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Query: UNIPROT:P06889 (Mol)
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The Rho-type GTPase Cdc42p is required for cell polarization and bud emergence in Saccharomyces cerevisiae. To identify genes whose functions are linked to CDC42, we screened for (i) multicopy suppressors of a Ts- cdc42 mutant, (ii) mutants that require multiple copies of CDC42 for survival, and (iii) mutations that display synthetic lethality with a partial-loss-of-function allele of CDC24, which encodes a guanine nucleotide exchange factor for Cdc42p. In all three screens, we identified a new gene, BEM4. Cells from which BEM4 was deleted were inviable at 37 degrees C. These cells became unbudded, large, and round, consistent with a model in which Bem4p acts together with Cdc42p in polarity establishment and bud emergence. In some strains, the ability of CDC42 to serve as a multicopy suppressor of the Ts- growth defect of deltabem4 cells required co-overexpression of Rho1p, which is an essential Rho-type GTPase necessary for cell wall integrity. This finding suggests that Bem4p also affects Rho1p function. Bem4p displayed two-hybrid interactions with Cdc42p, Rho1p, and two of the three other known yeast Rho-type GTPases, suggesting that Bem4p can interact with multiple Rho-type GTPases. Models for the role of Bem4p include that it serves as a chaperone or modulates the interaction of these GTPases with one or more of their targets or regulators.
Mol Cell Biol 1996 Aug
PMID:Identification of the bud emergence gene BEM4 and its interactions with rho-type GTPases in Saccharomyces cerevisiae. 875 39

Studies were undertaken to determine the effect of the Ras suppressor Rsu-1 on Ras signal transduction pathways in two different cell backgrounds. An expression vector containing the mouse rsu-1 cDNA under the control of a mouse mammary tumor virus promoter was introduced into NIH 3T3 cells and the pheochromocytoma cell line PC12. Cell lines developed in the NIH 3T3 background expressed p33rsu-1 at approximately twice the normal endogenous level. However, PC12 cell clones which expressed p33rsu-1 at an increased level in a regulatable fashion in response to dexamethasone were isolated. Analysis of proteins involved in regulation of Ras and responsive to Ras signal transduction revealed similar changes in the two cell backgrounds in the presence of elevated p33rsu-1. There was an increase in the level of SOS, the guanine nucleotide exchange factor, and an increase in the percentage of GTP-bound Ras. In addition, there was an increase in the amount of p120 Ras-specific GTPase-activating protein (GAP) and GAP-associated p190. However, a decrease in Ras GTPase-activating activity was detected in lysates of the Rsu-1 transfectants, and immunoprecipitated p120 GAP from the Rsu-1 transfectants showed less Ras GTPase-activating activity than GAP from control cells. Activation of Erk-2 kinase by growth factor and tetradecanyol phorbol acetate was greater in the Rsu-1 transfectants than in control cells. However, c-Jun amino-terminal kinase activity (Jun kinase) was not activatable by epidermal growth factor in Rsu-1 PC12 cell transfectants, in contrast to the PC12 vector control cell line. Transient expression of p33rsu-1 in Cos1 cells following cotransfection with either hemagglutinin-tagged Jun kinase or hemagglutinin-tagged Erk-2 revealed that Rsu-1 expression inhibited constitutive Jun kinase activity while enhancing Erk-2 activity. Detection of in vitro binding of Rsu-1 to Raf-1 suggested that in Rsu-1 transfectants, increased activation of the Raf-1 pathway occurred at the expense of activation of signal transduction leading to Jun kinase. These results indicate that inhibition of Jun kinase activation was sufficient to inhibit Ras transformation even in the presence of activated Erk-2.
Mol Cell Biol 1996 Oct
PMID:Increased expression of the Ras suppressor Rsu-1 enhances Erk-2 activation and inhibits Jun kinase activation. 881 60

