserved bipartite motifs in yeast eIF5 and eIF2Bepsilon, GTPase-activating and GDP-GTP exchange factors in translation initiation, mediate binding to their common substrate eIF2.
In the initiation phase of eukaryotic translation, eIF5 stimulates the hydrolysis of GTP bound to eIF2 in the 40S ribosomal pre-initiation complex, and the resultant GDP on eIF2 is replaced with GTP by the complex nucleotide exchange factor, eIF2B. Bipartite motifs rich in aromatic and acidic residues are conserved at the C-termini of eIF5 and the catalytic (epsilon) subunit of eIF2B. Here we show that these bipartite motifs are important for the binding of these factors, both in vitro and in vivo, to the beta subunit of their common substrate eIF2. We also find that three lysine-rich boxes in the N-terminal segment of eIF2beta mediate the binding of eIF2 to both eIF5 and eIF2B. Thus, eIF5 and eIF2Bepsilon employ the same sequence motif to facilitate interaction with the same segment of their common substrate. In agreement with this, archaea appear to lack eIF5, eIF2B and the lysine-rich binding domain for these factors in their eIF2beta homolog. The eIF5 bipartite motif is also important for its interaction with the eIF3 complex through the NIP1-encoded subunit of eIF3. Thus, the bipartite motif in eIF5 appears to be multifunctional, stimulating its recruitment to the 40S pre-initiation complex through interaction with eIF3 in addition to binding of its substrate eIF2. (+info
Structural basis for the inhibitory effect of brefeldin A on guanine nucleotide-exchange proteins for ADP-ribosylation factors.
Protein secretion through the endoplasmic reticulum and Golgi vesicular trafficking system is initiated by the binding of ADP-ribosylation factors (ARFs) to donor membranes, leading to recruitment of coatomer, bud formation, and eventual vesicle release. ARFs are approximately 20-kDa GTPases that are active with bound GTP and inactive with GDP bound. Conversion of ARF-GDP to ARF-GTP is regulated by guanine nucleotide-exchange proteins. All known ARF guanine nucleotide-exchange proteins contain a Sec7 domain of approximately 200 amino acids that includes the active site and fall into two classes that differ in molecular size and susceptibility to inhibition by the fungal metabolite brefeldin A (BFA). To determine the structural basis of BFA sensitivity, chimeric molecules were constructed by using sequences from the Sec7 domains of BFA-sensitive yeast Sec7 protein (ySec7d) and the insensitive human cytohesin-1 (C-1Sec7). Based on BFA inhibition of the activities of these molecules with recombinant yeast ARF2 as substrate, the Asp965-Met975 sequence in ySec7d was shown to be responsible for BFA sensitivity. A C-1Sec7 mutant in which Ser199, Asn204, and Pro209 were replaced with the corresponding ySec7d amino acids, Asp965, Gln970, and Met975, exhibited BFA sensitivity similar to that of recombinant ySec7d (rySec7d). Single replacement in C-1Sec7 of Ser199 or Pro209 resulted in partial inhibition by BFA, whereas replacement of Gln970 in ySec7d with Asn (as found in C-1Sec7) had no effect. As predicted, the double C-1Sec7 mutant with S199D and P209M was BFA-sensitive, demonstrating that Asp965 and Met975 in ySec7d are major molecular determinants of BFA sensitivity. (+info