Previously, ceramide-1-phosphate (C1P) was demonstrated to be a potent and specific activator of group IV cytosolic phospholipase A 2 ␣ (cPLA 2 ␣) via interaction with the C2 domain. In this study, we hypothesized that the specific interaction site for C1P was localized to the cationic ␤-groove (Arg 57 , Lys 58 , Arg 59 ) of the C2 domain of cPLA 2 ␣. In this regard, mutants of this region of cPLA 2 ␣ were generated (R57A/K58A/R59A, R57A/R59A, K58A/R59A, R57A/K58A, R57A, K58A, and R59A) and examined for C1P affinity by surface plasmon resonance. The triple mutants (R57A/K58A/ R59A), the double mutants (R57A/R59A, K58A/R59A, and R57A/K58A), and the single mutant (R59A) demonstrated significantly reduced affinity for C1P-containing vesicles as compared with wild-type cPLA 2 ␣. Examining these mutants for enzymatic activity demonstrated that these five mutants of cPLA 2 ␣ also showed a significant reduction in the ability of C1P to: 1) increase the V max of the reaction; and 2) significantly decrease the dissociation constant (K s A ) of the reaction as compared with the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA 2 ␣ demonstrated normal basal activity as well as normal affinities for phosphatidylcholine and phosphatidylinositol-4,5-bisphosphate as compared with wild-type cPLA 2 ␣. This study, for the first time, demonstrates a novel C1P interaction site mapped to the cationic ␤-groove of the C2 domain of cPLA 2 ␣.

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The general receptor for phosphoinositides isoform 1 (GRP1) is recruited to the plasma membrane in response to activation of phosphoinositide 3-kinases and accumulation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ]. GRP1ʼs pleckstrin homology (PH) domain recognizes PtdIns(3,4,5)P 3 with high specificity and affinity, however, the precise mechanism of its association with membranes remains unclear. Here, we detail the molecular basis of membrane anchoring by the GRP1 PH domain. Our data reveal a multivalent membrane docking involving PtdIns(3,4,5)P 3 binding, regulated by pH and facilitated by electrostatic interactions with other anionic lipids. The specific recognition of PtdIns(3,4,5)P 3 triggers insertion of the GRP1 PH domain into membranes. An acidic environment enhances PtdIns(3,4,5)P 3 binding and increases membrane penetration as demonstrated by NMR and monolayer surface tension and surface plasmon resonance experiments. The GRP1 PH domain displays a 28 nM affinity for POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/PtdIns(3,4,5)P 3 vesicles at pH 6.0, but binds 22-fold weaker at pH 8.0. The pH sensitivity is attributed in part to the His355 residue, protonation of which is required for the robust interaction with PtdIns (3,4,5)P 3 and significant membrane penetration, as illustrated by mutagenesis data. The binding affinity of the GRP1 PH domain for PtdIns(3,4,5)P 3 -containing vesicles is further amplified (by ?6-fold) by nonspecific electrostatic interactions with phosphatidylserine/phosphatidylinositol. Together, our results provide new insight into the multivalent mechanism of the membrane targeting and regulation of the GRP1 PH domain.-He, J., R. M. Haney, M. Vora, V. V. Verkhusha, R. V. Stahelin, and T. G. Kutateladze. The signaling lipid phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] is produced in plasma membranes in response to stimulation of cell surface receptors by growth factors and hormones (1). Class I phosphoinositide (PI) 3-kinases phosphorylate the inositol headgroup of the relatively abundant phosphatidylinositol 4,5-bisphosphate [Ptdns(4,5)P 2 ], transiently elevating the level of PtdIns (3,4,5)P 3 from undetectable to nearly 10% of the PtdIns (4,5)P 2 level (2-4). The concentration of PtdIns(3,4,5)P 3 is tightly regulated by the activity of PI 5-and 3-phosphatases, such as SHIP1/2 and PTEN, which dephosphorylate the inositol ring generating PtdIns(3,4)P 2 and PtdIns (4,5)P 2 (5, 6). Despite the transitory accumulation and low concentrations in the plasma membrane, PtdIns (3,4,5)P 3 is implicated in fundamental biological processes including growth, proliferation, migration, and survival of cells (1, 7). The PtdIns(3,4,5)P 3 -mediated signals are primarily recognized and transduced by pleckstrin homology (PH) domain-containing proteins that bind strongly and in some cases specifically to PtdIns(3,4,5)P 3 . Mutations in the PH domains that disrupt or promote PtdIns (3,4,5)P 3 binding cause various signaling disarrays leading to sever...

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 ...

The sphingolipid ceramide‐1‐phosphate (C1P) plays a critical role in the cellular signaling that mediates inflammation, cell proliferation and phagocytosis. C1P has been shown to increase the activity of cytosolic phospholipase A2 α (cPLA2α) as well regulate its translocation to cellular membranes, a process that promotes inflammation through the production of arachidonic acid. In this study we have resolved the first C1P binding site of a peripheral protein. Using NMR we demonstrate the cPLA2α C2 domain interaction site for C1P by mapping chemical shifts. Subsequently, this novel‐binding site is confirmed with in vitro biophysical and biochemical analysis as well as molecular dynamics simulations. To search the human genome for other C1P binding proteins we have performed proteomic studies to began high throughput characterization of the conserved nature of C1P binding. Funding source: American Heart Association SDG0735350N (R.V.S)