Rameh L E, Arvidsson A k, Carraway K L, Couvillon A D, Rathbun G, Crompton A, VanRenterghem B, Czech M P, Ravichandran K S, Burakoff S J, Wang D S, Chen C S, Cantley L C
Department of Cell Biology, Harvard Medical School and Division of Signal Transduction, Beth Israel Hospital, Boston, Massachusetts 02115, USA.
J Biol Chem. 1997 Aug 29;272(35):22059-66. doi: 10.1074/jbc.272.35.22059.
Pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains are structurally related regulatory modules that are present in a variety of proteins involved in signal transduction, such as kinases, phospholipases, GTP exchange proteins, and adapter proteins. Initially these domains were shown to mediate protein-protein interactions, but more recently they were also found to bind phosphoinositides. Most studies to date have focused on binding of PH domains to phosphatidylinositol (PtdIns)-4-P and PtdIns-4,5-P2 and have not considered the lipid products of phosphoinositide 3-kinase: PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3. Here we have compared the phosphoinositide specificity of six different PH domains and the Shc PTB domain using all five phosphoinositides. We show that the Bruton's tyrosine kinase PH domain binds to PtdIns-3,4, 5-P3 with higher affinity than to PtdIns-4,5-P2, PtdIns-3,4-P2 or inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4). This selectivity is decreased by the xid mutation (R28C). Selective binding of PtdIns-3,4,5-P3 over PtdIns-4,5-P2 or PtdIns-3,4-P2 was also observed for the amino-terminal PH domain of T lymphoma invasion and metastasis protein (Tiam-1), the PH domains of Son-of-sevenless (Sos) and, to a lesser extent, the PH domain of the beta-adrenergic receptor kinase. The oxysterol binding protein and beta-spectrin PH domains bound PtdIns-3,4,5-P3 and PtdIns-4,5-P2 with similar affinities. PtdIns-3,4,5-P3 and PtdIns-4,5-P2 also bound to the PTB domain of Shc with similar affinities and lipid binding was competed with phosphotyrosine (Tyr(P)-containing peptides. These results indicate that distinct PH domains select for different phosphoinositides.
普列克底物蛋白同源（PH）结构域和磷酸酪氨酸结合（PTB）结构域是结构相关的调节模块，存在于多种参与信号转导的蛋白质中，如激酶、磷脂酶、GTP交换蛋白和衔接蛋白。最初这些结构域被证明可介导蛋白质-蛋白质相互作用，但最近也发现它们能结合磷酸肌醇。迄今为止，大多数研究集中在PH结构域与磷脂酰肌醇（PtdIns）-4-P和PtdIns-4,5-P2的结合，而未考虑磷酸肌醇3-激酶的脂质产物：PtdIns-3-P、PtdIns-3,4-P2和PtdIns-3,4,5-P3。在此，我们使用所有五种磷酸肌醇比较了六种不同PH结构域和Shc PTB结构域的磷酸肌醇特异性。我们发现，布鲁顿酪氨酸激酶PH结构域与PtdIns-3,4,5-P3的结合亲和力高于与PtdIns-4,5-P2、PtdIns-3,4-P2或肌醇1,3,4,5-四磷酸（Ins-1,3,4,5-P4）的结合亲和力。这种选择性因xid突变（R28C）而降低。在T淋巴瘤侵袭和转移蛋白（Tiam-1）的氨基末端PH结构域、七号染色体失活蛋白（Sos）的PH结构域以及程度稍低的β-肾上腺素能受体激酶的PH结构域中，也观察到PtdIns-3,4,5-P3相对于PtdIns-4,5-P2或PtdIns-3,4-P2的选择性结合。氧化固醇结合蛋白和β-血影蛋白PH结构域与PtdIns-3,4,5-P3和PtdIns-4,5-P2的结合亲和力相似。PtdIns-3,4,5-P3和PtdIns-4,5-P2与Shc的PTB结构域的结合亲和力也相似，且脂质结合可被磷酸酪氨酸（含Tyr(P)的肽段）竞争。这些结果表明，不同的PH结构域对不同的磷酸肌醇具有选择性。
