Phosphorylation of Dok1 by Abl family kinases inhibits CrkI transforming activity.

The Crk SH2/SH3 adaptor and the Abl nonreceptor tyrosine kinase were first identified as oncoproteins, and both can induce tumorigenesis when overexpressed or mutationally activated. We previously reported the surprising finding that inhibition or knockdown of Abl family kinases enhanced transformation of mouse fibroblasts by CrkI. Abl family inhibitors are currently used or are being tested for treatment of human malignancies, and our finding raised concerns that such inhibitors might actually promote the growth of tumors overexpressing CrkI. Here, we identify the Dok1 adaptor as the key effector for the enhancement of CrkI transformation by Abl inhibition. We show that phosphorylation of tyrosines 295 and 361 of Dok1 by Abl family kinases suppresses CrkI transforming activity, and that upon phosphorylation these tyrosines bind the SH2 domains of the Ras inhibitor p120 RasGAP. Knockdown of RasGAP resulted in a similar enhancement of CrkI transformation, consistent with a critical role for Ras activity. Imaging studies using a FRET sensor of Ras activation revealed alterations in the localization of activated Ras in CrkI-transformed cells. Our results support a model in which Dok1 phosphorylation normally suppresses localized Ras pathway activity in Crk-transformed cells via recruitment and/or activation of RasGAP, and that preventing this negative feedback mechanism by inhibiting Abl family kinases leads to enhanced transformation by Crk.

Proteins that bind the Src homology 3 domain of CrkI have distinct roles in Crk transformation.

The v-Crk oncogene product consists of two protein interaction modules, a Src homology 2 (SH2) domain and a Src homology 3 (SH3) domain. Overexpression of CrkI, the cellular homolog of v-Crk, transforms mouse fibroblasts, and elevated CrkI expression is observed in several human cancers. The SH2 and SH3 domains of Crk are required for transformation, but the identity of the critical cellular binding partners is not known. A number of candidate Crk SH3-binding proteins have been identified, including the nonreceptor tyrosine kinases c-Abl and Arg, and the guanine nucleotide exchange proteins C3G, SOS1 and DOCK180. The aim of this study is to determine which of these are required for transformation by CrkI. We found that short hairpin RNA-mediated knockdown of C3G or SOS1 suppressed anchorage-independent growth of NIH-3T3 cells overexpressing CrkI, whereas knockdown of SOS1 alone was sufficient to suppress tumor formation by these cells in nude mice. Knockdown of C3G was sufficient to revert morphological changes induced by CrkI expression. By contrast, knockdown of Abl family kinases or their inhibition with imatinib enhanced anchorage-independent growth and tumorigenesis induced by Crk. These results show that SOS1 is essential for CrkI-induced fibroblast transformation, and also reveal a surprising negative role for Abl kinases in Crk transformation.

Requirement of Nck adaptors for actin dynamics and cell migration stimulated by platelet-derived growth factor B.

The Nck family of Src homology (SH) 2/SH3 domain adaptors functions to link tyrosine phosphorylation induced by extracellular signals with downstream regulators of actin dynamics. We investigated the role of mammalian Nck adaptors in signaling from the activated platelet-derived growth factor (PDGF) receptor (PDGFbetaR) to the actin cytoskeleton. We report here that Nck adaptors are required for cytoskeletal reorganization and chemotaxis stimulated by PDGF-B. Analysis of tyrosine-phosphorylated proteins demonstrated that Crk-associated substrate (p130(Cas)), not the activated PDGFbetaR itself, is the major Nck SH2 domain-binding protein in PDGF-B-stimulated cells. Both Nck- and p130(Cas)-deficient cells fail to display cytoskeletal rearrangements, including the formation of membrane ruffles and the disassembly of actin bundles, typically shown by their WT counterparts in response to PDGF-B. Furthermore, Nck and p130(Cas) colocalize in phosphotyrosine-enriched membrane ruffles induced by PDGF-B in NIH 3T3 cells. These results suggest that Nck adaptors play an essential role in linking the activated PDGFbetaR with actin dynamics through a pathway that involves p130(Cas).

