Coupling of ras p21 signalling and GTP hydrolysis by GTPase activating proteins.

Ras p21 proteins cycle between inactive, GDP-bound forms and active GTP-bound forms. Hydrolysis of bound GTP to GDP is mediated by proteins referred to as GAPs, two forms of which have been described. The first, p120-GAP, contains regions of homologies with tyrosine kinase oncogenes, and interacts with tyrosine phosphoproteins as well as with ras proteins; p120-GAP may therefore connect signalling pathways that involve tyrosine kinase and ras p21 proteins. The second type of GAP is the product of the neurofibromatosis type 1 gene (NF1-GAP). This is a protein of 325,000 Da that is defective in patients with NF1; NF1-GAP is regulated by signalling lipids, and may serve to connect ras p21 with phospholipid second messenger systems. The significance of ras p21 interaction with distinct GAPs is discussed.     

Inhibition of the ras p21 GTPase-activating protein-stimulated GTPase activity of c-Ha-ras p21 by smg p21 having the same putative effector domain as ras p21s

ras p21 GTPase-activating protein (GAP) has been proposed to interact with the putative effector domain of ras p21s, and smg p21, a ras p21-like guanine nucleotide binding protein (G protein), has been shown to have the same amino acid sequence as ras p21s in this region. In the present studies, we examined the effects of ras p21 GAP on the GTPase activity of smg p21 purified from human platelets, of smg p21 on the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 purified from Escherichia coli, and of c-Ha-ras p21 on the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. ras p21 GAP stimulated the GTPase activity of c-Ha-ras p21 but not that of smg p21. The GTP-bound form of smg p21, however, inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 in a dose-dependent manner. The half-maximum inhibition by smg p21 was obtained at 0.4 microM which was more potent than previously observed for ras p21 (2-200 microM). The GDP-bound form also inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21, but the efficiency was 40-50% that of the GTP-bound form. smg p21 GAP1 and -2 stimulated the GTPase activity of smg p21 but not that of c-Ha-ras p21. c-Ha-ras p21 did not inhibit the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. These results indicate that ras p21 GAP interacts with smg p21 without the subsequent stimulation of its GTPase activity.     

Regulation of the GTPase activity of the ras-related rap2 protein.

The small GTP-binding protein rap2A exhibits a high level of identity with rap1 and ras proteins (60% and 46%, respectively). Nevertheless, its intrinsic GTPase activity is not stimulated by ras-GAP, and unlike the rap1A protein, it cannot compete with ras proteins for their interaction with ras-GAP. In addition, rap1-GAPm that is highly active on the GTPase activity of the rap1A product, also stimulates the GTPase activity of the rap2A protein but with a 30-40-fold lower efficiency. An activity that greatly stimulated the GTPase activity of the rap2 protein (rap2-GAP) was found in bovine brain cytosol and purified. However, it copurified with the cytosolic form of rap1-GAP and was more efficient at stimulating the GTPase activity of the rap1 protein; this 55 kD polypeptide, that is recognized by an antibody raised against rap1-GAPm, likely represents a degraded and soluble form of the full size 89 kD molecule. In bovine brain membranes, a weak GAP activity toward the rap2A protein was also detected; however, it was also attributable to the membrane-associated rap1-GAPm. Thus, it appears that a single rap-GAP protein, complete or degraded, is able to stimulate the GTPase activity of both rap1 and rap2 proteins.     

A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity.

The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M-1 s-1 at 24 degrees C) as GAP purified from bovine brain or full-length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products. This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein.     

Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras.

A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21ras. This accumulation could be caused either by an increase in guanine nucleotide exchange on p21ras or by a decrease in the GTPase activity of p21ras. To investigate whether insulin and EGF affect nucleotide exchange on p21ras, we measured binding of [alpha-32P]GTP to p21ras in cells permeabilized with streptolysin O. For this purpose, we used a cell line which expressed elevated levels of p21 H-ras and which was highly responsive to insulin and EGF. Stimulation with insulin or EGF resulted in an increase in the rate of nucleotide binding to p21ras. To determine whether this increased binding rate is due to the activation of a guanine nucleotide exchange factor, we made use of the inhibitory properties of a dominant negative mutant of p21ras, p21ras (Asn-17). Activation of p21ras by insulin and EGF in intact cells was abolished in cells infected with a recombinant vaccinia virus expressing p21ras (Asn-17). In addition, the enhanced nucleotide binding to p21ras in response to insulin and EGF in permeabilized cells was blocked upon expression of p21ras (Asn-17). From these data, we conclude that the activation of a guanine nucleotide exchange factor is involved in insulin- and EGF-induced activation of p21ras.     

