Moarefi I, LaFevre-Bernt M, Sicheri F, Huse M, Lee C H, Kuriyan J, Miller W T
Laboratories of Molecular Biophysics, Howard Hughes Medical Institute, The Rockefeller University, New York 10021, USA.
Nature. 1997 Feb 13;385(6617):650-3. doi: 10.1038/385650a0.
The protein Hck is a member of the Src family of non-receptor tyrosine kinases which is preferentially expressed in haematopoietic cells of the myeloid and B-lymphoid lineages. Src kinases are inhibited by tyrosine-phosphorylation at a carboxy-terminal site. The SH2 domains of these enzymes play an essential role in this regulation by binding to the tyrosine-phosphorylated tail. The crystal structure of the downregulated form of Hck has been determined and reveals that the SH2 domain regulates enzymatic activity indirectly; intramolecular interactions between the SH3 and catalytic domains appear to stabilize an inactive form of the kinase. Here we compare the roles of the SH2 and SH3 domains in modulating the activity of Hck in an investigation of the C-terminally phosphorylated form of the enzyme. We show that addition of the HIV-1 Nef protein, which is a high-affinity ligand for the Hck SH3 domain, to either the downregulated or activated form of Hck causes a large increase in Hck catalytic activity. The intact SH3-binding motif in Nef is crucial for Hck activation. Our results indicate that binding of the Hck SH3 domain by Nef causes a more marked activation of the enzyme than does binding of the SH2 domain, suggesting a new mechanism for regulation of the activity of tyrosine kinases.
蛋白质Hck是非受体酪氨酸激酶Src家族的成员，在髓系和B淋巴细胞系的造血细胞中优先表达。Src激酶在羧基末端位点被酪氨酸磷酸化所抑制。这些酶的SH2结构域通过与酪氨酸磷酸化的尾部结合在这种调节中起重要作用。已确定Hck下调形式的晶体结构，结果显示SH2结构域间接调节酶活性；SH3和催化结构域之间的分子内相互作用似乎稳定了激酶的无活性形式。在此，我们在对该酶C末端磷酸化形式的研究中比较了SH2和SH3结构域在调节Hck活性中的作用。我们发现，将作为Hck SH3结构域高亲和力配体的HIV-1 Nef蛋白添加到Hck的下调形式或激活形式中，都会导致Hck催化活性大幅增加。Nef中完整的SH3结合基序对Hck激活至关重要。我们的结果表明，与SH2结构域结合相比，Nef与Hck SH3结构域的结合对该酶的激活作用更为显著，这提示了一种调节酪氨酸激酶活性的新机制。