Profiling the global tyrosine phosphorylation state by Src homology 2 domain binding.

Reversible tyrosine phosphorylation plays a crucial role in signal transduction, regulating many biological functions including proliferation, differentiation, and motility. The comprehensive characterization of the tyrosine phosphorylation state of a cell is of great interest for understanding the mechanisms that underlie signaling; however, current methods for analyzing tyrosine-phosphorylated proteins in crude protein extracts provide limited information, or are laborious and require relatively large amounts of protein. We have developed a simple, rapid, and flexible competitive binding assay based on the far-Western blot technique, in which a battery of Src homology 2 domain probes is used to detect patterns of specific tyrosine-phosphorylated sites. We demonstrate that distinct profiles of tyrosine phosphorylation can be detected with high sensitivity and specificity and low background. This proteomic approach can be used to rapidly profile the global tyrosine phosphorylation state of any cell of interest and has obvious applications as a molecular diagnostic tool, for example in the classification of tumors. The general strategy we describe here is not limited to Src homology 2 domains and could be used to profile the binding sites for any class of protein interaction domain.

Regulation of Cbl phosphorylation by the Abl tyrosine kinase and the Nck SH2/SH3 adaptor.

The Cbl proto-oncogene product is tyrosine phosphorylated in response to a wide variety of stimuli. Cbl and the Abl nonreceptor tyrosine kinase both bind to SH3 domains from the SH2/SH3 adaptor Nck, and are candidate effectors for Nck function. Numerous additional SH2- and SH3-domain-mediated interactions are also possible between Cbl, Abl, and Nck. We find that these three signaling proteins associate when overexpressed in mammalian cells and can regulate each other's activity. Co-expression of wt Cbl together with c-Abl, the activity of which is normally repressed in vivo, led to extensive Abl-dependent phosphorylation of Cbl. The major proline-rich region of Cbl was required for its phosphorylation by c-Abl, but not by a constitutively activated Abl mutant, suggesting Cbl activates c-Abl by engaging its SH3 domain. Efficient phosphorylation of Cbl and its stable association with Abl required the SH2 domain of Abl, suggesting that SH2-phosphotyrosine interactions prevent dissociation of active Abl from Cbl. We also show that overexpression of Nck could repress the phosphorylation of Cbl by Abl in vivo. Studies with Nck mutants suggested that the Nck SH2 domain is responsible for inhibiting the activity of Abl toward both Cbl and Nck itself, most likely by competing with the Abl SH2 for tyrosine-phosphorylated binding sites.

Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis.

C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G(-/-) homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase. From the embryo of this mouse, we prepared fibroblast cell lines, MEF-hC3G. Expression of Cre abolished the expression of C3G in MEF-hC3G and inhibited cell adhesion-induced activation of Rap1. The Cre-expressing MEF-hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R-Ras. Expression of Epac and CalDAG-GEFI, GEFs for Rap1, also suppressed the accelerated migration of the C3G-deficient cells. This observation indicated that Rap1 activation was sufficient to complement the C3G deficiency. In conclusion, C3G-dependent activation of Rap1 is required for adhesion and spreading of embryonic fibroblasts and for the early embryogenesis of the mouse.

Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway.

The Wiskott-Aldrich syndrome protein (WASP) and its relative neural WASP (N-WASP) regulate the nucleation of actin filaments through their interaction with the Arp2/3 complex and are regulated in turn by binding to GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate. The Nck Src homology (SH) 2/3 adaptor binds via its SH3 domains to a proline-rich region on WASP and N-WASP and has been implicated in recruitment of these proteins to sites of tyrosine phosphorylation. We show here that Nck SH3 domains dramatically stimulate the rate of nucleation of actin filaments by purified N-WASP in the presence of Arp2/3 in vitro. All three Nck SH3 domains are required for maximal activation. Nck-stimulated actin nucleation by N-WASP.Arp2/3 complexes is further stimulated by phosphatidylinositol 4,5-bisphosphate, but not by GTP-Cdc42, suggesting that Nck and Cdc42 activate N-WASP by redundant mechanisms. These results suggest the existence of an Nck-dependent, Cdc42-independent mechanism to induce actin polymerization at tyrosine-phosphorylated Nck binding sites.

