SH2 and SH3 domains as molecular adhesives: the interactions of Crk and Abl.

The Src homology domains SH2 and SH3 are modular components present in many signal transduction proteins. They allow rapid formation of stable protein complexes and may also regulate protein function through intramolecular binding events. SH2 domains recognize phosphotyrosyl residues in a specific sequence context, while SH3 domains recognize a PxxP motif and additional residues that mediate binding specificity.

Phosphorylation of Helicobacter pylori CagA by c-Abl leads to cell motility.

Helicobacter pylori induces a strong motogenic response in infected gastric epithelial host cells, which is enhanced by translocation of the pathogenic factor cytotoxin-associated gene A (CagA) into host cells via a specialized type IV secretion system. Once injected into the cytosol CagA is rapidly tyrosine phosphorylated by Src family kinases followed by Src inactivation. Hence, it remained unknown why CagA is constantly phosphorylated in sustained H. pylori infections to induce cell migration, whereas other substrates of Src kinases are dephosphorylated. Here, we identify the non-receptor tyrosine kinase c-Abl as a crucial mediator of H. pylori-induced migration and novel CagA kinase in epithelial cells. Upon H. pylori infection c-Abl directly interacts with CagA and localizes in focal adhesion complexes and membrane ruffles, which are highly dynamic cytoskeletal structures necessary for cell motility. Selective inhibition of c-Abl kinase activity by STI571 or shRNA abrogates sustained CagA phosphorylation and epithelial cell migration, indicating a pivotal role of c-Abl in H. pylori infection and pathogenicity. These results implicate c-Abl as a novel molecular target for therapeutic intervention in H. pylori-related gastric diseases.

Interaction of hematopoietic progenitor kinase 1 with adapter proteins Crk and CrkL leads to synergistic activation of c-Jun N-terminal kinase.

Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related protein kinase, is an upstream activator of c-Jun N-terminal kinase (JNK). In order to further characterize the HPK1-mediated JNK signaling cascade, we searched for HPK1-interacting proteins that could regulate HPK1. We found that HPK1 interacted with Crk and CrkL adaptor proteins in vitro and in vivo and that the proline-rich motifs within HPK1 were involved in the differential interaction of HPK1 with the Crk proteins and Grb2. Crk and CrkL not only activated HPK1 but also synergized with HPK1 in the activation of JNK. The HPK1 mutant (HPK1-PR), which encodes the proline-rich region alone, blocked JNK activation by Crk and CrkL. Dominant-negative mutants of HPK1 downstream effectors, including MEKK1, TAK1, and SEK1, also inhibited Crk-induced JNK activation. These results suggest that the Crk proteins serve as upstream regulators of HPK1. We further observed that the HPK1 mutant HPK1-KD(M46), which encodes the kinase domain with a point mutation at lysine-46, and HPK1-PR blocked interleukin-2 (IL-2) induction in Jurkat T cells, suggesting that HPK1 signaling plays a critical role in IL-2 induction. Interestingly, HPK1 phosphorylated Crk and CrkL, mainly on serine and threonine residues in vitro. Taken together, our findings demonstrate the functional interaction of HPK1 with Crk and CrkL, reveal the downstream pathways of Crk- and CrkL-induced JNK activation, and highlight a potential role of HPK1 in T-cell activation.

Structural basis for the specific interaction of lysine-containing proline-rich peptides with the N-terminal SH3 domain of c-Crk.

BACKGROUND: Proline-rich segments in the guanine nucleotide exchange factor C3G bind much more strongly to the N-terminal Src homology 3 domain (SH3-N) of the proto-oncogene product c-Crk than to other SH3 domains. The presence of a lysine instead of an arginine in the peptides derived from C3G appears to be crucial for this specificity towards c-Crk. RESULTS: In order to understand the chemical basis of this specificity we have determined the crystal structure of Crk SH3-N in complex with a high affinity peptide from C3G (PPPALPPKKR, Kd approximately 2 microM) at 1.5 A resolution. The peptide adopts a polyproline type II helix that binds, as dictated by electrostatic complementarity, in reversed orientation relative to the orientation seen in the earliest structures of SH3-peptide complexes. A lysine in the C3G peptide is tightly coordinated by three acidic residues in the SH3 domain. In contrast, the co-crystal structure of c-Crk SH3-N and a peptide containing an arginine at the equivalent position (determined at 1.9 A resolution) reveals non-optimal geometry for the arginine and increased disorder. CONCLUSIONS: The c-Crk SH3 domain engages in an unusual lysine-specific interaction that is rarely seen in protein structures, and which appears to be a key determinant of its unique ability to bind the C3G peptides with high affinity.

