Enhancement of guanine-nucleotide exchange activity of C3G for Rap1 by the expression of Crk, CrkL, and Grb2

Crk is an adaptor protein that consists almost entirely of SH2 and SH3 domains. We have previously demonstrated, by using in vivo and in vitro systems, that C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide exchange factor, specifically activates Rap1. C3G also binds to other adaptor proteins, including CrkL and Grb2. In the present study, we analyzed the effect of Crk, CrkL, and Grb2 on the C3G-Rap1 pathway. Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cells significantly increased the ratio of GTP/GDP bound to Rap1. Both the SH2 and SH3 domains of Crk were required for this activity. However, Crk did not stimulate the guanine nucleotide exchange activity of C3G for Rap1 in vitro, suggesting that Crk does not activate C3G by an allosteric mechanism. The requirement of the SH2 domain of Crk for the enhancement of guanine nucleotide exchange activity for Rap1 could be compensated for by the addition of a farnesylation signal to Crk, indicating that Crk enhanced the guanine nucleotide exchange activity of C3G by membrane recruitment of C3G. These results demonstrate that Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily by recruiting C3G to the cell membrane.     

Activation of the Rap1 GTPase by the B cell antigen receptor

The B cell antigen receptor (BCR) activates Ras, a GTPase that promotes cell proliferation by activating the Raf-1/MEK/ERK signaling module and other signaling enzymes. In its active GTP-bound form, the Rap1 GTPase may act as a negative regulator of Ras-mediated signaling by sequestering Ras effectors (e.g., Raf-1) and preventing their activation. In this report, we show that BCR engagement activates Rap1 and that this is dependent on production of diacylglycerol (DAG) by phospholipase C-gamma. Activation of Rap1 by the BCR was greatly reduced in phospholipase C-gamma-deficient B cells, whereas both a synthetic DAG and phorbol dibutyrate could activate Rap1 in B cells. We had previously shown that C3G, an activator of Rap1, associates with the Crk adaptor proteins in B cells and that BCR engagement causes Crk to bind to the Cas and Cbl docking proteins. However, the DAG-dependent pathway by which the BCR activates Rap1 apparently does not involve Crk signaling complexes since phorbol dibutyrate could activate Rap1 without inducing the formation of these complexes. Thus, the BCR activates Rap1 via a novel DAG-dependent pathway.     

Sos, Vav, and C3G participate in B cell receptor-induced signaling pathways and differentially associate with Shc-Grb2, Crk, and Crk-L adaptors

B cell antigen receptor (BCR)-mediated signal transduction controls B cell proliferation and differentiation. The BCR activates Ras, presumably by the formation of a Shc-Grb2 adaptor complex, which recruits the Grb2-associated guanine nucleotide exchange factor Sos to the plasma membrane. In order to reveal additional BCR-induced signaling events involving the Grb2 adaptor, we undertook the isolation of Grb2-binding proteins. Using the yeast two-hybrid system and bacterial fusion proteins, Vav and C3G were identified as Grb2 binders. Vav is a putative nucleotide exchange factor and a target for BCR-induced tyrosine phosphorylation. C3G exerts nucleotide exchange activity on the Ras-related Rap1 protein. While Sos binds to both Grb2 Src homology-3 (SH3) domains, Vav was found to associate selectively with the carboxyl-terminal SH3 domain, while C3G bound selectively to the amino-terminal SH3 domain of bacterially expressed Grb2. Despite the association of Vav with Grb2 in vitro, we could not demonstrate an interaction between endogenous Vav and Grb2 molecules in primary B cells. Instead, Vav was found to inducibly associate with the Grb2-related adaptor protein Crk upon BCR stimulation. C3G did not bind to either Grb2, Shc, or Crk in vivo. Instead, C3G was found in association with the Crk-L adaptor, both before and after BCR stimulation. We show that Crk-L also participates in BCR signaling, since it inducibly interacts with tyrosine-phosphorylated Cbl. We conclude that, in addition to Sos, Vav and C3G play a role in BCR-mediated signal transduction. These guanine nucleotide exchange factors selectively associate with Grb2, Crk, and Crk-L, respectively, which may serve to direct them to different target molecules. Since Cbl binds to Grb2, Crk, as well as Crk-L, we hypothesize that Cbl may affect the function of all three exchangers.     