SH3 domains: complexity in moderation.

The SH3 domain is perhaps the best-characterized member of the growing family of protein-interaction modules. By binding with moderate affinity and selectivity to proline-rich ligands, these domains play critical roles in a wide variety of biological processes ranging from regulation of enzymes by intramolecular interactions, increasing the local concentration or altering the subcellular localization of components of signaling pathways, and mediating the assembly of large multiprotein complexes. SH3 domains and their binding sites have cropped up in many hundreds of proteins in species from yeast to man, which suggests that they provide the cell with an especially handy and adaptable means of bringing proteins together. The wealth of genetic, biochemical and structural information available provides an intimate and detailed portrait of the domain, serving as a framework for understanding other modular protein-interaction domains. Processes regulated by SH3 domains also raise important questions about the nature of specificity and the overall logic governing networks of protein interactions.

c-Src signaling induced by the adapters Sin and Cas is mediated by Rap1 GTPase.

Oncogenic Src proteins have been extensively studied to gain insight into the signaling mechanisms of Src. To better understand signaling through wild-type Src, we used an approach that involves activation of Src signaling through the binding of physiologic ligands to the Src SH3 domain. To this end, we used full-length and truncated versions of the multiadapter molecules Cas and Sin to activate c-Src, and we examined the intracellular pathways that mediate Src signaling under these conditions. We show that although all proteins bind to and are phosphorylated by c-Src, quantitative differences exist in the ability of the different ligands to activate c-Src signaling. In addition, we show that Sin- and Cas-induced Src signaling, as assayed by transcriptional activation, is exclusively mediated through a pathway that involves the adapter Crk and the GTP-binding protein Rap1. These data are in contrast to previous observations showing Ras to mediate signaling downstream of transforming Src alleles. In our system, we found that signaling through the oncogenic SrcY527 mutant is indeed mediated by Ras. In addition, we found that Rap1 also mediates oncogenic Src signaling. Our results show for the first time that Rap1 mediates c-Src kinase signaling and reveal mechanistic differences in the signaling properties of wild-type and transforming Src proteins.

Abl family kinases and Cbl cooperate with the Nck adaptor to modulate Xenopus development.

We previously showed that overexpression of the Nck Src homology (SH) 2/SH3 adaptor in Xenopus embryos induced developmental defects including anterior truncation and mesoderm ventralization. Mutagenic analysis indicated that this was due to relocalization of endogenous proteins that bind the first two SH3 domains of Nck. We therefore screened a Xenopus expression library with Nck SH3 domains to identify Nck-interacting proteins, and evaluated candidate binding proteins for a potential role in Nck-induced anterior truncation/ventralization. Of 39 binding proteins analyzed, only the Abl-related kinase Arg and the Cbl proto-oncogene product bound preferentially to the first two SH3 domains in tandem compared with the individual domains, consistent with a role in the developmental phenotype. High level overexpression of c-Abl or Arg alone induced anterior truncation, as did lower levels of an activated form of Abl; Cbl alone had no effect. In a sensitized system where subthreshold amounts of a ventralizing Nck mutant were expressed, co-expression of the combination of Abl or Arg and Cbl at modest levels strongly potentiated anterior truncation, while Arg, Abl, or Cbl alone were without effect. These results suggest a role for both Cbl and Abl family kinases in patterning the Xenopus embryo.

Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch.