Crk family adaptors-signalling complex formation and biological roles.

Crk family adaptors are widely expressed and mediate the timely formation of signal transduction protein complexes upon a variety of extracellular stimuli, including various growth and differentiation factors. Selective formation of multi-protein complexes by the Crk and Crk-like (CRKL) proteins depends on specific motifs recognized by their SH2 and SH3 domains. In the case of the first SH3 domains [SH3(1)] a P-x-x-P-x-K motif is crucial for highly selective binding, while the SH2 domains prefer motifs which conform to the consensus pY-x-x-P. Crk family proteins are involved in the relocalization and activation of several different effector proteins which include guanine nucleotide releasing proteins like C3G, protein kinases of the Abl- and GCK-families and small GTPases like Rap1 and Rac. Crk-type proteins have been found not only in vertebrates but also in flies and nematodes. Major insight into the function of Crk within organisms came from the genetic model organism C. elegans, where the Crk-homologue CED-2 regulates cell engulfment and phagocytosis. Other biological outcomes of the Crk-activated signal transduction cascades include the modulation of cell adhesion, cell migration and immune cell responses. Crk family adaptors also appear to play a role in mediating the action of human oncogenes like the leukaemia-inducing Bcr-Abl protein. This review summarizes some key findings and highlights recent insights and open questions.

The Crk signaling pathway contributes to the bombesin-induced activation of the small GTPase Rap1 in Swiss 3T3 cells.

Rap1 is a small GTPase implicated in cell proliferation and differentiation. The mechanisms how endogenous Rap1 is activated by many mitogenic stimuli including the neuropeptide bombesin remained unclear. Here we analyse which signaling pathways are necessary for Rap1 activation. Bombesin-mediated Rap1 activation in Swiss 3T3 and primary mouse embryo fibroblasts requires signaling components similar to those being essential for complex formation between p130Cas and Crk adapter proteins. The Crk/CRKL-binding region of the Rap1-specific exchange factor C3G (CBR) inhibits the bombesin-stimulated Rap1 activity in transfected Swiss 3T3 cells. Further characterization in COS cells showed that the CBR or a c-Crk I SH3 mutant specifically reduces both the basal as well as the stimulated Rap1 activity in a dose-dependent manner, whereas Ras is not affected. The CBR is complexed with endogenous c-Crk II and CRKL and blocks the protein association with catalytically active C3G. Such suppressors of Crk signaling do not affect Erk-phosphorylation induced by bombesin. Embryonic fibroblasts from b-raf knockout mice showed a bombesin-inducible Erk-phosphorylation, providing evidence that B-Raf does not link Rap1 to Erk-activation in bombesin-stimulated fibroblasts. We conclude that cellular Crk/CRKL complexes, recruited to upstream signaling components, contribute to basal and bombesin-induced Rap1 activity, which is independent from the Ras-Raf-Erk pathway under these circumstances.

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.

Proline-rich sequences mediate the interaction of the Arg protein tyrosine kinase with Crk.