The arginine finger loop of yeast and human GAP is a determinant for the specificity toward Ras GTPase.

In this work, we have studied the role of the arginine finger region in determining the specificity of the GTPase activating proteins (GAPs) Saccharomyces cerevisiae Ira2p and human p120-GAP toward yeast Ras2p and human Ha-Ras p21. It is known that p120-GAP can enhance both Ras2p and Ha-Ras GTPase activities, whereas Ira2p is strictly specific for Ras2p and fails to activate Ha-Ras GTPase. Substitution in Ira2p of the arginine following the arginine finger with alanine, the residue found in the corresponding position of p120-GAP, or by glycine as found in neurofibromin, evokes a low but significant stimulation of Ha-Ras GTPase. The stimulatory activity of Ira2p on Ha-Ras increased by substituting segments of the finger loop region with p120-GAP residues, especially with the six residues forming the tip of the arginine loop. In p120-GAP, substitution of the entire finger loop with the corresponding region of Ira2p led to a construct completely inactive on Ha-Ras GTPase but active on yeast Ras2p GTPase. Analysis of these results and modeling of Ira2p.Ras complexes emphasize the importance of the finger loop region not only for the catalytic activity but also as a structural determinant involved in the specificity of GAPs toward Ras proteins from different organisms.     

Revisiting the structural flexibility of the complex p21(ras)-GTP: the catalytic conformation of the molecular switch II.

The hydrolysis of GTP in p21(ras) triggers conformational changes that regulate the ras/ERK signaling pathway. An important active site residue is Gln61, which has been found to be mutated in 30% of human tumors. The dynamics of the active site conformation is studied by using molecular dynamics simulation of two independent structures of the GTP-bound uncomplexed enzyme. Two distinct conformations of the enzyme are observed, in which the side-chain residue Gln61 is in different orientations. Essential dynamics analysis is used to describe the essential motions in the transition between the two conformations. Results are compared with earlier simulations of p21(ras) and its complex with GTPase activating protein p21-GAP.     

Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras.

Shc proteins are phosphorylated on tyrosine residues and associate with growth factor receptor-bound protein 2 (Grb2) upon treatment of cells with epidermal growth factor (EGF) or insulin. We have studied the role of Shc in insulin- and EGF-induced activation of p21ras in NIH 3T3 cells overexpressing human insulin receptors (A14 cells). A14 cells are equally responsive to insulin and EGF with respect to activation of p21ras. Analysis of Shc immunoprecipitates revealed that (i) both insulin and EGF treatment resulted in Shc tyrosine phosphorylation and (ii) Shc antibodies coimmunoprecipitated both Grb2 and mSOS after insulin and EGF treatment. The induction of tyrosine phosphorylation of Shc and the presence of Grb2 and mSOS in Shc immunoprecipitates followed similar time courses, with somewhat higher levels after EGF treatment. In mSOS immunoprecipitates, Shc could be detected as well. Furthermore, Shc immune complexes contained guanine nucleotide exchange activity toward p21ras in vitro. From these results, we conclude that after insulin and EGF treatment, Shc associates with both Grb2 and mSOS and therefore may mediate, at least in part, insulin- and EGF-induced activation of p21ras. In addition, we investigated whether the Grb2-mSOS complex associates with the insulin receptor or with insulin receptor substrate 1 (IRS1). Although we observed association of Grb2 with IRS1, we did not detect complex formation between mSOS and IRS1 in experiments in which the association of mSOS with Shc was readily detectable. Furthermore, whereas EGF treatment resulted in the association of mSOS with the EGF receptor, insulin treatment did not result in the association of mSOS with the insulin receptor. These results indicate that the association of Grb2-nSOS with Shc may be an important event in insulin-induced, mSOS-mediated activation of p21ras.     

