Differential roles of phosphatidylserine, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 in plasma membrane targeting of C2 domains -: Molecular dynamics simulation, membrane binding, and cell translocation studies of the PKCαC2 domain

Many cytosolic proteins are recruited to the plasma membrane ( PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine ( PS), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca2+-dependent C2 domain of protein kinase C alpha(PKC alpha) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKC alpha-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P-2 and PtdIns(3,4,5)P-3, but that PS and Ca2+ binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P-2 or PtdIns(3,4,5)P-3 augments the Ca2+- and PS-dependent membrane binding of PKC alpha-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca2+-dependent PS binding is essential for membrane interactions of PKC alpha-C2. PtdIns(4,5)P-2 alone cannot drive the membrane attachment of the domain but further stabilizes the Ca2+- and PS-dependent membrane binding. When the fluorescence protein-tagged PKC alpha-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P-2 and/or PtdIns(3,4,5)P-3 from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of PtdIns( 4,5) P2 or PtdIns( 3,4,5) P3 at the PM slowed membrane dissociation of PKC alpha-C2. Collectively, these studies show that PtdIns(4,5)P-2 and PtdIns(3,4,5)P-3 augment the Ca2+- and PS-dependent membrane binding of PKC alpha-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKC alpha-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.