Interaction of protein kinase C with phosphatidylserine. 2. Specificity and regulation

Abstract

The roles of specific and nonspecific interactions in the regulation of protein kinase C by lipid have been examined. Binding and activity measurements reveal two mechanisms by which protein kinase C interacts with membranes: (1) a specific binding to the activating lipid phosphatidylserine and (2) a nonspecific binding to nonactivating, acidic lipids. The specific interaction with phosphatidylserine is relatively insensitive to ionic strength, surface charge, and the presence of nonactivating lipids. The two second messengers of the kinase, diacylglycerol and Ca2+, increase markedly the affinity of the kinase for phosphatidylserine. In contrast, the nonspecific interaction is sensitive to ionic strength and surface charge, and is unaffected by diacylglycerol. These results suggest that electrostatic interactions promote the binding of protein kinase C to membranes but the cooperative and selective binding of phosphatidylserine is the dominant driving force in a productive protein-lipid interaction.

Mutagenesis of the C2 Domain of Protein Kinase C-α

“…Second, we measured the Ca 2ϩ dependences of binding of PKC-␣ and R249A/R252A to POPC/POPG/DG vesicles. It has been shown that PKC-␣ binds, albeit less tightly, to phosphatidyl glycerol-containing vesicles via mainly nonspecific electrostatic interactions even in the presence of DG (17,34). As shown in Fig.…”

A Novel Phosphatidylserine-binding Peptide Motif Defined by an Anti-idiotypic Monoclonal Antibody

“…The present consensus sequence motif of rat PKC␥ (amino acids 227-238) resides in the C2 homology domain, and the similar sequence motif is also identified in PSD2 (amino acids 561-572) but not in synaptotagmins. Since PKC (14,15) and PSD2 (29) were shown to interact specifically with PS but synaptotagmin binds nonspecifically to various acidic phospholipids (16), the consensus sequence between PKC and PSD, FXFX(I/V/L)(K/R)XX(D/Q)K, is likely to mediate the specific interaction of these proteins with PS.…”

“…Recent analyses have shown that it regulates the activity of various enzymes such as c-Raf-1 protein kinase (7), nitric oxide synthase (8), Na ϩ /K ϩ -ATPase (9), Dynamin GTPase (10), and diacylglycerol kinase (11) and acts as a ligand in recognition of apoptotic cells (12,13). PKC is a family of phospholipid-dependent kinases, and its enzymatic activity is allosterically regulated by 1,2-diacyl-sn-glycero-3-phospho-Lserine (PS) (14,15). Analyses of the interaction of conventional PKC (cPKC) with membrane lipids support a two-step model for the activation of cPKC, which includes initial binding to membranes that does not require PS since cPKC binds various acidic phospholipids in a Ca 2ϩ -dependent manner and allosteric activation by specific interaction with PS and diacylglycerol (15).…”

“…PKC is a family of phospholipid-dependent kinases, and its enzymatic activity is allosterically regulated by 1,2-diacyl-sn-glycero-3-phospho-Lserine (PS) (14,15). Analyses of the interaction of conventional PKC (cPKC) with membrane lipids support a two-step model for the activation of cPKC, which includes initial binding to membranes that does not require PS since cPKC binds various acidic phospholipids in a Ca 2ϩ -dependent manner and allosteric activation by specific interaction with PS and diacylglycerol (15). The initial binding of cPKC to membranes is believed to be mediated by the Ca 2ϩ -dependent phospholipid-binding (CaLB) domain (amino acids 186 -233 of PKC␥), a sequence motif that is also found in other cytosolic proteins such as cytosolic phospholipase A 2 , GTPase-activating protein, and synaptotagmin (16,17).…”

Structural Characterization of the C2 Domains of Classical Isozymes of Protein Kinase C and Novel Protein Kinase Cε by using Infrared Spectroscopy

“…Several studies have shown that activation of classical PKCs involves conformational changes, including the removal of the pseudosubstrate region from the active site of PKC [60,61]. Thus, ours results suggest that the C2 domain might also be involved in this conformational change during lipid binding which leads to protein activation…”