Mind the GAP, Rho, Rab and GDI.
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Animals↗
Biomedical subjects
L Van Aelst
Publications and source records attributed to L Van Aelst.
Rac regulates integrin-mediated spreading and increased adhesion of T lymphocytes.
Leukocyte adhesion to the extracellular matrix (ECM) is tightly controlled and is vital for the immune response. Circulating lymphocytes leave the bloodstream and adhere to ECM components at sites of inflammation and lymphoid tissues. Mechanisms for regulating T-lymphocyte-ECM adhesion include (i) an alteration in the affinity of cell surface integrin receptors for their extracellular ligands and (ii) an alteration of events following postreceptor occupancy (e.g., cell spreading). Whereas H-Ras and R-Ras were previously shown to affect T-cell adhesion by altering the affinity state of the integrin receptors, no signaling molecule has been identified for the second mechanism. In this study, we demonstrated that expression of an activated mutant of Rac triggered dramatic spreading of T cells and their increased adhesion on immobilized fibronectin in an integrin-dependent manner. This effect was not mimicked by expression of activated mutant forms of Rho, Cdc42, H-Ras, or ARF6, indicating the unique role of Rac in this event. The Rac-induced spreading was accompanied by specific cytoskeletal rearrangements. Also, a clustering of integrins at sites of cell adhesion and at the peripheral edges of spread cells was observed. We demonstrate that expression of RacV12 did not alter the level of expression of cell surface integrins or the affinity state of the integrin receptors. Moreover, our results indicate that Rac plays a role in the regulation of T-cell adhesion by a mechanism involving cell spreading, rather than by altering the level of expression or the affinity of the integrin receptors. Furthermore, we show that the Rac-mediated signaling pathway leading to spreading of T lymphocytes did not require activation of c-Jun kinase, serum response factor, or pp70(S6 kinase) but appeared to involve a phospholipid kinase.
Cell Adhesion↗
Rho GTPases and signaling networks.
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Animals↗
A role for POR1, a Rac1-interacting protein, in ARF6-mediated cytoskeletal rearrangements.
The ARF6 GTPase, the least conserved member of the ADP ribosylation factor (ARF) family, associates with the plasma membrane and intracellular endosome vesicles. Mutants of ARF6 defective in GTP binding and hydrolysis have a marked effect on endocytic trafficking and the gross morphology of the peripheral membrane system. Here we report that expression of the GTPase-defective mutant of ARF6, ARF6(Q67L), remodels the actin cytoskeleton by inducing actin polymerization at the cell periphery. This cytoskeletal rearrangement was inhibited by co-expression of ARF6(Q67L) with deletion mutants of POR1, a Rac1-interacting protein involved in membrane ruffling, but not with the dominant-negative mutant of Rac1, Rac1(S17N). A synergistic effect between POR1 and ARF6 for the induction of actin polymerization was detected. Furthermore, we observed that ARF6 interacts directly with POR1 and that this interaction was GTP dependent. These findings indicate that ARF6 and Rac1 function on distinct signaling pathways to mediate cytoskeletal reorganization, and suggest a role for POR1 as an important regulatory element in orchestrating cytoskeletal rearrangements at the cell periphery induced by ARF6 and Rac1.
ADP-Ribosylation Factors↗
RhoGDIgamma: a GDP-dissociation inhibitor for Rho proteins with preferential expression in brain and pancreas.
