Regulation of the nonreceptor tyrosine kinase Brk by autophosphorylation and by autoinhibition
Regulation of the nonreceptor tyrosine kinase Brk by autophosphorylation and by autoinhibition
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DOI：
10.1074/jbc.m203877200
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发表时间：
2002-09-13
影响因子：
4.8
通讯作者：
Miller, WT
中科院分区：
文献类型：
--
作者：
Qiu, HQ;Miller, WT
BRK是一种非受体酪氨酸激酶，与Frk家族的亲缘关系最密切，与Src家族的亲缘关系较远。BRK最初是在人类转移性乳腺肿瘤中过度表达的酪氨酸激酶的筛查中发现的。为了阐明BRK及其相关酪氨酸激酶的活性和调控，我们利用Sf9/杆状病毒系统对BRK进行了表达和纯化。我们用合成的多肽表征了BRK的底物专一性，我们发现动力学参数K-m和k(CAT)都对专一性起作用。我们进行了质谱学实验，以表明BRK在预测的激酶激活环内和在N末端的其他位置发生自磷酸化。自动磷酸化可增加野生型BRK的酶活性，但不能增加Y342A突变形式的BRK的酶活性。我们还进行了实验，以确定BRK的Src同源(SH)2和SH3结构域可能参与酶调节。C端酪氨酸(Tyr-447)的突变增加了酶的活性和SH2结构域的可及性，这与该残基在自身抑制中的作用一致。富含脯氨酸的多肽激活BRK，表明SH3结构域也参与维持BRK的非活性形式。BRK的这些生化结果可能有助于理解Frk家族中的其他酪氨酸激酶。
Brk (breast tumor kinase) is a nonreceptor tyrosine kinase that is most closely related to the Frk family of kinases, and more distantly to Src family kinases. Brk was originally identified in a screen for tyrosine kinases that are overexpressed in human metastatic breast tumors. To shed light on the activity and regulation of Brk and related tyrosine kinases, we expressed and purified Brk using the Sf9/baculovirus system. We characterized the substrate specificity of Brk using synthetic peptides, and we show that the kinetic parameters K-m and k(cat) both play a role in specificity. We carried out mass spectrometry experiments to show that Brk autophosphorylates within the predicted kinase activation loop and at additional sites in the N terminus. Autophosphorylation increases enzyme activity of wild-type Brk but not of a Y342A mutant form of Brk. We also carried out experiments to address the possible involvement of the Src homology (SH) 2 and SH3 domains of Brk in enzyme regulation. Mutation of a C-terminal tyrosine (Tyr-447) increases enzyme activity and SH2 domain accessibility, consistent with a role for this residue in autoinhibition. A proline-rich peptide activates Brk, suggesting that the SH3 domain is also involved in maintaining an inactive form of Brk. These biochemical results for Brk may aid in the understanding of other tyrosine kinases in the Frk family.