Differing rate dependence and temporal distribution of repolarization alternans in patients with and without ventricular tachycardia

The Shc protein helps to transmit signals from receptor and cytoplasmic tyrosine kinases to Ras. We have shown that several breast cancer cell lines (MDA-MB-453, BT474, MDA-MB-361, and SKBR3), which overexpress the ErbB2 receptor tyrosine kinase, contain constitutively tyrosine phosphorylated Shc. To investigate the role of Shc in these cells, we transfected them with a Shc-Y317F dominant-negative mutant defective in signaling to Ras. The transfectants were unable to form stable colonies, suggesting a critical role for Shc in the proliferation of these cells. In contrast, dominant-negative Shc transfectants of the nontransformed breast epithelial cell line HBL-100 grew normally. Surprisingly, cell cycle analysis of transfected SKBR3 cells suggested that the cells were blocked not only in G0-G1, but also in G2-M. The G2-M block was unexpected because Shc-Y317 is downstream of receptor tyrosine kinases that drive the early events in the cell cycle. Both the G0-G1 and G2-M arrest were rescued by transfection with wild-type Shc or oncogenic Ras 12V. Rescue by Ras suggests that Shc Y317 signals upstream of Ras, and that Shc to Ras effector pathways are involved in G2-M, although confirmation awaits a detailed molecular analysis. Most importantly, this work provides the first evidence for Shc involvement in G2-M.     

Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP

Rap1 is a small, Ras-like GTPase that was first identified as a protein that could suppress the oncogenic transformation of cells by Ras. Rap1 is activated by several extracellular stimuli and may be involved in cellular processes such as cell proliferation, cell differentiation, T-cell anergy and platelet activation. At least three different second messengers, namely diacylglycerol, calcium and cyclic AMP, are able to activate Rap1 by promoting its release of the guanine nucleotide GDP and its binding to GTP. Here we report that activation of Rap1 by forskolin and cAMP occurs independently of protein kinase A (also known as cAMP-activated protein kinase). We have cloned the gene encoding a guanine-nucleotide-exchange factor (GEF) which we have named Epac (exchange protein directly activated by cAMP). This protein contains a cAMP-binding site and a domain that is homologous to domains of known GEFs for Ras and Rap1. Epac binds cAMP in vitro and exhibits in vivo and in vitro GEF activity towards Rap1. cAMP strongly induces the GEF activity of Epac towards Rap1 both in vivo and in vitro. We conclude that Epac is a GEF for Rap1 that is regulated directly by cAMP and that Epac is a new target protein for cAMP.     

Fibroblast transformation by Fps/Fes tyrosine kinases requires Ras, Rac, and Cdc42 and induces extracellular signal-regulated and c-Jun N-terminal kinase activation

The small GTP-binding proteins Ras, Rac, and Cdc42 link protein-tyrosine kinases with mitogen-activated protein kinase (MAPK) signaling cascades. Ras controls the activation of extracellular signal-regulated kinases (ERKs), while Rac and Cdc42 regulate the c-Jun N-terminal kinases (JNKs). In this study, we investigated whether small G protein/MAPK cascades contribute to signal transduction by transforming variants of c-Fes, a nonreceptor tyrosine kinase implicated in cytokine signaling and myeloid differentiation. First, we investigated the effects of dominant-negative small G proteins on Rat-2 fibroblast transformation by a retroviral homolog of c-Fes (v-Fps) and by c-Fes activated via N-terminal addition of the v-Src myristylation signal (Myr-Fes). We observed that dominant-negative Ras, Rac, and Cdc42 inhibited v-Fps- and Myr-Fes-induced growth of Rat-2 cells in soft agar, indicating that activation of these small GTP-binding proteins is required for fibroblast transformation by Fps/Fes tyrosine kinases. To determine whether MAPK pathways are activated downstream of these small G proteins, we measured ERK and JNK activity in the v-Fps- and Myr-Fes-transformed Rat-2 cells. Both ERK and JNK activities were elevated in the transformed cells, suggesting that these pathways are involved in cellular transformation. Dominant-negative mutants of Ras (but not Rac or Cdc42) specifically inhibited ERK activation by v-Fps and Myr-Fes, demonstrating that ERK activation occurs exclusively downstream of Ras. All three dominant-negative small G proteins inhibited JNK activation by v-Fps and Myr-Fes, indicating that JNK activation by these tyrosine kinases requires both Ras and Rho family GTPases. These data demonstrate that multiple small G protein/MAPK cascades are involved in downstream signal transduction by Fps/Fes tyrosine kinases.     