GDP-dissociation inhibitors (GDIs) play a primary role in modulating the activation of GTPases and may also be critical for the cellular compartmentalization of GTPases. RhoGDI and GDI/D4 are two currently known GDIs for the Rho-subfamily of GTPases. Using their cDNAs to screen a human brain cDNA library under low stringency, we have cloned a homologous cDNA preferentially expressed at high levels in brain and pancreas. The predicted protein, named RhoGDIgamma, is approximately 50% identical to GDI/D4 and RhoGDI. It binds to CDC42 and RhoA with less affinity compared with RhoGDI and does not bind with Rac1, Rac2, or Ras. RhoGDIgamma functions as a GDI for CDC42 but with approximately 20 times less efficiency than RhoGDI. Immunohistochemical studies showed a diffuse punctate distribution of the protein in the cytoplasm with concentration around the nucleus in cytoplasmic vesicles. Overexpression of the protein in baby hamster kidney cells caused the cells to round up with loss of stress fibers. A distinct hydrophobic amino terminus in RhoGDIgamma, not seen in the other two RhoGDIs, could provide a mechanism for localization of the GDI to specific membranous compartment thus determining function distinct from RhoGDI or GDI/D4. Our results provide evidence that there is a family of GDIs for the Rho-related GTPases and that they differ in binding affinity, target specificity, and tissue expression. We propose that RhoGDI be renamed RhoGDIalpha and GDID4 be renamed RhoGDIbeta. The new GDI should widen the scope of investigation of this important class of regulatory protein.
Amino Acid Sequence↗
Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways.
Rac1 and RhoA are members of the Rho family of Ras-related proteins and function as regulators of actin cytoskeletal organization, gene expression, and cell cycle progression. Constitutive activation of Rac1 and RhoA causes tumorigenic transformation of NIH 3T3 cells, and their functions may be required for full Ras transformation. The effectors by which Rac1 and RhoA mediate these diverse activities, as well as the interrelationship between these events, remain poorly understood. Rac1 is distinct from RhoA in its ability to bind and activate the p65 PAK serine/threonine kinase, to induce lamellipodia and membrane ruffling, and to activate the c-Jun NH2-terminal kinase (JNK). To assess the role of PAK in Rac1 function, we identified effector domain mutants of Rac1 and Rac1-RhoA chimeric proteins that no longer bound PAK. Surprisingly, PAK binding was dispensable for Rac1-induced transformation and lamellipodium formation, as well as activation of JNK, p38, and serum response factor (SRF). However, the ability of Rac1 to bind to and activate PAK correlated with its ability to stimulate transcription from the cyclin D1 promoter. Furthermore, Rac1 activation of JNK or SRF, or induction of lamellipodia, was neither necessary nor sufficient for Rac1 transforming activity. Finally, the signaling pathways that mediate Rac1 activation of SRF or JNK were distinct from those that mediate Rac1 induction of lamellipodia. Taken together, these observations suggest that Rac1 regulates at least four distinct effector-mediated functions and that multiple pathways may contribute to Rac1-induced cellular transformation.
3T3 Cells↗
Lck regulates Vav activation of members of the Rho family of GTPases.
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.
Animals↗
RAC regulation of actin polymerization and proliferation by a pathway distinct from Jun kinase.
The RAC guanine nucleotide binding proteins regulate multiple biological activities, including actin polymerization, activation of the Jun kinase (JNK) cascade, and cell proliferation. RAC effector loop mutants were identified that separate the ability of RAC to interact with different downstream effectors. One mutant of activated human RAC protein, RACV12H40 (with valine and histidine substituted at position 12 and 40, respectively), was defective in binding to PAK3, a Ste20-related p21-activated kinase (PAK), but bound to POR1, a RAC-binding protein. This mutant failed to stimulate PAK and JNK activity but still induced membrane ruffling and mediated transformation. A second mutant, RACV12L37 (with leucine substituted at position 37), which bound PAK but not POR1, induced JNK activation but was defective in inducing membrane ruffling and transformation. These results indicate that the effects of RAC on the JNK cascade and on actin polymerization and cell proliferation are mediated by distinct effector pathways that diverge at the level of RAC itself.
3T3 Cells↗
Identification of a novel Rac1-interacting protein involved in membrane ruffling.
The Rac GTP binding proteins are implicated in actin cytoskeleton-membrane interaction in mammalian cells. In fibroblast cells, Rac has been shown to mediate growth factor-induced polymerization of actin to form membrane ruffles and lamellipodia. We report here the isolation of a noval Rac1-interacting protein, POR1. POR1 binds directly to Rac1, and the interaction of POR1 with Rac1 is GTP dependent. A mutation in the Rac1 effector binding loop shown to abolish membrane ruffling also abolishes interaction with POR1. Truncated versions of POR1 inhibit the induction of membrane ruffling by an activated mutant of Rac1, V12Rac1, in quiescent rat embryonic fibroblast REF52 cells. Furthermore, POR1 synergizes with an activated mutant of Ras, V12Ras, in the induction of membrane ruffling. These results suggest a potential role for POR1 in Rac1-mediated signaling pathways.
