Selective binding of activated pp60c-src by an immobilized synthetic phosphopeptide modeled on the carboxyl terminus of pp60c-src.
Selective binding of activated pp60c-src by an immobilized synthetic phosphopeptide modeled on the carboxyl terminus of pp60c-src.
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以 pp60c-src 羧基末端为模型的固定化合成磷酸肽选择性结合激活的 pp60c-src。
DOI：
10.1073/pnas.88.23.10696
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发表时间：
1991
影响因子：
11.1
通讯作者：
Laudano,AP
中科院分区：
文献类型：
--
作者：
Roussel,RR;Brodeur,SR;Shalloway,D;Laudano,AP
pp 60 c-src是c-src原癌基因的产物，其羧基末端在Tyr-527处的磷酸化抑制其酪氨酸激酶活性和转化潜力。已经提出pp 60 c-src的磷酸化羧基末端通过与SH 2（src同源2）结构域结合来抑制激酶活性。我们已经合成了对应于在Tyr-527处磷酸化和非磷酸化的pp 60 c-src的羧基末端13个残基的肽。高度转化突变体pp 60 c-src（F527），其中Tyr-527突变为Phe，与固定在Affi-Gel 10上的磷酸化肽结合。通过缺失SH 2结构域中的残基144-175而不是通过缺失残基93-143（其去除了大部分SH 3结构域）来消除磷酸化肽的结合。磷酸化的肽也结合到Rous肉瘤病毒的转化蛋白pp 60 v-src。只有痕量的pp 60 v-src和pp 60 c-src（F527）与相应的非磷酸化c-src肽结合。正常pp 60 c-src与磷酸化肽的结合效率远低于pp 60 c-src（F527）。对应于c-fgr蛋白的羧基末端的磷酸化肽也与pp 60 c-src（F527）结合，但具有较弱的亲和力。此外，磷酸化的合成羧基末端pp 60 c-src肽显着抑制磷酸化的pp 60 c-src（F527）在细胞骨架激酶测定。这些结果为pp 60 c-src的磷酸化羧基末端与c-src蛋白的SH 2结构域分子内或分子间结合的模型提供了直接证据。
Phosphorylation of the carboxyl terminus of pp60c-src, the product of the c-src protooncogene, at Tyr-527 suppresses its tyrosine kinase activity and transforming potential. It has been proposed that the phosphorylated carboxyl terminus of pp60c-src inhibits kinase activity by binding to the SH2 (src homology 2) domain. We have synthesized peptides corresponding to the carboxyl-terminal 13 residues of pp60c-src phosphorylated and nonphosphorylated at Tyr-527. A highly transforming mutant, pp60c-src(F527), in which Tyr-527 is mutated to Phe, bound to the phosphorylated peptide immobilized to Affi-Gel 10. Binding of the phosphorylated peptide was abolished by deletion of residues 144-175 in the SH2 domain but not by deletion of residues 93-143, which removes most of the SH3 domain. The phosphorylated peptide also bound to pp60v-src, the transforming protein of Rous sarcoma virus. Only traces of pp60v-src and pp60c-src(F527) bound to the corresponding nonphosphorylated c-src peptide. Normal pp60c-src bound much less efficiently to the phosphorylated peptide than did pp60c-src(F527). A phosphorylated peptide corresponding to the carboxyl terminus of the c-fgr protein also bound to pp60c-src(F527), but with weaker affinity. Furthermore, the phosphorylated synthetic carboxyl-terminal pp60c-src peptide markedly inhibited phosphorylation of pp60c-src(F527) during cytoskeletal kinase assays. These results provide direct evidence for models in which the phosphorylated carboxyl terminus of pp60c-src binds intramolecularly or intermolecularly to the SH2 domain of the c-src protein.