Arg is a ubiquitously expressed member of the Abelson family of nonreceptor protein-tyrosine kinases. Defining the Arg sequences that mediate its interaction with other proteins is essential to elucidating its role in cellular signaling. In this report we demonstrate that Arg associates with c-Crk, an adaptor protein composed of an SH2 domain and two SH3 domains, and examine the molecular mechanism of the interaction. In vitro experiments revealed that three proline-rich sequences with distinct specificities for SH3 domains are located in the Arg C-terminal domain, just C-terminal to the kinase domain, and that two of these sequences bind to the Crk N-terminal SH3 domain. These two sequences conform to the PxLPxK/R motif that has been observed in other proteins that bind the Crk N-terminal SH3 domain. The interaction of Arg with c-Crk in living cells was confirmed by the detection of coimmunoprecipitation in coinfected Sf9 cells. In addition, increased phosphorylation of c-Crk was observed in cotransfected COS cells, indicating that Crk is an Arg substrate. The site of c-Crk phosphorylation by Arg was identified as tyrosine 221, a residue whose modification has been shown to result in an intramolecular SH2 interaction and a folded conformation. These experiments extend the known Arg protein interacting motifs to include SH3 binding sites and suggest that Arg may function as an effector as well as a regulator of Crk activity.

The germinal center kinase (GCK)-related protein kinases HPK1 and KHS are candidates for highly selective signal transducers of Crk family adapter proteins.

Adapter proteins function by mediating the rapid and specific assembly of multi-protein complexes during the signal transduction which guards proliferation, differentiation and many functions of higher eukaryotic cells. To understand their functional roles in different cells it is important to identify the selectively interacting proteins in these cells. Two novel candidates for signalling partners of Crk family adapter proteins, the hematopoietic progenitor kinase 1 (HPK1) and the kinase homologous to SPS1/STE20 (KHS), were found to bind with great selectivity to the first SH3 domains of c-Crk and CRKL. While KHS bound exclusively to Crk family proteins, HPK1 also interacted with both SH3 domains of Grb2 and weakly with Nck, but not with more than 25 other SH3 domains tested. The interaction of HPK1 with c-Crk and CRKL was studied in more detail. HPK1-binding to the first SH3 domain of CRKL is direct and occurs via proline-rich motifs in the C-terminal, non-catalytic portion of HPK1. In vitro complexes were highly stable and in vivo complexes of c-Crk and CRKL with HPK1 were detectable by co-immunoprecipitation with transiently transfected cells but also with endogenous proteins. Furthermore, c-Crk II and, to a lesser extent, CRKL were substrates for HPK1. These results make it likely that HPK1 and KHS participate in the signal transduction of Crk family adapter proteins in certain cell types.

The C-terminal SH3 domain of the adapter protein Grb2 binds with high affinity to sequences in Gab1 and SLP-76 which lack the SH3-typical P-x-x-P core motif.

The adapter Grb2 is an important mediator of normal cell proliferation and oncogenic signal transduction events. It consists of a central SH2 domain flanked by two SH3 domains. While the binding specificities of the Grb2 SH2 and N-terminal SH3 domain [Grb2 SH3(N)] have been studied in detail, binding properties of the Grb2 SH3(C) domain remained poorly defined. Gab1, a receptor tyrosine kinase substrate which associates with Grb2 and the c-Met receptor, was previously shown to bind Grb2 via a region which lacks a Grb2 SH3(N)-typical motif (P-x-x-P-x-R). Precipitation experiments with the domains of Grb2 show now that Gab1 can bind stably to the Grb2 SH3(C) domain. For further analyses, Gab1 mutants were generated by PCR to test in vivo residues thought to be crucial for Grb2 SH3(C) binding. The Grb2 SH3(C) binding region of Gab1 has significant homology to a region of the adapter protein SLP-76. Peptides corresponding to epitopes SLP-76, Gab1, SoS and other proteins with related sequences, as well as mutant peptides were synthesized and analysed by tryptophan-fluorescence spectrometry and by in vitro competition experiments. These experiments define a 13 amino acid sequence with the unusual consensus motif P-x-x-x-R-x-x-K-P as required for a stable binding to the SH3(C) domain of Grb2. Additional analyses point to a distinct binding specificity of the Grb2-homologous adapter protein Mona (Gads), indicating that the proteins of the Grb2 adapter family may have partially overlapping, yet distinct protein binding properties.