spi1p of Schizosaccharomyces pombe is a structural homolog of the mammalian GTPase Ran. The distribution between the GTP- and GDP-bound forms of the protein is regulated by evolutionarily conserved gene products, rna1p and pim1p, functioning as GTPase-activating protein (GAP) and guanine nucleotide exchange factor (GEF), respectively. Antibodies to spi1p, pim1p, and rna1p were generated and used to demonstrate that pim1p is exclusively nuclear, while rna1p is cytoplasmic. A loss of pim1p GEF activity or an increase in the rna1p GAP activity correlates with a change in the localization of the GTPase from predominantly nuclear to uniformly distributed, suggesting that the two forms are topologically segregated and that the nucleotide-bound state of spi1p may dictate its intracellular localization. We demonstrate that the phenotype of cells overproducing the GAP resembles the previously reported phenotype of mutants with alterations in the GEF: the cells are arrested in the cell cycle as septated, binucleated cells with highly condensed chromatin, fragmented nuclear envelopes, and abnormally wide septa. Consistent with the expectation that either an increased dosage of the GAP or a mutation in the GEF would lead to an increase of the spi1p-GDP/spi1p-GTP ratio relative to that of wild-type cells, overexpression of the GAP together with a mutation in the GEF is synthetically lethal. The similar phenotypic consequences of altering the functioning of the nuclear GEF or the cytoplasmic GAP suggest that there is a single pool of the spi1p GTPase that shuttles between the nucleus and the cytoplasm. Phenotypically, rna1 null mutants, in which spi1p-GTP would be expected to accumulate, resemble pim1(ts) and rna1p-overproducing cells, in which spi1p-GDP would be expected to accumulate. Taken together, these results support the hypothesis that the balance between the GDP- and GTP-bound forms of spi1p mediates the host of nuclear processes that are adversely affected when the functioning of different components of this system is perturbed in various organisms.
Mol Cell Biol 1996 Nov
PMID:Perturbations in the spi1p GTPase cycle of Schizosaccharomyces pombe through its GTPase-activating protein and guanine nucleotide exchange factor components result in similar phenotypic consequences. 888 64

eIF2B is a five-subunit guanine nucleotide exchange factor that is negatively regulated by phosphorylation of the alpha subunit of its substrate, eIF2, leading to inhibition of translation initiation. To analyze this regulatory mechanism, we have characterized 29 novel mutations in the homologous eIF2B subunits encoded by GCD2, GCD7, and GCN3 that reduce or abolish inhibition of eIF2B activity by eIF2 phosphorylated on its alpha subunit [eIF2(alphaP)]. Most, if not all, of the mutations decrease sensitivity to eIF2(alphaP) without excluding GCN3, the nonessential subunit, from eIF2B; thus, all three proteins are critical for regulation of eIF2B by eIF2(alphaP). The mutations are clustered at both ends of the homologous region of each subunit, within two segments each of approximately 70 amino acids in length. Several mutations alter residues at equivalent positions in two or all three subunits. These results imply that structurally similar segments in GCD2, GCD7, and GCN3 perform related functions in eIF2B regulation. We propose that these segments form a single domain in eIF2B that makes multiple contacts with the alpha subunit of eIF2, around the phosphorylation site, allowing eIF2B to detect and respond to phosphoserine at residue 51. Most of the eIF2 is phosphorylated in certain mutants, suggesting that these substitutions allow eIF2B to accept phosphorylated eIF2 as a substrate for nucleotide exchange.
Mol Cell Biol 1997 Mar
PMID:Homologous segments in three subunits of the guanine nucleotide exchange factor eIF2B mediate translational regulation by phosphorylation of eIF2. 903 57

Vav is a member of a family of oncogene proteins that share an approximately 250-amino-acid motif called a Dbl homology domain. Paradoxically, Dbl itself and other proteins containing a Dbl domain catalyze GTP-GDP exchange for Rho family proteins, whereas Vav has been reported to catalyze GTP-GDP exchange for Ras proteins. We present Saccharomyces cerevisiae genetic data, in vitro biochemical data, and animal cell biological data indicating that Vav is a guanine nucleotide exchange factor for Rho-related proteins, but in similar genetic and biochemical experiments we fail to find evidence that Vav is a guanine nucleotide exchange factor for Ras. Further, we present data indicating that the Lck kinase activates the guanine nucleotide exchange factor and transforming activity of Vav.
Mol Cell Biol 1997 Mar
PMID:Lck regulates Vav activation of members of the Rho family of GTPases. 903 61

