Structural differences between repressed and derepressed forms of p60c-src.

Abstract

The kinase activity of p60c-rc is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c.src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60csrc. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60csrc that change conformation upon kinase activation and suggest functions for the amino-terminal region.

Conformational Changes Induced in the Protein Tyrosine Kinase p72syk by Tyrosine Phosphorylation or by Binding of Phosphorylated Immunoreceptor Tyrosine-Based Activation Motif Peptides

“…6). There were minimal effects with the monophosphorylated synthetic ␥ ITAM pep- tivity to proteolytic cleavage (35). Therefore, we tested the effect of the conformation of Syk on its sensitivity to proteolytic digestion.…”

“…Partial proteolysis was as previously described (35). Briefly, cell lysates immunoprecipitated with anti-SykI antibody were washed three times with wash buffer without protease inhibitors and then once with protease buffer (1 mM CaCl 2 , 0.5 mM Na 3 VO 4 , 10 mM Tris [pH 8.1]).…”

Identification of two juxtamembrane autophosphorylation sites in the PDGF beta-receptor; involvement in the interaction with Src family tyrosine kinases.

“…8. Comparison of the amino acid sequences surrounding the juxtamembrane tyrosine residues of the human PDGF a-and (3-receptors (PDGF-aR and PDGF-,BR), the human CSF-1R and the human Kit, and surrounding the carboxy-terminal tyrosine residue of the three Src family kinases, chicken Src (Takeya and Hanafusa, 1983), human Fyn (Semba et al, 1986) and human Yes (Sukegawa et al, 1987 (Cooper, 1990); several lines of evidence (Koch et al, 1989;MacAuley and Cooper, 1989;Matsuda et al, 1990) If the SH2 domain of the Src family kinases can interact with both their own carboxy-terminal tail (Roussel et al, 1991) and the juxtamembrane segment of the PDGF flreceptor, one would expect a similarity in the amino acid sequences in these regions. As shown in Figure 8, such a similarity is found, though it is weak: the consensus is Y(P)XXXD/E where X is not a charged amino acid residue.…”

c-Src enhances the spreading of src-/- fibroblasts on fibronectin by a kinase-independent mechanism.

“…Autophosphorylation at tyrosine 416 IY416) is required for full kinase activity (Piwnica-Worms et al 1987;Kmiecik et al 1988), and phosphorylation at tyrosine 527 (Y527) inhibits activity (Courtneidge 1985;Kmiecik and Shalloway 1987). Normally, Y527 is phosphorylated extensively in the cell by the carboxy-terminal Src kinase (CSK) (Imamoto and Soriano 1993), resulting in an intramolecular interaction between phospho-Y527 and the SH2 domain ("closed" conformation) thought to inhibit kinase activity (MacAuley and Cooper 1989). Deleting Y527 (as in v-Src), mutating it to a phenylalanine (Y527F), and presumably dephosphorylating Y527 in vivo disrupts this interaction and results in a highly active kinase with exposed amino terminal domains ("open" conformation) (Roussel et al 1991;Liu et al 1993) that now may interact with target proteins.…”