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-gamma 1 signaling in light of its unique interactions with actin and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2]. Here we raise evidence that the affinity of profilin with the D-3 phosphoinositides is substantially higher than that of PtdIns(4,5)P-2. The dissociation constants (K-d) are estimated to be 1.1 mu M, 5.7 mu M, and 11 mu M for phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P-2], phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P-3], and PtdIns(4,5)P-2, 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)P-2, PtdIns(3,4,5)P-3, and PtdIns(3,4)P-2 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)P-3, Ins(1,3,4,5)P-4, or Ins(1,3,4)P-3 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)P-2 turnover. The relative potency of individual phosphoinositides in offsetting the inhibitory effect of profilin on actin assembly is PtdIns(3,4)P-2, PtdIns(3,4,5)P-3 > PtdIns(4,5)P-2, consistent with their relative binding affinity with profilin. Moreover, the inhibitory effect of profilin on PLC-gamma 1-mediated PtdIns(4,5)P-2 hydrolysis is overcome by PtdIns(3,4)P-2 and PtdIns(3,4,5)P-3 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)P-2 turnover by PLC-gamma 1.