Shoelson S E, Sivaraja M, Williams K P, Hu P, Schlessinger J, Weiss M A
Joslin Diabetes Center, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215.
EMBO J. 1993 Feb;12(2):795-802. doi: 10.1002/j.1460-2075.1993.tb05714.x.
SH2 (src-homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein-effector coupling is provided by the association of phosphatidylinositol 3-kinase (PI 3-kinase) with specific phosphorylation sites within the PDGF receptor, the c-Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS-1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3-kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor-, IRS-1- and c-Src-derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3-kinase. Although phosphotyrosine and both activating and non-activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non-local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism.
SH2(src同源性2)结构域定义了一种新识别的结合基序,该基序介导靶磷酸酪氨酸蛋白与下游效应酶的物理缔合。磷脂酰肌醇3激酶(PI 3激酶)与血小板衍生生长因子受体(PDGF受体)、c-Src/多瘤病毒中T抗原复合物以及胰岛素受体底物IRS-1内的特定磷酸化位点的缔合,提供了这种磷蛋白-效应器偶联的一个例子。值得注意的是,磷蛋白与p85的SH2结构域的缔合也会刺激PI 3激酶p110亚基的催化活性增加,这可以被对应于靶磷蛋白磷酸化位点的磷酸肽所模拟。为了研究磷蛋白与p85 SH2结构域的结合如何刺激p110催化激活,我们研究了磷酸酪氨酸以及源自PDGF受体、IRS-1和c-Src的磷酸肽对PI 3激酶分离的SH2结构域构象产生的不同影响。尽管磷酸酪氨酸以及激活和非激活磷酸肽都能与SH2结构域结合,但激活磷酸肽以更高的亲和力结合,并诱导出定性上不同的构象变化,这通过圆二色光谱(CD)和核磁共振光谱(NMR)监测。酰胺质子交换和蛋白酶保护试验进一步表明,高亲和力、特异性磷酸肽结合诱导非局部动态SH2结构域稳定。基于这些发现,我们提出,与p85亚基的特异性磷蛋白结合会诱导SH2结构域结构发生变化,该变化会传递到p110亚基,并通过变构机制调节酶活性。