GTPase activating proteins for the smg-21 GTP-binding protein having the same effector domain as the ras proteins in human platelets

Two proteins stimulating the GTPase activity of the smg-21 GTP-binding protein (smg p21) having the same effector domain as the ras proteins (ras p21s) are partially purified from the cytosol fraction of human platelets. These proteins, designated as smg p21 GTPase activating protein (GAP) 1 and 2, do not stimulate the GTPase activity of c-Ha-ras p21. The GAP activity for c-Ha-ras p21 is also detected in the cytosol fraction of human platelets. smg p21 GAP1 and 2 are separated from c-Ha-ras p21 GAP by column chromatographies. The activity of smg p21 GAP1 and 2 is killed by tryptic digestion or heat boiling. The Mr values of smg p21 GAP1 and 2 are similar and are estimated to be 2.5-3.5 x 10(5) by gel filtration analysis. These results indicate that there are two GAPs for smg p21 in addition to a GAP for c-Ha-ras p21 in human platelets.     

Calcium inhibits epidermal growth factor-induced activation of p21ras in human primary keratinocytes.

Human primary keratinocytes are an elegant model system to study the balance between proliferation and differentiation. Both epidermal growth factor (EGF) and extracellular calcium have been implicated to function in the control of this balance, although the molecular mechanism underlying this process is poorly understood. In this study, we measured the effect of both EGF and calcium treatment on activation of p21ras and ERK2. We found that addition of EGF stimulated the activity of ERK2. This stimulation was dependent on p21ras activity, since it was completely abolished by expression of a dominant negative mutant of p21ras (p21ras(Asn-17)). Raising the level of extracellular calcium (1.8 mM) did not result in activation of ERK2. On the contrary, calcium treatment inhibited EGF-induced stimulation of ERK2 activity. In order to determine the site at which calcium treatment interferes in EGF-induced signaling, we analyzed the effect of calcium on the various steps that are involved in EGF-induced, p21ras-dependent activation of ERK2. We observed that calcium treatment inhibited EGF-induced p21ras activation. Calcium treatment, however, did not interfere with EGF-induced EGF receptor autophosphorylation or association of mammalian SOS with the EGF receptor and Shc. This, together with the observation that calcium treatment alone decreased the basal level of p21ras activity, indicates that calcium treatment interferes in EGF-mediated signaling at the level of p21ras. This type of cross talk may play a role in the decision between proliferation and differentiation in human primary keratinocytes.     

Diversity and versatility of GTPase activating proteins for the p21rho subfamily of ras G proteins detected by a novel overlay assay.

The p21ras superfamily, involved in diverse processes including cell growth and intracellular trafficking, possesses intrinsic GTPase activity and cycles between GTP-bound active and GDP-bound quiescent states. This intrinsic activity, which results in down-regulation, is accelerated by GTPase activating proteins (GAPs). Other proteins regulating the GDP/GTP cycle include exchange proteins and dissociation inhibitors. The p21s rho, rac, and cdc42Hs constitute a subfamily implicated in cytoskeletal organization. BCR and n-chimaerin are prototypes of a new GAP family for these p21s. To investigate proteins modulating GTP hydrolysis of the three p21s, we developed a novel overlay assay applicable to tissue extracts. Diverse GAPs with different specificities were identified in all rat tissues. Brain contained rac1 GAPs of 45, 50, 85, 100, and 150 kDa. The p50 and p150 GAPs also act on rhoA and cdc42Hs and are ubiquitous, while the p45-GAP, n-chimaerin, is brain- and testis-specific and acts preferentially on rac1; the p100 GAP acts on both rac1 and cdc42Hs and is brain-specific. A new class of p21-interacting proteins was also identified. This diversity, versatility, and tissue specificity of GAPs may be required for fine control of the down-regulation of GTP-bound p21s and the suggested specific downstream effects of individual GAPs, which could involve "cross-talk" between GAPs and p21s.     

NGF and EGF rapidly activate p21ras in PC12 cells by distinct, convergent pathways involving tyrosine phosphorylation.

Activation of p21ras, demonstrated directly as an increase in p21ras-associated GTP, was induced rapidly but transiently by both nerve growth factor (NGF) and epidermal growth factor (EGF) in PC12 cells. The factors activate p21ras to equal extents and with virtually identical time courses. Growth factor-induced p21ras activation and tyrosine phosphorylation have similar time courses and sensitivities to genistein inhibition, indicating that p21ras activation is a result of tyrosine kinase activity. Furthermore, PC12 mutants lacking the Trk NGF receptor tyrosine kinase also lack NGF-inducible p21ras activation. The protein kinase inhibitor K252a and the methyltransferase inhibitor MTA abolish NGF-induced, but not EGF-induced, p21ras activation--effects correlated with inhibition only of NGF-induced tyrosine phosphorylation. In spite of differences in sensitivity to genistein, MTA, and K252a, EGF- and NGF-stimulated p21ras activation are not additive, implying that they do share at least one step in common.     

Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein.

The p21ras GTPase-activating protein (GAP) down-regulates p21ras by stimulating its intrinsic GTPase activity. GAP is found predominantly as a monomer in the cytosol of normal cells. However, in cells expressing an activated cytoplasmic protein-tyrosine kinase, p60v-src, or stimulated with epidermal growth factor, GAP becomes phosphorylated on tyrosine and serine and forms distinct complexes with two phosphoproteins of 62 and 190 kDa (p62 and p190). In v-src-transformed Rat-2 cells, a minor fraction of GAP associates with the highly tyrosine phosphorylated p62 to form a complex that is localized at the plasma membrane and in the cytosol. In contrast, the majority of GAP enters a distinct complex with p190 that is exclusively cytosolic and contains predominantly phosphoserine. Epidermal growth factor stimulation also induces a marked conversion of monomeric GAP to higher-molecular-weight species in rat fibroblasts. The GAP-p190 complex is dependent on phosphorylation and shows reduced GAP activity. These results indicate that protein-tyrosine kinases induce GAP to form multiple heteromeric complexes, which are strong candidates for regulators or targets of p21ras.     

Fluorescence approaches to the study of the p21ras GTPase mechanism

The use of ribose-modified guanine nucleotides and tryptophan mutants of p21ras, neither of which have significant effect on the kinetic mechanism of the p21ras GTPase and the GAP-activated p21ras GTPase, will now allow a detailed kinetic study of how GAP and other regulatory proteins interact with p21ras. This will lead to a better understanding of how the relative concentrations of 'active' p21ras. GTP and 'inactive' p21ras. GDP are regulated in the cell.     

p21ras mediates control of IL-2 gene promoter function in T cell activation.

It has been shown previously in T cells that stimulation of protein kinase C or the T cell antigen receptor leads to a rapid and persistent activation of p21ras as measured by a dramatic increase in the amount of bound GTP. These stimuli are also known to induce the expression of the T lymphocyte growth factor, interleukin-2 (IL-2), an essential growth factor for the immune system. Receptor induced activation of p21ras has been demonstrated in several cell types but involvement of protein kinase C as an upstream activator of p21ras appears to be unique to T cells. In this study we show that p21ras acts as a component of the protein kinase C and T cell antigen receptor downstream signalling pathway controlling IL-2 gene expression. In the murine T cell line EL4, constitutively active p21ras greatly potentiates the phorbol ester and T cell receptor agonist induced production of IL-2 as measured both by biological assay for the cytokine and by the use of a reporter construct. Active p21ras also partially replaces the requirement for protein kinase C activation in synergizing with a calcium ionophore to induce production of IL-2. Furthermore, using a dominant negative mutant of ras, Ha-rasN17, we show that endogenous ras function is essential for induction of IL-2 expression in response to protein kinase C or T cell receptor stimulation. Activation of ras proteins is thus a necessary but not sufficient event in the induction of IL-2 synthesis. Ras proteins are therefore pivotal signalling molecules in T cell activation.     

CD2 antigen mediated activation of the guanine nucleotide binding proteins p21ras in human T lymphocytes.

T cell stimulation via the TCR complex (TCR/CD3 complex) results in activation of the guanine nucleotide binding proteins encoded by the ras protooncogenes (p21ras). In the present study we show that the activation state of p21ras in T lymphocytes can also be controlled by triggering of the CD2 Ag. The activation state of p21ras is controlled by GTP levels on p21ras. In T cells stimulation of protein kinase C is able to induce an accumulation of "active" p21ras-GTP complexes due to an inhibitory effect of protein kinase C stimulation on the intrinsic GTPase activity of p21ras. The regulatory effect of protein kinase C on p21ras GTPase activity appears to be mediated via regulation of GAP, the GTPase activating protein of p21ras. In the present report, we demonstrate that the TCR/CD3 complex and the CD2 Ag control the accumulation of p21ras-GTP complexes via a regulatory effect on p21ras GTPase activity. The TCR/CD3 complex and CD2 Ag are also able to control the cellular activity of GAP. These data demonstrate that p21ras is part of the signal transduction responses controlled by the CD2 Ag, and reveal that the TCR/CD3 complex and CD2 Ag control the activation state of p21ras via a similar mechanism.     