The p21-activated kinases (PAKs), stimulated by binding with GTP-liganded forms of Cdc42 or Rac, modulate cytoskeletal actin assembly and activate MAP-kinase pathways. The 2.3 A resolution crystal structure of a complex between the N-terminal autoregulatory fragment and the C-terminal kinase domain of PAK1 shows that GTPase binding will trigger a series of conformational changes, beginning with disruption of a PAK1 dimer and ending with rearrangement of the kinase active site into a catalytically competent state. An inhibitory switch (IS) domain, which overlaps the GTPase binding region of PAK1, positions a polypeptide segment across the kinase cleft. GTPase binding will refold part of the IS domain and unfold the rest. A related switch has been seen in the Wiskott-Aldrich syndrome protein (WASP).

Cdc42 is required for PIP(2)-induced actin polymerization and early development but not for cell viability.

BACKGROUND: Cdc42 and other Rho GTPases are conserved from yeast to humans and are thought to regulate multiple cellular functions by inducing coordinated changes in actin reorganization and by activating signaling pathways leading to specific gene expression. Direct evidence implicating upstream signals and components that regulate Cdc42 activity or for required roles of Cdc42 in activation of downstream protein kinase signaling cascades is minimal, however. Also, whereas genetic analyses have shown that Cdc42 is essential for cell viability in yeast, its potential roles in the growth and development of mammalian cells have not been directly assessed. RESULTS: To elucidate potential functions of Cdc42 mammalian cells, we used gene-targeted mutation to inactivate Cdc42 in mouse embryonic stem (ES) cells and in the mouse germline. Surprisingly, Cdc42-deficient ES cells exhibited normal proliferation and phosphorylation of mitogen- and stress-activated protein kinases. Yet Cdc42 deficiency caused very early embryonic lethality in mice and led to aberrant actin cytoskeletal organization in ES cells. Moreover, extracts from Cdc42-deficient cells failed to support phosphatidylinositol 4,5-bisphosphate (PIP(2))-induced actin polymerization. CONCLUSIONS: Our studies clearly demonstrate that Cdc42 mediates PIP(2)-induced actin assembly, and document a critical and unique role for Cdc42 in this process. Moreover, we conclude that, unexpectedly, Cdc42 is not necessary for viability or proliferation of mammalian early embryonic cells. Cdc42 is, however, absolutely required for early mammalian development.

Phosphorylation of c-Crk II on the negative regulatory Tyr222 mediates nerve growth factor-induced cell spreading and morphogenesis.

The Crk family of adaptor proteins participate in diverse signaling pathways that regulate growth factor-induced proliferation, anchorage-dependent DNA synthesis, and cytoskeletal reorganization, important for cell adhesion and motility. Using kidney epithelial 293T cells for transient co-transfection studies and the nerve growth factor (NGF)-responsive PC12 cell line as a model system for neuronal morphogenesis, we demonstrate that the non-receptor tyrosine kinase c-Abl is an intermediary for NGF-inducible c-Crk II phosphorylation on the negative regulatory Tyr(222). Transient expression of a c-Crk II Tyr(222) point mutant (c-Crk Y222F) in 293T cells induces hyperphosphorylation of paxillin on Tyr(31) and enhances complex formation between c-Crk Y222F and paxillin as well as c-Crk Y222F and c-Abl, suggesting that c-Crk II Tyr(222) phosphorylation induces both the dissociation of the Crk SH2 domain from paxillin and the Crk SH3 domain from c-Abl. Interestingly, examination of the early kinetics of NGF stimulation in PC12 cells showed that c-Crk II Tyr(222) phosphorylation preceded paxillin Tyr(31) phosphorylation, followed by a transient initial dissociation of the c-Crk II paxillin complex. PC12 cells overexpressing c-Crk Y222F manifested a defect in cellular adhesion and neuritogenesis that led to detachment of cells from the extracellular matrix, thus demonstrating the biological significance of c-Crk II tyrosine phosphorylation in NGF-dependent morphogenesis. Whereas previous studies have shown that Crk SH2 binding to paxillin is critical for cell adhesion and migration, our data show that the phosphorylation cycle of c-Crk II determines its dynamic interaction with paxillin, thereby regulating turnover of multiprotein complexes, a critical aspect of cytoskeletal plasticity and actin dynamics.

