Direct demonstration of an intramolecular SH2-phosphotyrosine interaction in the Crk protein

ArticleinNature 374(6521):477-9 · April 1995with3 Reads
Impact Factor: 41.46 · DOI: 10.1038/374477a0 · Source: PubMed

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.

    • "The two SH3 domains are connected via a linker region containing a single, highly conserved, protein conformation-regulating tyrosine residue, plus several proline residues [9,10]. Phosphorylation of the tyrosine residue (Tyr 211 and Tyr 207 in human CrkII and CrkL, respectively) by Abl, or other protein tyrosine kinases (PTKs), promotes intramolecular binding of the linker region to the self-SH2, thereby sequestering the SH2 and SH3N domains [10,11]. Recent in vitro studies demonstrated that the conformation of a GlyePro motif within the CrkII linker region is under the control of peptidyl-prolyl cis-trans isomerases (PPIases) [12e15]. "
    [Show abstract] [Hide abstract] ABSTRACT: Members of the Crk family of adaptor proteins are key players in signal transduction from a variety of cell surface receptors. CrkI and CrkII are two alternative-spliced forms of a single gene which possess an N-terminal SH2 domain and an SH3 domain that mediate interaction with other proteins. CrkII possesses an additional C-terminal linker region plus an extra SH3 domain, which does not interact with other proteins, but operates as regulatory moiety. Utilizing human Jurkat T cells, we demonstrate that CrkII-SH3N binding of C3G is inhibited by cyclosporin A (CsA) plus FK506 that inhibit the cyclophilin A (CypA) and FK506 binding protein (FKBP) peptidyl-prolyl cis-trans isomerases (PPIases; also termed immunophilins), respectively. Jurkat T cells were found to express ∼5-fold lower levels of CrkI protein compared to CrkII, but the efficiency of C3G binding by CrkI was ∼5-fold higher than that of CrkII, suggesting that the majority of cellular CrkII proteins adopt a conformation that is inaccessible for C3G. Treatment of Jurkat T cells with CsA plus FK506 led to a time-dependent conformational change in overexpressed human CrkII1-236 protein-containing FRET-based biosensor, supporting the accumulation of cis conformers of human CrkII1-236 in the presence of PPIase inhibitors. Our data suggest that the Gly219-Pro-Tyr motif in the human CrkII linker region serves as the recognition and isomerization site of PPIases, and raise the possibility that CsA and FK506 might interfere with selected effector T cell functions via a CrkII-, but not CrkI-dependent mechanisms.
    Full-text · Article · Jan 2016 · Biochemical and Biophysical Research Communications
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    • "This conformation is induced by active Abl kinase, which phosphorylates Crk at Y221 in the SH3 linker region (Feller et al. 1994 ). The SH2 domain of Crk then binds to this pTyr and in doing so prevents its binding to intermolecular targets (Feller et al. 1994; Rosen et al. 1995). "
    [Show abstract] [Hide abstract] ABSTRACT: Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events.
    Full-text · Article · Dec 2013 · Cold Spring Harbor perspectives in biology
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    • "While there are several plausible explanations for this, one interesting possibility is that there may be energetic strain associated with achieving conformational closure in the intramolecular case of 4p-PTEN not associated with a phospho-tail–t-PTEN intermolecular interaction. Long-range intramolecular protein conformational switching induced by phosphorylation has been observed in several well-established cases including CrkL, Src, and SHP-1/2 (Lu et al., 2001; Rosen et al., 1995; Sicheri et al., 1997; Xu et al., 1997; Zhang et al., 2003). Each of these examples involves an SH2 domain interacting with a phosphotyrosine. "
    [Show abstract] [Hide abstract] ABSTRACT: eLife digest PTEN is an enzyme that is found in almost every tissue in the body, and its job is to stop cells dividing. If it fails to perform this job, the uncontrolled proliferation of cells can lead to the growth of tumors. PTEN stops cells dividing by localizing at the plasma membrane of a cell and removing a phosphate group from a lipid called PIP3: this sends a signal, via the PI3K pathway, that suppresses the replication and survival of cells. Three regions of PTEN are thought to be central to its biological functions: one of these regions, the phosphatase domain, is directly responsible for removing a phosphate group from the lipid PIP3; a second region, called the C2 domain, is known to be critical for PTEN binding to the cell membrane; however, the role of third region, called the C-terminal domain, is poorly understood. Many proteins are regulated by the addition and removal of phosphate groups, and PTEN is no exception. In particular, it seems as if the addition of phosphate groups to four amino acid residues in the C-terminal domain can switch off the activity of PTEN, but the details of this process have been elusive. Now, Bolduc et al. have employed a variety of biochemical and biophysical techniques to explore this process, finding that the addition of the phosphate groups reduced PTEN’s affinity for the plasma membrane. At the same time, interactions between the C-terminal and C2 domains of the PTEN cause the shape of the enzyme to change in a way that ‘buries’ the residues to which the phosphate groups have been added. In addition to offering new insights into PTEN, the work of Bolduc et al. could help efforts to identify compounds with clinical anti-cancer potential. DOI:
    Full-text · Article · Jul 2013 · eLife Sciences
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