Profilin binding to sub-micellar concentrations of phosphatidylinositol (4,5) bisphosphate and phosphatidylinositol (3,4,5) trisphosphate.
Profilin is a small (12-15 kDa) actin binding protein which promotes filament turnover. Profilin is also involved in the signaling pathway linking receptors in the cell membrane to the microfilament system within the cell. Profilin is thought to play critical roles in this signaling pathway through its interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] (P.J. Lu, W.R. Shieh, S.G. Rhee, H.L. Yin, C.S. Chen, Lipid products of phosphoinositide 3-kinase bind human profilin with high affinity, Biochemistry 35 (1996) 14027-14034). To date, profilin's interaction with polyphosphoinositides (PPI) has only been studied in micelles or small vesicles. Profilin binds with high affinity to small clusters of PI(4,5)P(2) molecules. In this work, we investigated the interactions of profilin with sub-micellar concentrations of PI(4,5)P(2) and PI(3,4,5)P(3). Fluorescence anisotropy was used to determine the relevant dissociation constants for binding of sub-micellar concentrations of fluorescently labeled PPI lipids to profilin and we show that these are significantly different from those determined for profilin interaction with micelles or small vesicles. We also show that profilin binds more tightly to sub-micellar concentrations of PI(3,4,5)P(3) (K(D)=720 microM) than to sub-micellar concentrations of PI(4,5)P(2) (K(D)=985 microM). Despite the low affinity for sub-micellar concentration of PI(4,5)P(2), profilin was shown to bind to giant unilamellar vesicles in presence of 0.5% mole fraction of PI(4,5)P(2) The implications of these findings are discussed.
Moens
Pierre D J
PD
School of Biological, Biomedical and Molecular Sciences, The University of New England, Armidale, NSW 2351 Australia. pmoens@une.edu.au
Bagatolli
Luis A
LA
D001792:Blood Platelets / Q000737:chemistry; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008027:Light; D008051:Lipid Bilayers; D008823:Micelles; D008956:Models, Chemical; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D051304:Profilins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence
D008051:Lipid Bilayers; D008823:Micelles; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D051304:Profilins; C060974:phosphatidylinositol 3,4,5-triphosphate