Direct demonstration of an intramolecular SH2-phosphotyrosine interaction in the Crk protein
Protein Engineering Network Centres of Excellence, University of Toronto, Ontario, Canada. Nature
(Impact Factor: 41.46).
04/1995; 374(6521):477-9. DOI: 10.1038/374477a0
Many signal transduction processes are mediated by the binding of Scr-homology-2 (SH2) domains to phosphotyrosine (pTyr)-containing proteins. Although most SH2-pTyr interactions occur between two different types of molecules, some appear to involve only a single molecular type. It has been proposed that the enzymatic activity and substrate recognition of the Src-family kinases, and the protein-binding and transforming activity of Crk-family adaptor proteins, are regulated by intramolecular SH2-pTyr interactions. In addition, the DNA-binding activity of Stat transcription factors seems to be regulated by SH2-mediated homodimerization. Here we examine the phosphorylated and non-phosphorylated forms of murine Crk II (p-mCrk and mCrk, respectively) using a combination of physical techniques. The Crk protein contains a single SH2 domain and two SH3 domains in the order SH2-SH3-SH3. There is a tyrosine-phosphorylation site between the two SH3 domains at residue 221 which is phosphorylated in vivo by the Abl tyrosine kinase. Using NMR spectroscopic analysis, we show here that the SH2 domain of purified p-mCrk is bound to pTyr, and by hydrodynamic measurements that the phosphorylated protein is monomeric. These results provide direct demonstration of an intramolecular SH2-pTyr interaction in a signalling molecule.
Available from: Masahiro Maruoka
- "The initial and an essential event is the interaction between the Crk SH3 (N) domain and three of the four PR motifs of Abl, inducing transactivation of c-Abl to phosphorylate CrkII at Y221 in the spacer region [98, 99]. This phosphorylation induces an intramolecular SH2-pTyr clamp and suppression of the c-Abl kinase activity as well, resulting in the disassembly of Crk-mediated signaling complexes and abrogates downstream signaling . In contrast, v-Crk lacks Y221 and hence is presumed to take an open structure constitutively . "
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ABSTRACT: Abl is a nonreceptor tyrosine kinase and plays an essential role in the modeling and remodeling of F-actin by transducing extracellular signals. Abl and its paralog, Arg, are unique among the tyrosine kinase family in that they contain an unusual extended C-terminal half consisting of multiple functional domains. This structural characteristic may underlie the role of Abl as a mediator of upstream signals to downstream signaling machineries involved in actin dynamics. Indeed, a group of SH3-containing accessory proteins, or adaptor proteins, have been identified that bind to a proline-rich domain of the C-terminal portion of Abl and modulate its kinase activity, substrate recognition, and intracellular localization. Moreover, the existence of signaling cascade and biological outcomes unique to each adaptor protein has been demonstrated. In this paper, we summarize functional roles and mechanisms of adaptor proteins in Abl-regulated actin dynamics, mainly focusing on a family of adaptor proteins, Abi. The mechanism of Abl's activation and downstream signaling mediated by Abi is described in comparison with those by another adaptor protein, Crk.
Available from: Tamjeed Saleh
- "The ability of Crk to function as an adaptor protein is negatively regulated and terminated by phosphorylation on Y221, which results in an intramolecular SH2-pTyr clamp, thereby resulting in the disassembly of Crk-mediated signaling complexes (Feller et al., 1994; Rosen et al., 1995; Kobashigawa et al., 2007). Here, we show that in addition to Y221, another tyrosine residue, namely Y251, located within the highly conserved RT loop of the SH3C domain of Crk is also concomitantly phosphorylated by the Abl and Bcr-Abl tyrosine kinases. "
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ABSTRACT: Here, we report the identification and characterization of a novel tyrosine phosphorylation site in the carboxy-terminal Src Homology 3 (SH3) (SH3C) domain of the Crk adaptor protein. Y251 is located in the highly conserved RT loop structure of the SH3C, a region of Crk involved in the allosteric regulation of the Abl kinase. Exploiting kinase assays to show that Y251 is phosphorylated by Abl in vitro, we generated affinity-purified antisera against phosphorylated Y251 in Crk and showed that Abl induces phosphorylation at Y251 in vivo, and that the kinetics of phosphorylation at Y251 and the negative regulatory Y221 site in vitro are similar. Y251 on endogenous Crk was robustly phosphorylated in chronic myelogenous leukemia cell lines and in A431 and MDA-MB-468 cells stimulated with epidermal growth factor. Using streptavidin-biotin pull downs and unbiased high-throughput Src Homology 2 (SH2) profiling approaches, we found that a pY251 phosphopeptide binds specifically to a subset of SH2 domains, including Abl and Arg SH2, and that binding of pY251 to Abl SH2 induces transactivation of Abl 1b. Finally, the Y251F Crk mutant significantly abrogates Abl transactivation in vitro and in vivo. These studies point to a yet unrealized positive regulatory role resulting from tyrosine phosphorylation of Crk, and identify a novel mechanism by which an adaptor protein activates a non-receptor tyrosine kinase by SH2 domain displacement.
Available from: Tamar Geiger
- "These changes are attributable to the enzymatic activity of pp60 src , since the addition of an inhibitor that blocks the kinase activity restores the original fibroblastic morphology (Fig. 2). Similarly , tyrosine phosphorylation of the adaptor protein CRK by Abl , locks the molecule in a folded structure, while its dephosphorylation by a protein tyrosine phosphatase, PTPN1, opens up CRK, rendering its SH2 domain available for binding to one of several phospho-proteins (e.g., paxillin, cbl, shc), and thus affecting the adhesome network. "
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ABSTRACT: Over the years, malignant transformation has been investigated on multiple levels, ranging from clinical pathology to the underlying molecular mechanisms. In "zooming in" on this process, cancer biologists have focused their attention on the molecular and cellular manifestations of the "transformed phenotype", including the genomic instability of cancer cells, their deregulated transcriptional activity, their aberrant morphology and dynamics, and the altered signaling networks activated in them. Attempts to elucidate the mechanisms underlying malignant and metastatic transformation are primarily motivated by the desire to identify specific molecules and signaling pathways that can serve as targets for novel therapies. In recent years, such studies were reinforced by major technological and conceptual developments: novel and powerful tools for genomic and proteomic analysis have been developed, and advanced computational approaches offer "systems-level" integration of rich and complex biological datasets into meaningful functional networks. In this article, we consider the current and potential impact of these new experimental approaches and, in particular, the recent progress made in quantitative proteomics, to elucidate the mechanisms underlying the "transformed phenotype". We will primarily focus on the adhesion and migration of cancer cells, and their relationships to the deregulated growth, metastatic dissemination, and anchorage independence associated with malignant transformation.
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