The Cbl phosphotyrosine-binding domain selects a D(N/D)XpY motif and finds to the Tyr292 negative regulatory phosphorylation site of ZAP-70
The Cbl phosphotyrosine-binding domain selects a D(N/D)XpY motif and finds to the Tyr292 negative regulatory phosphorylation site of ZAP-70
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DOI：
10.1074/jbc.272.52.33140
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发表时间：
1997-12-26
影响因子：
4.8
通讯作者：
Band, H
中科院分区：
文献类型：
--
作者：
Lupher, ML;Zhou, SY;Band, H
Cbl原癌基因产物通过目前未确定的机制作为受体和非受体酪氨酸激酶的新型负调节剂出现。因此，确定Cbl如何与酪氨酸激酶物理相互作用具有重大意义。我们最近确定了一个磷酸酪氨酸结合（PTB）域内的N-末端转化区的Cbl（Cbl-N），介导直接结合ZAP-70酪氨酸激酶。在这里，我们已经筛选了一个简并磷酸肽库，并显示Cbl-PTB结构域选择D（N/D）XpY基序，让人想起，但不同于由PTB结构域的Shc和IRS-1/2识别的NPXpY基序。由该基序预测的对应于ZAP-70的体内负调节磷酸化位点的磷酸肽（Tyr（P）（292））特异性抑制ZAP-70与Cbl-N的结合，ZAP-70/Y292 F突变体不能与Cbl-N结合，而D290 A突变体导致结合降低64%，证实了Tyr（P）和Y-2残基在Cbl-PTB结构域识别中的重要性。最后，ZAP-70/Y292 F突变体在体内也不能与Cbl-N或全长Cbl缔合。这些结果鉴定了Cbl在ZAP-70的负调控中的潜在的Cbl-PTB结构域依赖性作用，并预测了已知与Cbl相互作用的其他蛋白酪氨酸激酶上的潜在Cbl-PTB结构域结合位点。
The Cbl protooncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases through currently undefined mechanisms. Therefore, determining how Cbl physically interacts with tyrosine kinases is of substantial interest. We recently identified a phosphotyrosine binding (PTB) domain residing within the N-terminal transforming region of Cbl (Cbl-N), which mediated direct binding to ZAP-70 tyrosine kinase. Here, we have screened a degenerate phosphopeptide library and show that the Cbl-PTB domain selects a D(N/D)XpY motif, reminiscent of but distinct from the NPXpY motif recognized by the PTB domains of Shc and IRS-1/2. A phosphopeptide predicted by this motif and corresponding to the in vivo negative regulatory phosphorylation site of ZAP-70 (Tyr(P)(292)) specifically inhibited binding of ZAP-70 to Cbl-N, A ZAP-70/Y292F mutant failed to bind to Cbl-N, whereas a D290A mutant resulted in a 64% decrease in binding, confirming the importance of the Tyr(P) and Y-2 residues in Cbl-PTB domain recognition. Finally the ZAP-70/Y292F mutant also failed to associate with Cbl-N or full-length Cbl in vivo. These results identify a potential Cbl-PTB domain-dependent role for Cbl in the negative regulation of ZAP-70 and predict potential Cbl-PTB domain binding sites on other protein tyrosine kinases known to interact with Cbl.