The leukaemic oncoproteins Bcr-Abl and Tel-Abl (ETV6/Abl) have altered substrate preferences and activate similar intracellular signalling pathways.

Inappropriate activation of Abl family kinases plays a crucial role in different human leukaemias. In addition to the well known oncoproteins p190Bcr-Abl and p210Bcr-Abl, Tel-Abl, a novel fusion protein resulting from a different chromosomal translocation, has recently been described. In this study, the kinase specificities of the Bcr-Abl and Tel-Abl proteins were compared to the physiological Abl family kinases c-Abl and Arg (abl related gene). Using short peptides which correspond to the target epitopes in known substrate proteins of Abl family kinases, we found a higher catalytic promiscuity of Bcr-Abl and Tel-Abl. Similar to Bcr-Abl, Tel-Abl was found in complexes with the adapter protein CRKL. In addition, c-Crk II and CRKL are tyrosine phosphorylated and complexed with numerous other tyrosine phosphorylated proteins in Tel-Abl expressing Ba/F3 cells. GTPase analysis with a Ras-GTP-specific precipitation assay showed constitutive elevation of GTP-loaded Ras in cells expressing the leukaemic Abl proteins. The mitogenic MAPK/Erk kinases as well as Akt/PKB, a kinase implicated to negatively regulate apoptosis, were also constitutively activated by both Bcr-Abl and Tel-Abl. The results indicate that the leukaemic Abl-fusion proteins have catalytic specificities different from the normal kinases c-Abl and Arg and that Tel-Abl is capable to activate at least some pathways which are also upregulated by Bcr-Abl.

Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G.

Many Src Homology 3 (SH3) domains function as molecular adhesives in intracellular signal transduction. Based on previous ultrastructural studies, short motifs which bind to the first SH3 domains of the adapters Crk and CRKL were selectively mutagenised to generate Crk/CRKL SH3-binding peptides of very high affinity and selectivity. Affinities were increased up to 20-fold compared to the best wildtype sequences, while the selectivity against a similar SH3 domain [Grb2SH3(N)] was not only retained, but sometimes increased. Blot techniques with GST-fusion peptides and in solution precipitation assays with biotinylated high affinity Crk binding peptides (HACBPs) were subsequently used to analyse the binding of these sequences to a large panel of SH3 domain-containing fusion proteins. Only those proteins which contained the CrkSH3(1) or CRKLSH3(1) domains bound efficiently to the HACBPs. A GST-HACBP fusion protein precipitated Crk and CRKL proteins out of 35S-labelled and unlabelled cell lysates. Very little binding of other cellular proteins to HACBP was detectable, indicative of a great preference for Crk and CRKL when compared to the wide variety of other endogenous cellular proteins. Moreover, HACBP disrupted in vitro preexisting Crk-complexes with DOCK180 and the exchange factors SoS and C3G, which are known targets of Crk adapters, in a concentration dependent manner. HACBP-based molecules should therefore be useful as highly selective inhibitors of intracellular signalling processes involving Crk and CRKL.

Multiple interactions of the cytosolic polyproline region of the CD95 ligand: hints for the reverse signal transduction capacity of a death factor.

The CD95/Fas/Apo-1 ligand is expressed on activated lymphocytes, NK cells, platelets, certain immune-privileged cells and some tumor cells and induces apoptosis through the death receptor CD95/Fas/Apo-1. In murine T cells, membrane-bound CD95L (Fas ligand) also acts as a costimulatory receptor to coordinate activation and function in vivo. The molecular basis for this reverse signal transduction is yet unknown. In the present report, we identify individual interaction domains of enzymes and adapter molecules that selectively interact with full-length CD95L from transfectants and human T cells. These results may help to explain the costimulatory capacity of CD95L.

Identification and characterization of a cytosolic protein tyrosine kinase of HeLa cells.