Nucleocytoplasmic transport of mRNA is essential for eukaryotic gene expression. However, how mRNA is exported from the nucleus is mostly unknown. To elucidate the mechanisms of mRNA transport, we took a genetic approach to identify genes, the products of which play a role in that process. From about 1000 temperature -sensitive (ts- or cs-) mutants, we identified five ts- mutants that are defective in poly(A)+ RNA transport by using a situ hybridization with an oligo(dT)50 as a probe. These mutants accumulate poly(A)+ RNA in the nuclei when shifted to a nonpermissive temperature. All five mutations are tightly linked to the ts- growth defects, are recessive, and fall into four different groups designated as ptr 1-4 (poly(A)+ RNA transport). Interestingly, each group of mutants has a differential localization pattern of poly(A)+ RNA in the nuclei at the nonpermissive temperature, suggesting that they have defects at different steps of the mRNA transport pathway. Localization of a nucleoplasmin-green fluorescent protein fusion suggests that ptr2 and ptr3 have defects also in nuclear protein import. Among the isolated mutants, only ptr2 showed a defect in pre-mRNA splicing. We cloned the ptr2+ and ptr3+ genes and found that they encode Schizosaccharomyces pombe homologues of the mammalian RCC1, a guanine nucleotide exchange factor for RAN/TC4, and the ubiquitin-activating enzyme E1 involved in ubiquitin conjugation, respectively. The ptr3+ gene is essential for cell viability, and Ptr3p tagged with green fluorescent protein was localized in both the nucleus and the cytoplasm. This is the first report suggesting that the ubiquitin system plays a role in mRNA export.
Mol Biol Cell 1997 May
PMID:Isolation and molecular characterization of mRNA transport mutants in Schizosaccharomyces pombe. 916 69

Cross-linking of the B-cell antigen receptor (BCR) induces tyrosine phosphorylation of Shc, which is believed to lead to the activation of Ras. Previous work has shown that tyrosine-phosphorylated Shc forms complexes with another adapter protein, Grb2, and the Ras guanine nucleotide exchange factor SOS. Here, we demonstrate that phosphorylation of Shc by the hematopoietic cell-specific tyrosine kinase Syk induces binding of Grb2 to Shc, suggesting that Syk phosphorylates Shc in stimulated B cells. Surprisingly, Syk-phosphorylated Shc possesses two Grb2 binding sites rather than the one site that has been previously reported. Both of these sites are required for efficient formation of Shc-Grb2-SOS complexes in vitro and in vivo. We suggest that two Grb2 proteins anchored by a single Shc protein bind simultaneously to one SOS molecule, resulting in a complex that is more stable than a complex containing only a single Grb2 protein bound to one SOS molecule. This model is consistent with our observation that BCR stimulation greatly increases the amount of SOS associated with Grb2.
Mol Cell Biol 1997 Jul
PMID:Shc contains two Grb2 binding sites needed for efficient formation of complexes with SOS in B lymphocytes. 919 44

The solution structure of the pleckstrin homology (PH) domain of mouse Son-of-sevenless 1 (mSos1), a guanine nucleotide exchange factor for Ras, was determined by multidimensional NMR spectroscopy. The structure of the mSos1 PH domain involves the fundamental PH fold, consisting of seven beta-strands and one alpha-helix at the C terminus, as determined for the PH domains of other proteins. By contrast, the mSos1 PH domain showed two major characteristic features. First, the N-terminal region, whose amino acid sequence is highly conserved among Sos proteins, was found to form an alpha-helix, which interacts with the beta-sheet structure of the fundamental PH fold. Second, there is a long unstructured loop between beta3 and beta4. Furthermore, the mSos1 PH domain was found to bind phosphatidylinositol-4,5-bisphosphate by a centrifugation assay. The addition of inositol-1,4,5-trisphosphate to the mSos1 PH domain induced backbone amide chemical shift changes mainly in the beta1/beta2 loop and the N- and C-terminal parts of the long beta3/beta4 loop. This inositol-1,4,5-trisphosphate-binding mode of the mSos1 PH domain is somewhat similar to those of the PH domains of pleckstrin and phospholipase Cdelta1, and is clearly different from those of other PH domains.
J Mol Biol 1997 Jun 20
PMID:The solution structure of the pleckstrin homology domain of mouse Son-of-sevenless 1 (mSos1). 921 62