Effector domain mutations dissociate p21ras effector function and GTPase-activating protein interaction.

The GTPase activity of p21ras is stimulated by GTPase-activating proteins (GAPs) such as p120GAP and the product of the neurofibromatosis 1 gene, which may negatively regulate p21 function. GAPs are also proposed effectors of ras. We have sought activating substitutions in c-H-ras in the region encoding the effector domain, on the rationale that such mutations would dissociate effector function from negative regulation by GAP. One such activating mutation, Pro-34-->Arg, encodes protein that is substantially bound to GTP in vivo. In vitro, this protein is not stimulated by GAPs, and its binding to p120GAP is grossly impaired. The results support the idea that the p21 structural requirements for effector function and GAP interaction are quite different and suggest that some molecule(s) other than p120GAP serves as the ras effector. In contrast to the results obtained with p120GAP, the Pro-34-->Arg p21 species is effectively coupled to the raf-1 product, as judged from electrophoretic mobility shifts of the Raf-1 phosphoprotein.     

Regulation of p21ras activity.

The ras genes encode GTP/GDP-binding proteins that participate in mediating mitogenic signals from membrane tyrosine kinases to downstream targets. The activity of p21ras is determined by the concentration of GTP-p21ras, which is tightly regulated by a complex array of positive and negative control mechanisms. GAP and NF1 can negatively regulate p21ras activity by stimulating hydrolysis of GTP bound to p21ras. Other cellular factors can positively regulate p21ras by stimulating GDP/GTP exchange.     

Role of the C-terminal region of smg p21, a ras p21-like small GTP-binding protein, in membrane and smg p21 GDP/GTP exchange protein interactions

Limited proteolysis with trypsin of smg p21B, a ras p21-like small GTP-binding protein having the same putative effector domain as ras p21s, produced the N-terminal fragment and the C-terminal tail of Lys-Lys-Ser-Ser-geranylgeranyl-Cys methyl ester. The Mr values of the intact smg p21B, the N-terminal fragment, and the C-terminal tail were estimated to be about 22,000, 20,500, and less than 1,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the GDP- and GTP-bound forms of the intact smg p21B bound to various membranes and phosphatidylserine-linked Affi-Gel. However, both the GDP- and GTP-bound forms of the N-terminal fragment failed to bind to membranes and phosphatidylserine-linked Affi-Gel. In contrast, the C-terminal tail bound to membranes and phosphatidylserine-linked Affi-Gel. The N-terminal fragment contained a GDP/GTP-binding and GTPase domain and exhibited these two activities, but the C-terminal tail did not show any such activity. A GTPase-activating protein for smg p21 stimulated the GTPase activity of both the intact smg p21B and the N-terminal fragment. In contrast, a GDP/GTP exchange protein for smg p21, named GDP dissociation stimulator, stimulated the GDP/GTP exchange reaction of the intact smg p21B but not that of the N-terminal fragment. These results indicate 1) that smg p21B is composed of at least two functionally different domains, the N-terminal GDP/GTP-binding and GTPase domain and the C-terminal membrane-binding domain, 2) that smg p21B binds to membranes through its C-terminal hydrophobic and basic domain, and 3) that this C-terminal domain is also essential for the smg p21 GDP dissociation stimulator action but not for the smg p21 GTPase-activating protein action.     

The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21

The neurofibromatosis type 1 (NF1) protein contains a region of significant sequence similarity to ras p21 GTPase-activating protein (GAP) and the yeast IRA1 gene product. A fragment of NF1 cDNA encoding the GAP-related domain (NF1 GRD) was expressed, immunoaffinity purified, and assayed for effects on N-ras p21 GTPase activity. The GTPase of wild-type ras p21 was stimulated by NF1 GRD, but oncogenic mutants of ras p21 (Asp-12 and Val-12) were unaffected, and the GTPase of an effector mutant (Ala-38) was only weakly stimulated. NF1 GRD also down-regulated RAS function in S. cerevisiae. The affinity of NF1 GRD for ras p21 was estimated to be 250 nM: this is more than 20-fold higher than the affinity of GAP for ras p21. However, its specific activity was about 30 times lower. These kinetic measurements suggest that NF1 may be a significant regulator of ras p21 activity, particularly at low ras p21 concentrations.