Differential Roles of Phosphatidylserine, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 in Plasma Membrane Targeting of C2 Domains

Abstract

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 Ca 2؉ -dependent C2 domain of protein kinase C␣ (PKC␣) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKC␣-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 , but that PS and Ca 2؉ binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P 2 or PtdIns(3,4,5)P 3 augments the Ca 2؉ -and PSdependent membrane binding of PKC␣-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca 2؉ -dependent PS binding is essential for membrane interactions of PKC␣-C2. PtdIns(4,5)P 2 alone cannot drive the membrane attachment of the domain but further stabilizes the Ca 2؉ -and PS-dependent membrane binding. When the fluorescence protein-tagged PKC␣-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)P 2 or PtdIns(3,4,5)P 3 at the PM slowed membrane dissociation of PKC␣-C2. Collectively, these studies show that PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 augment the Ca 2؉ -and PS-dependent membrane binding of PKC␣-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKC␣-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.One of the hallmarks of cell signaling proteins is their reversible recruitment to the plasma membrane (PM) 5 in response to receptor activation (1-4). Although some of these proteins are known to translocate to the PM through protein-protein interactions, a large number of proteins are recruited to the PM by directly interacting with lipids present in the PM (4). The inner leaflet of the PM of mammalian cells is known to be rich in anionic phospholipids, PS (about 20 mol %) (5) and inositol phospholipids, including PtdIns(4,5)P 2 (about 1 mol %) (6 -8). These anionic lipids have been shown to play key roles in PM recruitment of a wide variety of cytosolic proteins. A series of in vitro membrane binding and cellular translocation studies of various proteins, including protein kinase C (PKC) (9) and sphingosine kinase (10), as well as their isolated lipid binding domains (11), have indicated that PS-selective proteins are targeted to the PM through direct ...

Structural and mechanistic insights into the association of PKCα-C2 domain to PtdIns(4,5)P 2

“…This orientation would be similar to the one found in the crystal structure presented here and thus would be compatible with a perfect docking of the C2 domain in the membrane interface being PtdIns(4,5)P 2 , the target molecule. Furthermore, this orientation would also explain the biochemical results showing that PtdIns(3,4,5)P 3 can also bind (although with lower affinity) to this C2 domain (10,20,28), because the phosphate group in the C3 of the inositol ring would point to the membrane interface not being directly involved in the protein interaction (see Fig. 1C and the docking model proposed in Fig.…”

Phosphoinositide 3-Kinase Regulates Plasma Membrane Targeting of the Ras-specific Exchange Factor RasGRP1

“…As a result, the PT domain can weakly bind to the plasma membrane in the absence of receptor ligation by detecting a set of constitutively present phosphoinositides, composed of PI(4,5)P 2 plus low levels of PI(3,4)P 2 and PI(3,4,5)P 3 . Basic/hydrophobic clusters in Rit, Rin, and PKC␣ are similarly targeted to the plasma membrane by PI(4,5)P 2 in combination with PI(3,4)P 2 and/or PI(3,4,5)P 3 (30,69). Given the large excess of PI(4,5)P 2 over PI(3,4,5)P 3 plus (PI(3,4)P 2 in cells, it is surprising that PI(4,5)P 2 does not completely dictate the localization of basic hydrophobic clusters due to their broad specificities for phosphoinositides, but it is possible that the ratio of PI(4,5)P 2 to PI(3,4,5)P 2 plus PI(3,4)P 2 available for BHC binding at the plasma membrane is lower than whole cell quantities would suggest, perhaps due to selective sequestration of PI(4,5)P 2 (70,71).…”

Characterization of a Binding Site for Anionic Phospholipids on KCNQ1

“…Interestingly, the presence of PS in the liposomes used for surface plasmon resonance was required for binding of the KCNQ1 fusion proteins and without PS no binding of the full-length KCNQ1 was possible. PS has previously been shown to be required for the membrane recruitment of the C2 domain of PKC␣ (53). PIP 2 and PtdIns(3,4,5)P 3 make up only a fraction of the negative charge in the membrane and PS is a predominantly negatively charged lipid that has been shown to be involved in recruiting proteins to this location (54).…”