Reciprocal regulation of Hck activity by phosphorylation of Tyr527 and Tyr416 -: Effect of introducing a high affinity intramolecular SH2 ligand
Reciprocal regulation of Hck activity by phosphorylation of Tyr527 and Tyr416 -: Effect of introducing a high affinity intramolecular SH2 ligand
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DOI：
10.1074/jbc.275.4.2721
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发表时间：
2000-01-28
影响因子：
4.8
通讯作者：
Miller, WT
中科院分区：
文献类型：
--
作者：
Porter, M;Schindler, T;Miller, WT
Src家族酪氨酸激酶Hck具有两个磷酸化位点，Tyr（527）和Tyr(416)，它们以相反的方式影响催化活性。当被磷酸化时，Tyr（527）及其c末端残基参与了与SH2结构域的抑制分子内相互作用。然而，该序列不符合高亲和力SH2配体pYEEI的序列，我们将该序列突变为YEEI，并表明这种突变形式的Hck不能被外源SH2配体激活。Hck的SH3结构域也参与了与催化结构域的抑制相互作用。SH3配体Nef以与野生型Hck相似的方式结合并激活yei -Hck突变体，表明破坏SH3相互作用会覆盖加强的SH2相互作用。另一个磷酸化位点Tyr（416）是激活环中的自磷酸化位点。Tyr（416）的磷酸化是Hck激活所必需的。我们将该残基突变为丙氨酸，并对其催化活性进行了表征。与自磷酸化野生型Hck相比，Y416A突变体表现出更高的肽K-m值和更低的V-max。我们还提供了激活环与SH2和SH3结构域分子内结合之间的串扰的证据。
The Src family tyrosine kinase Hck possesses two phosphorylation sites,Tyr(527) and Tyr(416), that affect the catalytic activity in opposite ways. When phosphorylated, Tyr(527) and residues C-terminal to it are involved in an inhibitory intramolecular interaction with the SH2 domain. However, this sequence does not conform to the sequence of the high affinity SH2 ligand, pYEEI, We mutated this sequence to YEEI and show that this mutant form of Hck cannot be activated by exogenous SH2 ligands. The SH3 domain of Hck is also involved in an inhibitory interaction with the catalytic domain. The SH3 ligand Nef binds to and activates YEEI-Hck mutant in a similar manner to wild-type Hck, indicating that disrupting the SH3 interaction overrides the strengthened SH2 interaction. The other phosphorylation site, Tyr(416), is the autophosphorylation site in the activation loop. Phosphorylation of Tyr(416) is required for Hck activation. We mutated this residue to alanine and characterized its catalytic activity. The Y416A mutant shows a higher K-m value for peptide and a lower V-max than autophosphorylated wild-type Hck. We also present evidence for cross-talk between the activation loop and the intramolecular binding of the SH2 and SH3 domains.