Burks D J, Wang J, Towery H, Ishibashi O, Lowe D, Riedel H, White M F
Howard Hughes Medical Institute, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
J Biol Chem. 1998 Nov 20;273(47):31061-7. doi: 10.1074/jbc.273.47.31061.
Using a yeast two-hybrid system, we identified several proteins that interact with the PH domains in IRS-1 and IRS-2, including Lon protease, myeloblast protein, and nucleolin. Although the roles of these molecules in insulin action are not yet known, each protein contained an acidic motif that interacted with the PH domain of IRS-2. However, only the acidic motif in nucleolin bound to IRS-1, suggesting that the PH domain in IRS-1 and IRS-2 are not identical. Moreover, synthetic peptides based on the acidic motif in Lon protease and myeloblast protein inhibited the binding of nucleolin to the PH domain of IRS-2 but not to the PH domain of IRS-1, confirming the selectivity of these PH domains. The ability to bind acidic motifs may be a specific function of the PH domain in IRS proteins, because the PH domains in betaARK, phospholipase Cgamma, or spectrin did not bind nucleolin. In 32D cells, nucleolin bound to both IRS-1 and IRS-2, and expression of the acidic motif of nucleolin inhibited insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. These results suggest that the binding of acidic motifs to the PH domain of IRS-1 and IRS-2 disrupts coupling to the activated insulin receptor. Our results are consistent with the hypothesis that the PH domain in the IRS proteins may ordinarily bind acidic peptide motifs in membrane proteins or other acidic membrane elements that couple IRS proteins to activated membrane receptors.
利用酵母双杂交系统，我们鉴定出了几种与胰岛素受体底物-1（IRS-1）和胰岛素受体底物-2（IRS-2）中的PH结构域相互作用的蛋白质，包括Lon蛋白酶、成髓细胞蛋白和核仁素。尽管这些分子在胰岛素作用中的角色尚不清楚，但每种蛋白质都含有一个与IRS-2的PH结构域相互作用的酸性基序。然而，只有核仁素中的酸性基序与IRS-1结合，这表明IRS-1和IRS-2中的PH结构域并不相同。此外，基于Lon蛋白酶和成髓细胞蛋白中酸性基序的合成肽抑制了核仁素与IRS-2的PH结构域的结合，但不抑制与IRS-1的PH结构域的结合，证实了这些PH结构域的选择性。结合酸性基序的能力可能是IRS蛋白中PH结构域的一种特定功能，因为β-肾上腺素受体激酶（betaARK）、磷脂酶Cγ或血影蛋白中的PH结构域不与核仁素结合。在32D细胞中，核仁素与IRS-1和IRS-2都结合，并且核仁素酸性基序的表达抑制了胰岛素刺激的IRS-1和IRS-2的酪氨酸磷酸化。这些结果表明，酸性基序与IRS-1和IRS-2的PH结构域的结合破坏了与活化胰岛素受体的偶联。我们的结果与以下假设一致，即IRS蛋白中的PH结构域通常可能与膜蛋白或其他将IRS蛋白与活化膜受体偶联的酸性膜元件中的酸性肽基序结合。