Activation of nuclear factor of activated T cells-(NFAT) and activating protein 1 (AP-1) by oncogenic 70Z Cbl requires an intact phosphotyrosine binding domain but not Crk(L) or p85 phosphatidylinositol 3-kinase association

The Cbl proto-oncogene product is a complex adapter protein that functions as a negative regulator of protein tyrosine kinases. It is rapidly tyrosine-phosphorylated and associates with Crk(L) and p85 phosphatidylinositol 3-kinase (PI3K) upon engagement of numerous receptors linked to tyrosine kinases. Elucidation of the mechanism(s) underlying Cbl deregulation is therefore of considerable interest. The 70Z Cbl oncoprotein shows increased baseline tyrosine phosphorylation in fibroblasts and enhances nuclear factor of activated T cells (NFAT) activity in Jurkat T cells. Its transforming ability has been proposed to relate to its increased phosphotyrosine content. We demonstrate that 70Z Cbl shows increased basal and activation-induced tyrosine phosphorylation and association with Crk(L) and p85 PI3K in Jurkat T cells. 70Z Cbl, however, retains the ability to enhance NFAT and activating protein 1 (AP1) activity in the absence of Crk(L)/p85 PI3K association. In contrast, the G306E mutation, which inactivates the phosphotyrosine binding domain of Cbl, blocks NFAT/AP1 activation by 70Z Cbl. We conclude that 70Z Cbl-induced NFAT/AP1 activation requires the phosphotyrosine binding domain but not Crk(L)/p85 PI3K association. We hypothesize that 70Z Cbl acts as a dominant negative by blocking the negative regulatory function of the Cbl phosphotyrosine binding domain on protein-tyrosine kinases.     

The small G-proteins Rap 1 as potential targets of vasoactive intestinal peptide effects in the human clonic cancer cells HT29

We recently reported that the vasoactive intestinal peptide (VIP) potently inhibited proliferation and induced in parallel a strong cAMP rise, in the human colonic cancer cell line HT29. In this study, we investigated whether Rap 1 proteins could be potential targets of VIP effects in HT29 cells. These Ras-related proteins in which activity was demonstrated to be regulated by PKA phosphorylation, are considered as potential modulators of the Ras / Raf / MAP kinases cascade that governs cell growth control. Our data revealed that the Rap 1a isoform is highly expressed in HT29 cells and mainly localized in a late endosomal compartment. In these cells, VIP induces Rap 1 phosphorylation and a yet unidentified modification that leads to their acidification. This latter Rap 1 acidification seems to be, at least partially, cAMP-dependent. It is concluded that in HT29 cells, Rap 1 proteins may be part of a VIP-induced signaling cascade.     

Activity of Rap1 is regulated by bombesin, cell adhesion, and cell density in NIH3T3 fibroblasts

Rap1 and Ras are homologous GTPases that are implicated in cell proliferation and differentiation. At present, little is known about the regulation of Rap1 activity. Using a recently developed assay with activation-specific probes, we found increased activity of endogenous Rap1 in NIH3T3 cells after stimulation with the neuropeptide growth factor bombesin in a concentration- and time-dependent manner. The activity of endogenous Ras was unaffected. Analysis of putative effectors showed no activation of c-Raf-1 or B-Raf after bombesin stimulation. However, MAPK/Erk-phosphorylation and the proliferation rate was increased. In addition, Rap1 was activated during cell adhesion to coated and uncoated tissue culture plates, as well as in response to various mitogens. Surprisingly, the basal Rap1 activity was observed to be cell density-dependent, with low levels when cells were reaching confluency. The results suggest that Rap1 acts as an important mediator of mitogenic signals distinct to Ras activation.     

