Hunter S, Burton E A, Wu S C, Anderson S M
Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
J Biol Chem. 1999 Jan 22;274(4):2097-106. doi: 10.1074/jbc.274.4.2097.
We have investigated the interaction between Cbl and the Src-related tyrosine kinase Fyn. Fyn was observed to be constitutively associated with Cbl in lysates of several different cell types including the interleukin-3-dependent murine myeloid cell line 32Dcl3, and the prolactin-dependent rat thymoma cell line Nb2. Binding studies indicated that Cbl could bind to glutathione S-transferase (GST) fusion proteins encoding the unique, Src homology domain 3 (SH3), and SH2 domains of Fyn, Hck, or Lyn. Fusion proteins encoding either the SH3 or SH2 domains of Fyn bound to Cbl as effectively as the fusion protein encoding the unique, SH3, and SH2 domains of Fyn. The Fyn SH2 domain bound to both tyrosine-phosphorylated and nonphosphorylated Cbl, implying that this interaction might be phosphotyrosine-independent. Binding of the Fyn SH2 domain to Cbl was not disrupted by the addition of phosphotyrosine, phosphoserine, or phosphothreonine. A GST fusion protein encoding the proline-rich region of Cbl bound to Fyn present in a total cell lysate. Far Western blot analysis also indicated that the SH3 domain of Fyn bound preferentially to the proline-rich region of Cbl. The addition of [gamma-32P]ATP to either anti-Cbl immunoprecipitates or anti-Fyn immunoprecipitates resulted in the phosphorylation of both Cbl and Fyn as demonstrated by immunoprecipitation of the phosphorylated proteins with specific antisera. Fyn directly phosphorylated a GST fusion protein containing the C-terminal region of Cbl (GST-CBL-LZIP). In contrast, immunoprecipitated JAK2 was not able to phosphorylate this same region of Cbl. The GST-CBL-LZIP fusion protein contains a binding site for the SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase, which mapped to Tyr731, which is present in the sequence YEAM. Mutation of Tyr731 in GST-CBL-LZIP eliminated binding of the p85 subunit of phosphatidylinositol 3-kinase and substantially reduced the phosphorylation of this fusion protein by Fyn, despite the presence of four other tyrosine residues in this fusion protein. These data are consistent with the hypothesis that Cbl represents a substrate for Src-like kinases that are activated in response to the engagement of cell surface receptors, and that Src-like kinases are responsible for the phosphorylation of a tyrosine residue in Cbl that may regulate activation of phosphatidylinositol 3-kinase.
我们研究了Cbl与Src相关酪氨酸激酶Fyn之间的相互作用。在包括白细胞介素-3依赖的小鼠骨髓细胞系32Dcl3和催乳素依赖的大鼠胸腺瘤细胞系Nb2在内的几种不同细胞类型的裂解物中，观察到Fyn与Cbl持续相关。结合研究表明，Cbl可以与编码Fyn、Hck或Lyn的独特的Src同源结构域3（SH3）和SH2结构域的谷胱甘肽S-转移酶（GST）融合蛋白结合。编码Fyn的SH3或SH2结构域的融合蛋白与Cbl的结合效果与编码Fyn的独特、SH3和SH2结构域的融合蛋白相同。Fyn的SH2结构域与酪氨酸磷酸化和非磷酸化的Cbl都结合，这意味着这种相互作用可能不依赖于磷酸酪氨酸。加入磷酸酪氨酸、磷酸丝氨酸或磷酸苏氨酸不会破坏Fyn的SH2结构域与Cbl的结合。编码Cbl富含脯氨酸区域的GST融合蛋白与总细胞裂解物中的Fyn结合。Far Western印迹分析也表明，Fyn的SH3结构域优先与Cbl的富含脯氨酸区域结合。向抗Cbl免疫沉淀或抗Fyn免疫沉淀中加入[γ-32P]ATP，导致Cbl和Fyn都发生磷酸化，这通过用特异性抗血清免疫沉淀磷酸化蛋白得以证明。Fyn直接磷酸化了包含Cbl C末端区域的GST融合蛋白（GST-CBL-LZIP）。相比之下，免疫沉淀的JAK2不能磷酸化Cbl的同一区域。GST-CBL-LZIP融合蛋白包含磷脂酰肌醇3-激酶p85亚基SH2结构域的结合位点，该位点定位于YEAM序列中的Tyr731。GST-CBL-LZIP中Tyr731的突变消除了磷脂酰肌醇3-激酶p85亚基的结合，并显著降低了Fyn对该融合蛋白的磷酸化，尽管该融合蛋白中还存在其他四个酪氨酸残基。这些数据与以下假设一致：Cbl是细胞表面受体激活后被激活的Src样激酶的底物，并且Src样激酶负责Cbl中一个酪氨酸残基的磷酸化，该酪氨酸残基可能调节磷脂酰肌醇3-激酶的激活。
