Lipid products of phosphoinositide 3-kinase bind human profilin with high affinity

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-γ1 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 (K(d)) are estimated to be 1.1 μM, 5.7 μM, and 11 μM 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 α-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-γ1-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-γI 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-γI.