Mss4 and its yeast homologue, Dss4, have been proposed to function as guanine nucleotide exchange factors (GEFs) for a subset of Rab proteins in the secretory pathway. We have previously shown that Rab1A mutants defective in GTP-binding potently inhibit endoplasmic reticulum to Golgi transport, presumably by sequestering an unknown GEF regulating its function. We now demonstrate that these mutants stably associate with Mss4 both in vivo and in vitro and that Mss4 effectively neutralizes the inhibitory activity of the Rab1A mutants. An equivalent Rab3A mutant (Rab3A[N135I]), a Rab protein specifically involved in regulated secretion at the cell surface, associates with Mss4 as efficiently as the Rab1A[N124I] mutant. Although Rab3A[N135I] prevents the ability of Mss4 to neutralize the inhibitory effects of Rab1A mutants on transport, it has no effect on Rab1 function or endoplasmic reticulum to Golgi transport. Furthermore, quantitative immunodepletion of Mss4 fails to inhibit transport in vitro. We conclude that Mss4 and its yeast homologue, Dss4, are not GEFs mediating activation of Rab, but rather, they interact with the transient guanine nucleotide-free state, defining a new class of Ras-superfamily GTPase effectors that function as guanine nucleotide-free chaperones (GFCs).
Mol Biol Cell 1997 Jul
PMID:Mss4 does not function as an exchange factor for Rab in endoplasmic reticulum to Golgi transport. 924 9

The ability of insulin to stimulate protein synthesis and cellular growth is mediated through the insulin receptor (IR), which phosphorylates Tyr residues in the insulin receptor substrate-signaling proteins (IRS-1 and IRS-2), Gab-1, and Shc. These phosphorylated substrates directly bind and activate enzymes such as phosphatidylinositol 3'-kinase (PI3K) and the guanine nucleotide exchange factor for p21Ras (GRB-2/SOS), which are in turn required for insulin-stimulated protein synthesis, cell cycle progression, and prevention of apoptosis. We have now shown that one or more members of the atypical protein kinase C group, as exemplified by the zeta isoform (PKC zeta), are downstream of IRS-1 and P13K and mediate the effect of insulin on general protein synthesis. Ectopic expression of constitutively activated PKC zeta eliminates the requirement of IRS-1 for general protein synthesis but not for insulin-stimulated activation of 70-kDa S6 kinase (p70S6K), synthesis of growth-regulated proteins (e.g., c-Myc), or mitogenesis. The fact that PKC zeta stimulates general protein synthesis but not activation of p70S6K indicates that PKC zeta activation does not involve the proto-oncogene Akt, which is also activated by PI3K. Yet insulin is still required for the stimulation of general protein synthesis in the presence of constitutively active PKC zeta and in the absence of IRS-1, suggesting a requirement for the convergence of the IRS-1/PI3K/PKC zeta pathway with one or more additional pathways emanating from the IR, e.g., Shc/SOS/p21Ras/mitogen-activated protein kinase. Thus, PI3K appears to represent a bifurcation in the insulin signaling pathway, one branch leading through PKC zeta to general protein synthesis and one, through Akt and the target of rapamycin (mTOR), to growth-regulated protein synthesis and cell cycle progression.
Mol Cell Biol 1997 Sep
PMID:Requirement of protein kinase C zeta for stimulation of protein synthesis by insulin. 927 96


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