Singh S S, Chauhan A, Murakami N, Chauhan V P
NYS Institute For Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA.
Biochemistry. 1996 Dec 24;35(51):16544-9. doi: 10.1021/bi9609634.
Actin-binding proteins such as profilin and gelsolin bind to phosphatidylinositol (PI) 4,5-bisphosphate (PI 4,5-P2) and regulate the concentration of monomeric actin. We report here that profilin and gelsolin stimulate PI 3-kinase-mediated phosphorylation of PI 4,5-P2 (lipid kinase activity) in a concentration-dependent manner. This effect is specific to profilin and gelsolin because other cytoskeletal proteins such as tau or actin do not affect PI 3-kinase activity. In addition to lipid kinase activity, PI 3-kinase also has protein kinase activity: it phosphorylates proteins (p85 subunit of PI 3-kinase). However, the protein kinase activity of PI 3-kinase was not affected in the presence of profilin. Kinetic analysis, as a function of varying concentrations of ATP and PI 4,5-P2, showed that profilin affects the Vmax of PI 3-kinase without affecting k(m). Profilin may also affect PI 3-kinase activity by its direct association to the enzyme because dot-blot analysis using antibody to glutathione S-transferase (GST) suggested that GST-85 kDa, a fusion protein of PI 3-kinase, binds to profilin. However, PI 3-kinase did not affect the actin-sequestering ability of profilin (determined by pyrene-labeled actin), which indicates that actin and p85 do not share a common binding site on profilin. These studies suggest that profilin and gelsolin may control the generation of 3-OH phosphorylated phosphoinositides, which in turn may regulate the actin polymerization.
诸如丝切蛋白和凝溶胶蛋白等肌动蛋白结合蛋白可与磷脂酰肌醇（PI）4,5-二磷酸（PI 4,5-P2）结合，并调节单体肌动蛋白的浓度。我们在此报告，丝切蛋白和凝溶胶蛋白以浓度依赖性方式刺激PI 3激酶介导的PI 4,5-P2磷酸化（脂质激酶活性）。这种效应是丝切蛋白和凝溶胶蛋白所特有的，因为其他细胞骨架蛋白，如微管相关蛋白tau或肌动蛋白，不会影响PI 3激酶活性。除了脂质激酶活性外，PI 3激酶还具有蛋白激酶活性：它可使蛋白（PI 3激酶的p85亚基）磷酸化。然而，在丝切蛋白存在的情况下，PI 3激酶的蛋白激酶活性并未受到影响。作为不同浓度ATP和PI 4,5-P2的函数进行的动力学分析表明，丝切蛋白影响PI 3激酶的最大反应速度（Vmax），而不影响米氏常数（Km）。丝切蛋白还可能通过其与该酶的直接结合来影响PI 3激酶活性，因为使用谷胱甘肽S-转移酶（GST）抗体进行的斑点印迹分析表明，PI 3激酶的融合蛋白GST-85 kDa可与丝切蛋白结合。然而，PI 3激酶并不影响丝切蛋白的肌动蛋白隔离能力（由芘标记的肌动蛋白测定），这表明肌动蛋白和p85在丝切蛋白上不共享共同的结合位点。这些研究表明，丝切蛋白和凝溶胶蛋白可能控制3-OH磷酸化磷酸肌醇的生成，进而可能调节肌动蛋白聚合。
