Lupher M L, Songyang Z, Shoelson S E, Cantley L C, Band H
Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
J Biol Chem. 1997 Dec 26;272(52):33140-4. doi: 10.1074/jbc.272.52.33140.
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.
Cbl原癌基因产物已成为受体和非受体酪氨酸激酶的一种新型负调节因子，但其具体机制尚不清楚。因此，确定Cbl如何与酪氨酸激酶发生物理相互作用具有重要意义。我们最近在Cbl的N端转化区（Cbl-N）中鉴定出一个磷酸酪氨酸结合（PTB）结构域，该结构域介导了与ZAP-70酪氨酸激酶的直接结合。在此，我们筛选了一个简并磷酸肽文库，结果表明Cbl-PTB结构域选择了一个D（N/D）XpY基序，该基序与Shc和IRS-1/2的PTB结构域识别的NPXpY基序相似但不同。由该基序预测的、对应于ZAP-70体内负调节磷酸化位点（Tyr（P）292）的磷酸肽特异性抑制了ZAP-70与Cbl-N的结合。ZAP-70/Y292F突变体无法与Cbl-N结合，而D290A突变体导致结合减少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/Y292F突变体在体内也无法与Cbl-N或全长Cbl结合。这些结果确定了Cbl在ZAP-70负调节中潜在的依赖Cbl-PTB结构域的作用，并预测了已知与Cbl相互作用的其他蛋白酪氨酸激酶上潜在的Cbl-PTB结构域结合位点。
