INTERKINASE DOMAIN OF KIT CONTAINS THE BINDING-SITE FOR PHOSPHATIDYLINOSITOL 3' KINASE
INTERKINASE DOMAIN OF KIT CONTAINS THE BINDING-SITE FOR PHOSPHATIDYLINOSITOL 3' KINASE
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DOI：
10.1073/pnas.89.2.678
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发表时间：
1992-01-15
影响因子：
11.1
通讯作者：
YARDEN, Y
中科院分区：
文献类型：
--
作者：
LEV, S;GIVOL, D;YARDEN, Y
我们先前对c-kit编码的干细胞因子（SCF）受体所使用的信号转导途径的分析表明与I型磷脂酰肌醇3'激酶（PI 3 K）的有效偶联。在试图本地化的受体的网站与PI 3 K的相互作用，我们分别删除非催化68个氨基酸长的interkinase结构域或远端的催化序列的羧基末端部分。配体诱导的PI 3 K与前缺失突变体的关联的损失和由羧基末端缺失的受体保留的PI 3 K关联暗示PI 3 K与激酶插入物的相互作用。通过针对激酶插入物的抗肽抗体对缔合的部分抑制和针对SCF受体的羧基尾的抗体的作用的缺乏进一步支持了这一点。使用细菌表达的激酶插入结构域作为融合蛋白，以直接测试其作为PI 3 K缔合位点的假定功能。PI 3 K活性和相关的85 kDa磷蛋白证明，该蛋白质与细胞裂解物中的PI 3 K结合。该协会是依赖于磷酸化的酪氨酸残基上表达的激酶插入。基于这些观察结果，我们得出结论，SCF受体的激酶插入结构域选择性地与PI 3 K的p85调节亚基相互作用，并且这种关联需要激酶插入区域中的酪氨酸残基的磷酸化，显然不涉及受体的大量细胞质结构或酪氨酸激酶功能。
Our previous analysis of the signal transduction pathway used by the c-kit-encoded receptor for the stem cell factor (SCF) indicated efficient coupling to the type I phosphatidylinositol 3' kinase (PI3K). In an attempt to localize the receptor's site of interaction with PI3K, we separately deleted either the noncatalytic 68-amino-acid-long interkinase domain or the carboxyl-terminal portion distal to the catalytic sequences. Loss of ligand-induced association of PI3K with the former deletion mutant and retention of the PI3K association by the carboxyl-terminally deleted receptor implied interactions of PI3K with the kinase insert. This was further supported by partial inhibition of the association by an anti-peptide antibody directed against the kinase insert and lack of effect of an antibody directed to the carboxyl tail of the SCF receptor. A bacterially expressed kinase insert domain was used as a fusion protein to directly test its presumed function as a PI3K association site. This protein bound PI3K from cell lysate as demonstrated by PI3K activity and by an associated phosphoprotein of 85 kDa. The association was dependent on phosphorylation of the tyrosine residues on the expressed kinase insert. On the basis of these observations, we conclude that the kinase insert domain of the SCF receptor selectively interacts with the p85 regulatory subunit of PI3K and that this association requires phosphorylation of tyrosine residues in the kinase insert region, with apparently no involvement of the bulk cytoplasmic structure or tyrosine kinase function of the receptor.