Adaptor Proteins, Signal Transducing↗
Identification of Ste4 as a potential regulator of Byr2 in the sexual response pathway of Schizosaccharomyces pombe.
A conserved MAP kinase cascade is central to signal transduction in both simple and complex eukaryotes. In the yeast Schizosaccharomyces pombe, Byr2, a homolog of mammalian MAPK/ERK kinase kinase and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation. A screen for S. pombe proteins that interact with Byr2 in a two-hybrid system led to the isolation of Ste4, a protein that is known to be required for sexual function. Ste4 binds to the regulatory region of Byr2. This binding site is separable from the binding site for Ras1. Both Ste4 and Ras1 act upstream of Byr2 and act at least partially independently. Ste4 contains a leucine zipper and is capable of homotypic interaction. Ste4 has regions of homology with STE50, an S. cerevisiae protein required for sexual differentiation that we show can bind to STE11.
Binding Sites↗
Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation.
Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.
3T3 Cells↗
Differential interactions of human Sos1 and Sos2 with Grb2.
The guanine nucleotide exchange factor Son of sevenless (Sos) performs a crucial step in the coupling of receptor tyrosine kinases to Ras activation. Mammalian cells contain two related but distinct Sos proteins, Sos1 and Sos2. Although they share a high degree of overall similarity, it is not known to what extent their biological and biochemical properties overlap. In the present study, we have compared the interactions of the two human homologues of Sos, hSos1 and hSos2, with the adaptor protein Grb2. We show that hSos2 interacts with Grb2 via its proline-rich COOH-terminal domain and that this interaction is dependent on the SH3 domains of Grb2. In general, these characteristics are similar to the ones reported previously for the interaction of hSos1 with Grb2. However, the apparent binding affinity of hSos2 for Grb2 is significantly higher relative to that of hSos1 both in vitro and in vivo. The region conferring this higher binding affinity has been mapped to residues 1126-1242 of the hSos2 COOH-terminal domain. These results suggest that Sos1 and Sos2 may differentially contribute to receptor-mediated Ras activation.
Adaptor Proteins, Signal Transducing↗
Multiple Ras functions can contribute to mammalian cell transformation.
We have developed a generalized approach, using two hybrid interactions, to isolate Ha-Ras effector loop mutations that separate the ability of Ha-Ras to interact with different downstream effectors. These mutations attenuate or eliminate Ha-ras(G12V) transformation of mammalian cells, but retain complementary activity, as demonstrated by synergistic induction of foci of growth-transformed cells, and by the ability to activate different downstream components. The transformation defect of Ha-ras(G12V, E37G) is rescued by a mutant, raf1, that restores interaction. These results indicate that multiple cellular components, including Raf1, are activated by Ha-Ras and contribute to Ha-Ras-induced mammalian cell transformation.
Animals↗
Two types of RAS mutants that dominantly interfere with activators of RAS.
In the fission yeast Schizosaccharomyces pombe, ras1 regulates both sexual development (conjugation and sporulation) and cellular morphology. Two types of dominant interfering mutants were isolated in a genetic screen for ras1 mutants that blocked sexual development. The first type of mutation, at Ser-22, analogous to the H-rasAsn-17 mutant (L. A. Feig and G. M. Cooper, Mol. Cell. Biol. 8:3235-3243, 1988), blocked only conjugation, whereas a second type of mutation, at Asp-62, interfered with conjugation, sporulation, and cellular morphology. Analogous mutations at position 64 of Saccharomyces cerevisiae RAS2 or position 57 of human H-ras also resulted in dominant interfering mutants that interfered specifically and more profoundly than mutants of the first type with RAS-associated pathways in both S. pombe or S. cerevisiae. Genetic evidence indicating that both types of interfering mutants function upstream of RAS is provided. Biochemical evidence showing that the mutants are altered in their interaction with the CDC25 class of exchange factors is presented. We show that both H-rasAsn-17 and H-rasTyr-57, compared with wild-type H-ras, are defective in their guanine nucleotide-dependent release from human cdc25 and that this defect is more severe for the H-rasTyr-57 mutant. Such a defect would allow the interfering mutants to remain bound to, thereby sequestering RAS exchange factors. The more severe interference phenotype of this novel interfering mutant suggests that it functions by titrating out other positive regulators of RAS besides those encoded by ste6 and CDC25.