Concentration-dependent positive and negative regulation of a MAP kinase by a MAP kinase kinase.

There are at least three distinct MAP kinase signaling modules in mammalian cells, distinguished by the family of kinases (Erk, SAPK/JNK, or p38) that is ultimately activated. Many input signals activate multiple MAP kinase cascades, and the mechanisms that control the specificity of signal output are not well understood. We show that SEK1/MKK4, a MAP kinase kinase proposed to activate SAPK/JNK, is a very potent inhibitor of p54 SAPK beta/JNK3 both in vitro and in vivo if present at equimolar or higher ratios. In contrast SEK can activate SAPK when present in substoichiometric amounts, but this activation is slow, consistent with the rate-limiting step in activation being the dissociation of an inactive SEK:SAPK complex. The N-terminal unique region of SEK is both necessary and partially sufficient for inhibition of SAPK, and is also necessary for activation of SAPK by SEK in vitro. We have also used the p38 MAP kinase and its activator MKK6 to examine the regulatory relationships among different kinases involved in stress responses. We show using purified kinases that inhibitory activity is specific for the combination of SEK and SAPK: SEK can activate but not inhibit p38, and MKK6 can activate but not inhibit SAPK beta and p38. These results reveal a potential mechanism for regulating stress-activated kinases, adding to a growing body of evidence suggesting that MAP kinases are controlled by relatively stable interactions with their activators.

Engineering temperature-sensitive SH3 domains.

BACKGROUND: The ability to control specific protein-protein interactions conditionally in vivo would be extremely helpful for analyzing protein-protein interaction networks. SH3 (Src homology 3) modular protein binding domains are found in many signaling proteins and they play a crucial role in signal transduction by binding to proline-rich sequences. RESULTS: Random in vitro mutagenesis coupled with yeast two-hybrid screening was used to identify mutations in the second SH3 domain of Nck that render interaction with its ligand temperature sensitive. Four of the mutants were functionally temperature sensitive in mammalian cells, where temperature sensitivity was correlated with a pronounced instability of the mutant domains at the nonpermissive temperature. Two of the mutations affect conserved residues in the hydrophobic core (Val133 and Val160), suggesting a general strategy for engineering temperature-sensitive SH3-containing proteins. Indeed mutagenesis of the corresponding positions in another SH3 domain, that of Crk-1, rendered the full-length Crk-1 protein temperature sensitive for function and stability in mammalian cells. CONCLUSIONS: Construction of temperature-sensitive SH3 domains is a novel approach to regulating the function of SH3 domains in vivo. Such mutants will be valuable in dissecting SH3-mediated signaling pathways. Furthermore, the methodology described here to isolate temperature-sensitive domains should be widely applicable to any domain involved in protein-protein interactions.

Activation of the Abl tyrosine kinase in vivo by Src homology 3 domains from the Src homology 2/Src homology 3 adaptor Nck.

The nonreceptor tyrosine kinase c-Abl is tightly regulated in vivo, but the mechanisms that normally repress its activity are not well understood. We find that a construct encoding the first two Src homology 3 (SH3) domains of the Src homology 2/SH3 adaptor protein Nck can activate c-Abl in human 293T cells. A myristoylated Nck SH3 domain construct, which is expected to localize to membranes, potently activated Abl when expressed at low levels. An unmyristoylated Nck SH3 domain construct, which localizes to the cytosol and nucleus, also activated Abl but only at high levels of expression. Activation by both myristoylated and unmyristoylated Nck constructs required the C terminus of Abl; a C-terminally truncated form of Abl was not activated, although this construct could still be activated by deletion of its SH3 domain. Activation did not require the major binding sites in the Abl C terminus for Nck SH3 domains, however, suggesting that the mechanism of activation does not require direct binding to the C terminus. Activation of c-Abl by Nck SH3 domains provides a robust experimental system for analyzing the mechanisms that normally repress Abl activity and how that normal regulation can be perturbed.

NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling.

Although hepatitis C virus (HCV) infection is an emerging global epidemic causing severe liver disorders, the molecular mechanisms of HCV pathogenesis remain elusive. The NS5A nonstructural protein of HCV contains several proline-rich sequences consistent with Src homology (SH) 3-binding sites found in cellular signaling molecules. Here, we demonstrate that NS5A specifically bound to growth factor receptor-bound protein 2 (Grb2) adaptor protein. Immunoblot analysis of anti-Grb2 immune complexes derived from HeLa S3 cells infected with a recombinant vaccinia virus (VV) expressing NS5A revealed an interaction between NS5A and Grb2 in vivo. An inactivating point mutation in the N-terminal SH3 domain, but not in the C-terminal SH3 domain, of Grb2 displayed significant diminished binding to NS5A. However, the same mutation in both SH3 regions completely abrogated Grb2 binding to NS5A, implying that the two SH3 domains bind in cooperative fashion to NS5A. Further, mutational analysis of NS5A assigned the SH3-binding region to a proline-rich motif that is highly conserved among HCV genotypes. Importantly, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was inhibited in HeLa S3 cells infected with NS5A-expressing recombinant VV but not recombinant VV control. Additionally, HeLa cells stably expressing NS5A were refractory to ERK1/2 phosphorylation induced by exogenous epidermal growth factor. Moreover, the coupling of NS5A to Grb2 in these cells was induced by epidermal growth factor stimulation. Therefore, NS5A may function to perturb Grb2-mediated signaling pathways by selectively targeting the adaptor. These findings highlight a viral interceptor of cellular signaling with potential implications for HCV pathogenesis.

Mechanism of activation of Pak1 kinase by membrane localization.

Pak kinases are a family of serine/threonine protein kinases homologous to Ste20p of yeast. Paks can be activated in vivo and in vitro by binding to GTP-bound Cdc42 and Rac1, members of the Rho family of small GTPases implicated in regulating the organization of the actin cytoskeleton. We have previously reported that the SH2/SH3-containing adaptor protein Nck binds Pak kinase through its second SH3 domain. Pak1 can be targeted to the membrane by Nck in response to tyrosine phosphorylation, and membrane association of Pak1 is sufficient to increase its specific activity. The mechanism whereby Pak is activated by membrane localization, however, is unknown. We show here that expression of three proteins that inhibit Rho-family GTPases by different mechanisms (RhoGDI, Bcr and D57Y Cdc42) all block the activation of Pak by a membrane-targeted Nck SH3 domain, demonstrating that the in vivo activation of Pak1 induced by membrane localization is dependent on Rho-family GTPases. This implies that Pak activity can be regulated in cells both by the level of GTP loading of various Rho-family GTPases and the local concentration of Pak relative to these GTPases. Our data also suggest the existence of Rho-family GTPases in addition to Cdc42 and Rac1 that can activate Pak on membranes.

Endocytosis: EH domains lend a hand.

A number of proteins that have been implicated in endocytosis feature a conserved protein-interaction module known as an EH domain. The three-dimensional structure of an EH domain has recently been solved, and is likely to presage significant advances in understanding molecular mechanisms of endocytosis.

Protein-protein interactions in signaling cascades.

The process of signal transduction is dependent on specific protein-protein interactions. In many cases these interactions are mediated by modular protein domains that confer specific binding activity to the proteins in which they are found. Rapid progress has been made in the biochemical characterization of binding interactions, the identification of binding partners, and determination of the three-dimensional structures of binding modules and their ligands. The resulting information establishes the logical framework for our current understanding of the signal transduction machinery. In this overview a variety of protein interaction modules are discussed, and issues relating to binding specificity and the significance of a particular interaction are considered.