Lu P J, Shieh W R, Rhee S G, Yin H L, Chen C S
Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington 40536-0082, USA.
Biochemistry. 1996 Nov 5;35(44):14027-34. doi: 10.1021/bi961878z.
To gain insight into the physiological function of phosphoinositide 3-kinase (PI 3-kinase) lipid products, this study examines the interactions of the D-3 phosphoinositides with profilin and the consequent effects on actin dynamics and phosphoinositide turnover. Profilin, a ubiquitous actin-regulating protein, plays a putative role in regulating actin assembly and PLC-gamma 1 signaling in light of its unique interactions with actin and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. Here we raise evidence that the affinity of profilin with the D-3 phosphoinositides is substantially higher than that of PtdIns(4,5)P2. The dissociation constants (Kd) are estimated to be 1.1 microM, 5.7 microM, and 11 microM for phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2], phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], and PtdIns(4,5)P2, respectively. Spectroscopic data show that while all these phosphoinositides alter the tryptophan fluorescence of profilin in a similar fashion, the respective conformational effect on profilin is vastly different. Based on CD data, the alpha-helical contents of profilin in the presence of 8 molar equiv of PtdIns(4,5)P2, PtdIns(3,4,5)P3, and PtdIns(3,4)P2 are 17.4%, 11.5%, and 1.4%, respectively, vis-a-vis 9.4% for profilin alone. In contrast, no appreciable change in the fluorescence and CD spectra is observed when related inositol phosphates such as Ins(1,4,5)P3, Ins(1,3,4,5)P4, or Ins(1,3,4)P3 at comparable concentrations are tested. Evidence suggests that this differential recognition bears functional significance concerning the intricate roles of profilin and inositol lipids in modulating actin polymerization and PtdIns(4,5)P2 turnover. The relative potency of individual phosphoinositides in offsetting the inhibitory effect of profilin on actin assembly is PtdIns(3,4)P2 > PtdIns(3,4,5)P3 > PtdIns(4,5)P2, consistent with their relative binding affinity with profilin. Moreover, the inhibitory effect of profilin on PLC-gamma 1-mediated PtdIns(4,5)P2 hydrolysis is overcome by PtdIns(3,4)P2 and PtdIns(3,4,5)P3 through a combined effect of PLC-gamma 1 activation and preferential profilin binding. This D-3 phosphoinositide-mediated regulation may represent a new mechanism for controlling PtdIns(4,5)P2 turnover by PLC-gamma 1.
为深入了解磷酸肌醇3激酶（PI 3激酶）脂质产物的生理功能，本研究检测了D-3磷酸肌醇与丝切蛋白的相互作用以及对肌动蛋白动力学和磷酸肌醇周转的后续影响。丝切蛋白是一种普遍存在的肌动蛋白调节蛋白，鉴于其与肌动蛋白和磷脂酰肌醇4,5-二磷酸[PtdIns(4,5)P2]的独特相互作用，在调节肌动蛋白组装和PLC-γ1信号传导中发挥推定作用。在此我们提出证据表明，丝切蛋白与D-3磷酸肌醇的亲和力显著高于其与PtdIns(4,5)P2的亲和力。磷脂酰肌醇3,4-二磷酸[PtdIns(3,4)P2]、磷脂酰肌醇3,4,5-三磷酸[PtdIns(3,4,5)P3]和PtdIns(4,5)P2的解离常数（Kd）估计分别为1.1微摩尔、5.7微摩尔和11微摩尔。光谱数据表明，虽然所有这些磷酸肌醇都以类似方式改变丝切蛋白的色氨酸荧光，但对丝切蛋白各自的构象影响却大不相同。基于圆二色性（CD）数据，在存在8摩尔当量的PtdIns(4,5)P2、PtdIns(3,4,5)P3和PtdIns(3,4)P2时，丝切蛋白的α-螺旋含量分别为17.4%、11.5%和1.4%，而单独的丝切蛋白为9.4%。相比之下，当测试浓度相当的相关肌醇磷酸如Ins(1,4,5)P3、Ins(1,3,4,5)P4或Ins(1,3,4)P3时，未观察到荧光和CD光谱有明显变化。有证据表明，这种差异识别在丝切蛋白和肌醇脂质在调节肌动蛋白聚合和PtdIns(4,5)P2周转的复杂作用方面具有功能意义。各磷酸肌醇抵消丝切蛋白对肌动蛋白组装抑制作用的相对效力为PtdIns(3,4)P2 > PtdIns(3,4,5)P3 > PtdIns(4,5)P2，与其与丝切蛋白的相对结合亲和力一致。此外，PtdIns(3,4)P2和PtdIns(3,4,5)P3通过PLC-γ1激活和丝切蛋白优先结合的联合作用，克服了丝切蛋白对PLC-γ1介导的PtdIns(4,5)P2水解的抑制作用。这种D-3磷酸肌醇介导的调节可能代表了一种由PLC-γ1控制PtdIns(4,5)P2周转的新机制。