Base Sequence↗
Complex formation between RAS and RAF and other protein kinases.
We used a Saccharomyces cerevisiae genetic system to detect the physical interaction of RAS and RAF oncoproteins. We also observed interaction between RAS and byr2, a protein kinase implicated as a mediator of the Schizosaccharomyces pombe ras1 protein. Interaction with RAS required only the N-terminal domains of RAF or byr2 and was disrupted by mutations in either the guanine nucleotide-binding or effector-loop domains of RAS. We observed interaction between MEK (a kinase that phosphorylates mitogen-activated protein kinases) and the catalytic domain of RAF. RAS and MEK also interacted but only when RAF was overexpressed.
Base Sequence↗
Molecular cloning of a gene involved in glucose sensing in the yeast Saccharomyces cerevisiae.
Cells of the yeast Saccharomyces cerevisiae display a wide range of glucose-induced regulatory phenomena, including glucose-induced activation of the RAS-adenylate cyclase pathway and phosphatidylinositol turnover, rapid post-translational effects on the activity of different enzymes as well as long-term effects at the transcriptional level. A gene called GGS1 (for General Glucose Sensor) that is apparently required for the glucose-induced regulatory effects and several ggs1 alleles (fdp1, byp1 and cif1) has been cloned and characterized. A GGS1 homologue is present in Methanobacterium thermoautotrophicum. Yeast ggs1 mutants are unable to grow on glucose or related readily fermentable sugars, apparently owing to unrestricted influx of sugar into glycolysis, resulting in its rapid deregulation. Levels of intracellular free glucose and metabolites measured over a period of a few minutes after addition of glucose to cells of a ggs1 delta strain are consistent with our previous suggestion of a functional interaction between a sugar transporter, a sugar kinase and the GGS1 gene product. Such a glucose-sensing system might both restrict the influx of glucose and activate several signal transduction pathways, leading to the wide range of glucose-induced regulatory phenomena. Deregulation of these pathways in ggs1 mutants might explain phenotypic defects observed in the absence of glucose, e.g. the inability of ggs1 diploids to sporulate.
Alleles↗
A yeast homologue of the bovine lens fibre MIP gene family complements the growth defect of a Saccharomyces cerevisiae mutant on fermentable sugars but not its defect in glucose-induced RAS-mediated cAMP signalling.
Recently a new family of membrane proteins comprising the bovine lens fibre major intrinsic protein, soybean nodulin-26 protein and the Escherichia coli glycerol facilitator has been described [M.E. Baker and M.H. Saier, Jr (1990) Cell, 60, 185-186]. These proteins have six putative membrane spanning domains and one (probably intracellular) intermembrane fragment is particularly well conserved. We have identified a new member of this family in the yeast Saccharomyces cerevisiae. It also possesses the six transmembrane domains and the highly conserved intermembrane sequence. In contrast to the other three proteins which are all approximately 280 amino acids long, the yeast protein has an N-terminal extension of approximately 250 amino acids, which contains a string of 17 asparagine residues and a C-terminal extension of approximately 150 amino acids. The gene, which we called FPS1 (for fdp1 suppressor), suppresses in single copy the growth defect on fermentable sugars of the yeast fdp1 mutant but it is not allelic to FDP1. The deficiency of the fdp1 mutant in glucose-induced RAS-mediated cAMP signalling and in rapid glucose-induced changes in the activity of certain enzymes was not restored. Deletion of FPS1 does not cause any of the phenotypic deficiencies of the fdp1 mutant.
Amino Acid Sequence↗