Dominant-negative mutants of the SH2/SH3 adapters Nck and Grb2 inhibit MAP kinase activation and mesoderm-specific gene induction by eFGF in Xenopus

The SH2/SH3 adapters Nck, Grb2 and Crk promote the assembly of signaling complexes by binding to tyrosine phosphorylated proteins using their SH2 domains and to proline-rich sequences on effector molecules using their SH3 domains. FGF, which activates a receptor tyrosine kinase, induces mesoderm formation in Xenopus embryos through activation of the Ras/Raf/MAPK signaling pathway. We present evidence that dominant-negative mutants of Nck and Grb2, but not Crk1, can inhibit mesoderm-specific gene induction by eFGF in Xenopus animal cap explants. We also show that dominant-negative mutants of Grb2 and Nck can inhibit eFGF-induced Erk1 activation in Xenopus animal caps, and that targeting the first two SH3 domains of Nck to the membrane can activate Erk1 in the absence of eFGF. Furthermore, combinations of the dominant-negative Grb2 mutants with the inhibitory Nck mutant synergistically inhibited Erk1 activation by eFGF in Xenopus animal caps, suggesting that the dominant-negative Nck and Grb2 mutants inhibit Erk1 activation by binding to different proteins. By contrast only Grb2 mutants could inhibit eFGF-induced Erk1 activation in human 293 cells, demonstrating diversity in the specific mechanisms of signaling from FGF to MAP kinases in different cells.     

Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein

Rap1 small GTP-binding protein has the same amino acid sequence at its effector domain as that of Ras. Rap1 has been shown to antagonize the Ras functions, such as the Ras-induced transformation of NIH 3T3 cells and the Ras-induced activation of the c-Raf-1 protein kinase-dependent mitogen-activated protein (MAP) kinase cascade in Rat-1 cells, whereas we have shown that Rap1 as well as Ras stimulates DNA synthesis in Swiss 3T3 cells. We have established a cell-free assay system in which Ras activates bovine brain B-Raf protein kinase. Here we have used this assay system and examined the effect of Rap1 on the B-Raf activity to phosphorylate recombinant MAP kinase kinase (MEK). Recombinant Rap1B stimulated the activity of B-Raf, which was partially purified from bovine brain and immunoprecipitated by an anti-B-Raf antibody. The GTP-bound form was active, but the GDP-bound form was inactive. The fully post-translationally lipid-modified form was active, but the unmodified form was nearly inactive. The maximum B-Raf activity stimulated by Rap1B was nearly the same as that stimulated by Ki-Ras. Rap1B enhanced the Ki-Ras-stimulated B-Raf activity in an additive manner. These results indicate that not only Ras but also Rap1 is involved in the activation of the B-Raf-dependent MAP kinase cascade.     

Extracellular signal-regulated activation of Rap1 fails to interfere in Ras effector signalling

The small GTPase Rap1 has been implicated in both negative and positive control of Ras-mediated signalling events. We have investigated which extracellular signals can activate Rap1 and whether this activation leads to a modulation of Ras effector signalling, i.e. the activation of ERK and the small GTPase Ral. We found that Rap1 is rapidly activated following stimulation of a large variety of growth factor receptors. These receptors include receptor tyrosine kinases for platelet-derived growth factor (PDGF) and epithelial growth factor (EGF), and G protein-coupled receptors for lysophosphatidic acid (LPA), thrombin and endothelin. At least three distinct pathways may transduce a signal towards Rap1 activation: increase in intracellular calcium, release of diacylglycerol and cAMP synthesis. Surprisingly, activation of endogenous Rap1 fails to affect Ras-dependent ERK activation. In addition, we found that although overexpression of active Rap1 is able to activate the Ral pathway, activation of endogenous Rap1 in fibroblasts does not result in Ral activation. Rap1 also does not negatively influence Ras-mediated Ral activation. We conclude that activation of Rap1 is a common event upon growth factor treatment and that the physiological function of Rap1 is likely to be different from modulation of Ras effector signalling.     

The small Mr Ras-like GTPase Rap1 and the phospholipase C pathway act to regulate phagocytosis in Dictyostelium discoideum

The function of the small-Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes in Dictyostelium and the oxidative burst in mammalian neutrophils. To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type [Rap1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(+) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C-specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.     

Cellular proteins binding to the first Src homology 3 (SH3) domain of the proto-oncogene product c-Crk indicate Crk-specific signaling pathways

The widely expressed c-Crk protein, composed of one SH2 and two SH3 domains, lacks an apparent catalytic domain, suggesting that it functions through the formation of specific complexes with other proteins. Bacterially expressed c-Crk formed in vitro highly stable complexes via the first SH3 domain [SH3(N)]. Most prominent were a 185 kDa protein of unknown identity (p185), Sos- immunoreactive bands of 170 kDa (p170) and 145 to 155 kDa bands, corresponding to the recently cloned C3G protein. p170 also bound to Ash/Grb2 and Nck while p185 and C3G bound only to Crk. Additional Crk binding proteins were found in hematopoietic cells, particularly the myeloid-monocytic lineage. The protein binding properties of Crk were subsequently compared to CRKL, the product of a homologous but distinct gene, and found to be very similar. The binding of two guanine nucleotide exchange factors, Sos and C3G, to Crk and CRKL indicates that Ras or related proteins likely play a role in signaling through Crk family proteins.     

