Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (␣, I, II, ␥, ␦, ⑀, , , and ) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position ؉1 carboxyl-terminal of the phosphorylated Ser and basic residues at position ؊3. All isozymes, except PKC, selected peptides with basic amino acids at positions ؊6, ؊4, and ؊2. PKC␣, -I, -II, -␥, and -selected peptides with basic amino acid at positions ؉2, ؉3, and ؉4, but PKC␦, -⑀, -, and -preferred peptides with hydrophobic amino acid at these positions. At position ؊5, the selectivity was quite different among the various isozymes; PKC␣, -␥, and -␦ selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKC showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position ؊3 to ؉2 for PKC␣, -I, -II, -␥, -␦, and -were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKC␣, -I, -␦, -, and -were prepared and used for the determination of K m and V max for these isozymes. As judged by V max /K m values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft. Protein kinase C (PKC)1 family members play crucial roles in the signal transduction of a variety of extracellular stimuli, such as hormones and growth factors (1). To date, twelve isozymes of PKC have been identified in mammalian tissues and subdivided into conventional PKC (cPKC) members comprising ␣, I, II, and ␥ isoforms (activated by calcium, acidic phospholipid, and diacylglycerol (DAG)), novel PKCs (nPKC) comprising ␦, ⑀, , and (activated by DAG and acidic phospholipid but insensitive to calcium), and atypical PKCs (aPKC) / and (mechanism of regulation not clear) (1-6). Another subgroup of PKCs may be defined by PKC, which has a potential signal peptide and transmembrane domain (7). Since these PKC isozymes differ in their expression in different tissues and in their mode of activation (1), each isozyme may play some specific role in signal transduction processes. Recent investigations using various approaches such as overexpression and down-regulation of specific isozymes support this idea (1,5). A large number of proteins have been shown to be phosphorylated by PKC in ...
Phosphatidylserine (PS) is a relatively minor constituent of biological membranes. Despite its low abundance, PS in the plasma membrane (PM) plays key roles in various phenomena such as the coagulation cascade, clearance of apoptotic cells, and recruitment of signaling molecules. PS also localizes in endocytic organelles, but how this relates to its cellular functions remains unknown. Here we report that PS is essential for retrograde membrane traffic at recycling endosomes (REs). PS was most concentrated in REs among intracellular organelles, and evectin-2 (evt-2), a protein of previously unknown function, was targeted to REs by the binding of its pleckstrin homology (PH) domain to PS. X-ray analysis supported the specificity of the binding of PS to the PH domain. Depletion of evt-2 or masking of intracellular PS suppressed membrane traffic from REs to the Golgi. These findings uncover the molecular basis that controls the RE-to-Golgi transport and identify a unique PH domain that specifically recognizes PS but not polyphosphoinositides. cholera toxin | endocytosis
Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that release nucleoside 5-monophosphate from a variety of nucleotides and nucleotide derivatives. The mammalian NPP family comprises seven members, but only three of these (NPP1-3) have been studied in some detail. Previously we showed that lysophospholipase D, which hydrolyzes lysophosphatidylcholine (LPC) to produce lysophosphatidic acid, is identical to NPP2. More recently an uncharacterized novel NPP member (NPP7) was shown to have alkaline sphingomyelinase activity. These findings raised the possibility that other members of the NPP family act on phospholipids. Here we show that the sixth member of the NPP family, NPP6, is a choline-specific glycerophosphodiester phosphodiesterase. The sequence of NPP6 encodes a transmembrane protein containing an NPP domain with significant homology to NPP4, NPP5, and NPP7/ alkaline sphingomyelinase. When expressed in HeLa cells, NPP6 was detected in both the cells and the cell culture medium as judged by Western blotting and by enzymatic activity. Recombinant NPP6 efficiently hydrolyzed the classical substrate for phospholipase C, p-nitrophenyl phosphorylcholine, but not the classical nucleotide phosphodiesterase substrate, p-nitrophenyl thymidine 5-monophosphate. In addition, NPP6 hydrolyzed LPC to form monoacylglycerol and phosphorylcholine but not lysophosphatidic acid, showing it has a lysophospholipase C activity. NPP6 showed a preference for LPC with short (12:0 and 14:0) or polyunsaturated (18:2 and 20:4) fatty acids. It also hydrolyzed glycerophosphorylcholine and sphingosylphosphorylcholine efficiently. In mice, NPP6 mRNA was predominantly detected in kidney with a lesser expression in brain and heart, and in human it was detected in kidney and brain. The present results suggest that NPP6 has a specific role through the hydrolysis of polyunsaturated LPC, glycerophosphorylcholine, or sphingosylphosphorylcholine in these organs.Nucleotide pyrophosphatases/phosphodiesterases (NPPs) 1 are ubiquitous membrane-associated or secreted ectoenzymes that have a role in regulating extracellular nucleotide metabolism (1, 2). They act by hydrolyzing a variety of nucleotides and nucleotide derivatives such as ATP and ADP. The mammalian NPP family has had five members (NPP1-5) (1, 2) that fall within two subgroups. NPP1-3 are type II transmembrane glycoproteins (ϳ900 amino acids) that have similar modular structures composed of a short amino-terminal intracellular domain, a single transmembrane domain, two somatomedin B-like motifs, a conserved catalytic site, a nuclease-like sequence, and a putative carboxyl-terminal "EF-hand" motif (Fig. 1D). In contrast, NPP4 (1, 2) and NPP5 (1-3) have a shorter structure (ϳ450 amino acids), a predicted type I transmembrane orientation, a short intracellular carboxyl-terminal domain, and a conserved catalytic site. The extracellular domains of NPP4 and NPP5 contain only a phosphodiesterase motif (Fig. 1D). Recently two additional ...
Infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7, which causes diarrhea and hemorrhagic colitis in humans, often results in fatal systemic complications, such as neurological damage and hemolytic-uremic syndrome. Because Stx circulating in the blood is a major causative factor of these complications, the development of a Stx neutralizer that functions in the circulation holds promise as a viable therapy. Here we developed a series of carbosilane dendrimers, in which trisaccharides of globotriaosyl ceramide, a receptor for Stx, were variously oriented at their termini (referred to as SUPER TWIG), and identified a SUPER TWIG with six trisaccharides as a Stx neutralizer functioning in the circulation. This SUPER TWIG specifically bound to Stx with high affinity (Kd ؍ 1.1 ؋ 10 ؊6 M) and inhibited the incorporation of the toxin into target cells. Intravenous administration of the SUPER TWIG along with Stx to mice substantially reduced the fatal brain damage and completely suppressed the lethal effect of Stx. Moreover, the SUPER TWIG protected mice from challenge with a fatal dose of E. coli O157:H7, even when administered after the establishment of the infection. The SUPER TWIG neutralized Stx in vivo by a mechanism in which the accumulation and immediate degradation of Stx by phagocytic macrophages present in the reticuloendothelial system were induced. Taken together, our findings indicate that this SUPER TWIG is therapeutic agent against infections by Stx-producing E. coli.
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