Fractionation of a cytosolic extract of HeLa cells revealed the existence of a highly active protein tyrosine kinase. Chromatographic fractionation of the extract resulted in partial purification of a single enzymatic activity that coeluted with a 94-kDa polypeptide. In vitro phosphorylation of the isolated enzyme showed that p94 was the only polypeptide phosphorylated and only the tyrosine residue(s) was (were) modified. The fractionated enzyme (p94 kinase) also phosphorylated a number of other nonspecific substrates exclusively on tyrosine residues. Unlike other protein tyrosine kinases that have been characterized, p94 kinase is relatively insensitive to inhibition by the isoflavone genistein. Using two different antisera, we provided evidence that the HeLa p94 kinase is most likely the FER gene product, which was previously shown to be expressed in a wide variety of cell types. These results represent the first biochemical characterization of the cellular FER gene product and also provide a basis for studying the biochemistry of tyrosine kinase function in HeLa cells.

c-Abl kinase regulates the protein binding activity of c-Crk.

c-Crk is a proto-oncogene product composed largely of Src homology (SH) 2 and 3 domains. We have identified a kinase activity, which binds to the first Crk SH3 domain and phosphorylates c-Crk on tyrosine 221 (Y221), as c-Abl. c-Abl has a strong preference for c-Crk, when compared with common tyrosine kinase substrates. The phosphorylation of c-Crk Y221 creates a binding site for the Crk SH2 domain. Bacterially expressed c-Crk protein lacks phosphorylation on Y221 and can bind specifically to several proteins, while mammalian c-Crk, which is phosphorylated on tyrosine, remains uncomplexed. The protein binding activity of c-Crk is therefore likely regulated by a mechanism similar to that of the Src family kinases. v-Crk is truncated before c-Crk Y221 and forms constitutive complexes with c-Abl and other proteins. Our results suggest that c-Abl regulates c-Crk function and that it could be involved in v-Crk transformation.

Rapid lamellipodia formation in nerve growth factor-stimulated PC12 cells is dependent on Rac and PI3K activity.

Neuronal differentiation of PC12 cells is achieved by stimulation with nerve growth factor (NGF) but not by epidermal growth factor (EGF). However, features of differentiation such as neurite outgrowth are observable at the earliest after several hours. Using actin staining of the cells, we show here that NGF stimulation leads to lamellipodia formation within only 3 min at the periphery of the PC12 cells. EGF stimulation or microinjection of differentiation-inducing c-Crk I protein does not cause lamellipodia. The actin reorganization after NGF stimulation is blocked by microinjecting dominant negative Rac protein. The lamellipodia formation is also abolished by inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY 294002 in a concentration-dependent manner. Phase-contrast time-lapse microscopy was used to analyze membrane dynamics in real time and to confirm the induction of lamellipodia by NGF and their inhibition by pretreatment with both wortmannin and LY 294002. The results indicate that NGF, but not EGF, leads to rapid lamellipodia formation in PC12 cells via phosphatidylinositol 3-kinase and the small GTPase Rac, thereby defining a novel role for these factors in early NGF signaling.

Four proline-rich sequences of the guanine-nucleotide exchange factor C3G bind with unique specificity to the first Src homology 3 domain of Crk.

The widely expressed cellular Crk protein has the domain structure SH2-SH3-SH3. We have previously demonstrated that the more N-terminal SH3 domain of Crk (CrkSH3(N)) specifically binds several cytoplasmic proteins. A cDNA encoding one of these proteins was isolated and found to have two different splice forms. The sequence is virtually identical to C3G, a guanine-nucleotide exchange factor. The center region of the 145-155-kDa protein contains four similar proline-rich sequences which are capable of binding individually to the SH3(N) domains of c-Crk and v-Crk. Comparison of these sequences in C3G to proline-rich sequences in other Crk-binding proteins suggests that positively charged amino acids following the prolines play an important role in the binding to the CrkSH3(N) domain. The endogenous C3G could be coprecipitated with Crk from cell lysates of cells expressing high levels of c-Crk or v-Crk, suggesting high binding affinity and a possible interaction in vivo. Unlike many other SH3-binding proteins which interact with multiple SH3 domains, C3G from cell lysates binds preferentially to the CrkSH3(N) domain. This unique binding specificity supports the idea that C3G plays an important role in Crk signaling pathways.