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

Abstract

A monoclonal anti-idiotypic antibody, Id8F7, previously shown to bind to a phosphatidylserine (PS)-specific binding site on protein kinase C (PKC) has been used to identify a 12-amino acid consensus sequence shared by PKC and phosphatidylserine decarboxylase (PSD). The 14-amino acid synthetic peptide derived from the corresponding region of PSD (amino acids 351-364 of the enzyme from Chinese hamster ovary cells) bound effectively and specifically to PS, and that derived from rat PKC␥ (amino acids 227-240) bound weakly but specifically to PS. Analysis of binding of Id8F7 to various synthetic peptides revealed that the consensus sequence motif, FXFXLKXXXKXR, is responsible for the interaction with both Id8F7 and PS. The results suggest that the conserved amino acid residues represent a basic structural motif for the specific interaction with PS, and the corresponding regions of PKC and PSD form the PS-specific binding sites of these enzymes.Although it is generally accepted that phospholipids in membranes are essential for the catalytic activity of many membrane-bound enzymes, it is still unclear how phospholipids regulate the activity of the enzymes. Phosphatidylserine (PS) 1 in membranes is known to be an essential cofactor for the activation of protein kinase C (PKC) (1-3) and blood coagulation (4 -6). 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). 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). However, it has been difficult to validate the presence of the PS-specific binding site on PKC, since the enzyme interacts with multiple phospholipid molecules during activation. Our previous studies showed that the binding to PKC of the monoclonal anti-idiotypic antibody Id8F7 raised against the combining site of the PS-specific antibody is inhibited by PS but not by other phospholipids including the synthetic PS analogue, 1,2-diacyl-sn-glycero-3-phospho-D-serine (18,19). The binding of Id8F7 to PKC is significantly enhanced by the presence of diacylglycerol and is independent of the presence of Ca 2ϩ (19). These observa...

Identification of Novel Anionic Phospholipid Binding Domains in Neutral Sphingomyelinase 2 with Selective Binding Preference

“…As a result, the key positively charged amino acid residues of these binding domains can form electrostatic bonds with the negative changed heads of APLs while the HSs inserts into the membrane, resulting direct and specific interaction between nSMase2 and membrane. Finally, the identified domains required for APL binding resemble those of other PS-acting enzymes such as PKC and phosphatidylserine decarboxylase (42). Altogether, the above evidences support that the direct interactions exist between nSMase2 and APLs.…”

“…Of these sequences, the amino acids Arg-33, Arg-45, and Arg-48 were crucial for lipid binding to the first domain whereas Arg-92 and Arg-93 were critical residues in the second domain. In previous studies, the motif "FXFXLKXXXKXR" was found in the APL-binding C2 domain in PKC and other enzymes (42). Notably, a similar peptide sequence "FLFGRSEIR" was found in the C terminus of Isc1p and was also demonstrated to be important for PS binding (36).…”

Control of Protein Kinase C Activity, Phorbol Ester-induced Cytoskeletal Remodeling, and Cell Survival Signals by the Scaffolding Protein SSeCKS/GRAVIN/AKAP12

“…Here, we confirm that SSeCKS binds PS independently and at levels likely to be physiologically relevant. The affinity of SSeCKS for PS is likely facilitated by the potential PS-binding motif, 506 FSSSGLKKLSGKKQR 520 , which is homologous (bold residues) to the known PS-binding motif in PKC, FXFX-LKXXXXKXXR (53). Interestingly, SSeCKS also binds phosphatidic acid and phosphatidylglycerol albeit at affinities 2-4-fold lower than for PS.…”

Phosphatidylserine receptor is required for the engulfment of dead apoptotic cells and for normal embryonic development in zebrafish

“…This is similar to the corresponding molecular weight for the mouse species , slightly smaller than the corresponding figure for the gene products in humans (45.5 kDa) and D. melanogaster (45 kDa), but larger than the analogous figure for C. elegans (38.4 kDa). In addition, the consensus sequence for the PSR-binding motif (FxFxLKxxxKxR) found in protein kinase C isoforms indicates that a 12-amino acid peptide motif is responsible for the specific interaction with PSR (Igarashi et al, 1995). A potential tyrosine phosphorylation site is indicated by box A ( Fig.…”