Rap1 mediates sustained MAP kinase activation induced by nerve growth factor

Activation of mitogen-activated protein (MAP) kinase (also known as extracellular-signal-regulated kinase, or ERK) by growth factors can trigger either cell growth or differentiation. The intracellular signals that couple growth factors to MAP kinase may determine the different effects of growth factors: for example, transient activation of MAP kinase by epidermal growth factor stimulates proliferation of PC12 cells, whereas they differentiate in response to nerve growth factor, which acts partly by inducing a sustained activation of MAP kinase. Here we show that activation of MAP kinase by nerve growth factor involves two distinct pathways: the initial activation of MAP kinase requires the small G protein Ras, but its activation is sustained by the small G protein Rap1. Rap1 is activated by CRK adaptor proteins and the guanine-nucleotide-exchange factor C3G, and forms a stable complex with B-Raf, an activator of MAP kinase. Rap1 is required for at least two indices of neuronal differentiation by nerve growth factor: electrical excitability and the induction of neuron-specific genes. We propose that the activation of Rap1 by C3G represents a common mechanism to induce sustained activation of the MAP kinase cascade in cells that express B-Raf.     

Mitogenic and oncogenic properties of the small G protein Rap1b

It has been widely reported that the small GTP-binding protein Rap1 has an anti-Ras and anti-mitogenic activity. Thus, it is generally accepted that a normal physiological role of Rap1 proteins is to antagonize Ras mitogenic signals, presumably by forming nonproductive complexes with proteins that are typically effectors or modulators of Ras. Rap1 is activated by signals that raise intracellular levels of cAMP, a molecule that has long been known to exert both inhibitory and stimulatory effects on cell growth. We have now tested the intriguing hypothesis that Rap1 could have mitogenic effects in systems in which cAMP stimulates cell proliferation. The result of experiments addressing this possibility revealed that Rap1 has full oncogenic potential. Expression of Rap1 in these cells results in a decreased doubling time, an increased saturation density, and an unusual anchorage-dependent morphological transformation. Most significantly, however, Rap1-expressing cells formed tumors when injected into nude mice. Thus, we propose that the view that holds Rap1 as an antimitogenic protein should be restricted and conclude that Rap1 is a conditional oncoprotein.     

Differential interaction of Crkl with Cbl or C3G, Hef-1, and gamma subunit immunoreceptor tyrosine-based activation motif in signaling of myeloid high affinity Fc receptor for IgG (Fc gamma RI)

Cbl-Crkl and Crkl-C3G interactions have been implicated in T cell and B cell receptor signaling and in the regulation of the small GTPase, Rap1. Recent evidence suggests that Rap1 plays a prominent role in the regulation of immunoreceptor tyrosine-based activation motif (ITAM) signaling. To gain insight into the role of Crkl in myeloid ITAM signaling, we investigated Cbl-Crkl and Crkl-C3G interactions following Fc gamma RI aggregation in U937IF cells. Fc gamma RI cross-linking of U937IF cells results in the tyrosine phosphorylation of Cbl, Crkl, and Hef-1, an increase in the association of Crkl with Cbl via direct SH2 domain interaction and increased Crkl-Hef-1 binding. Crkl constitutively binds to the guanine nucleotide-releasing protein, C3G, via direct SH3 domain binding. Our data show that distinct Cbl-Crkl and Crkl-C3G complexes exist in myeloid cells, suggesting that these complexes may modulate distinct signaling events. Anti-Crkl immunoprecipitations demonstrate that the ITAM-containing gamma subunit of Fc gamma RI is induced to form a complex with the Crkl protein, and Crkl binds to the cytoskeletal protein, Hef-1. The induced association of Crkl with Cbl, Hef-1, and Fc gamma RI gamma after Fc gamma RI activation and the constitutive association between C3G and Crkl provide the first evidence that a Fc gamma RI gamma-Crkl-C3G complex may link ITAM receptors to the activation of Rap1 in myeloid cells.