Translocation of pleckstrin requires its phosphorylation and newly formed ligands

Pleckstrin is the major substrate of protein kinase C (PKC) in platelets. We sought to determine whether pleckstrin phosphorylation is sufficient to target the soluble protein to binding sites. Permeabilization of platelets by streptolysin O (SLO) was used to separate bound and soluble pleckstrin. Platelets were incubated with phorbol 12-myristate 13-acetate (PMA) and/or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in the presence of [gamma-(32)P]ATP and SLO. PMA stimulated pleckstrin phosphorylation, but this pleckstrin diffused from permeabilized platelets. Addition of GTP[S] with PMA caused up to 40-50% of pleckstrin to be retained within platelets and enhanced secretion of platelet 5-hydroxytryptamine. PKC alpha pseudosubstrate peptide inhibited pleckstrin phosphorylation, the binding of pleckstrin and secretion. After extraction of permeabilized platelets containing bound pleckstrin with Triton X-100, the protein was solubilized. Thus, phosphorylated pleckstrin was retained in platelets only after activation of GTP-binding proteins that stimulate the formation of membrane-bound pleckstrin ligands. Translocation of pleckstrin may facilitate the associated secretion.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 1.

Double-stranded cDNA of alfalfa mosaic virus (AlMV) RNA 1 has been cloned and sequenced. From clones with overlapping inserts, and other sequence data, the complete primary sequence of the 3644 nucleotides of RNA 1 was deduced: a long open reading frame for a protein of Mr 125,685 is flanked by a 5'-terminal sequence of 100 nucleotides and a 3' noncoding region of 163 nucleotides, including the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common. The two UGA-termination codons halfway RNA 1, that were postulated by Van Tol et al. (FEBS Lett. 118, 67-71, 1980) to account for partial translation of RNA 1 in vitro into Mr 58,000 and Mr 62,000 proteins, were not found in the reading frame of the Mr 125,685 protein.     

Isolation and characterization of bovine and mouse terminal deoxynucleotidyltransferase cDNAs expressible in mammalian cells.

We have isolated nearly full-length cDNA clones of terminal deoxynucleotidyltransferase (TdT) from calf thymus and mouse lymphoma cDNA libraries. The libraries were constructed using the pcD vector system which permits the expression of cDNA inserts in mammalian cells. The bovine TdT cDNA clone contains an open reading frame coding for 520 amino acids, Mr 59,678. The mouse TdT cDNA clone contains an open reading frame of 1,587 bp, whose translated cDNA encodes a 60,004 dalton protein. The mouse TdT cDNA clone contains 60 bp in the 3' end region of the coding sequence not found in the bovine TdT cDNA sequence, otherwise, the clones share about 80% homology. A possible nuclear-localization-sequence (Pro-Arg-Lys-Lys-Arg-Pro-Arg) was conserved in the N-terminal region in the mouse and bovine cDNA clones. Bovine and mouse cDNAs transfected into COS7 monkey fibroblasts directed the synthesis of enzymatically active protein of Mr 60,000 which was detected immunologically using polyclonal rabbit antibody against bovine TdT. Bovine TdT expressed in COS7 cells by nearly full-length cDNA clone was localized in the nucleus and the translational product of pOK103 lacking the nuclear-localization-sequence was localized in the cytoplasm.     

Structure and phosphatidylinositol-(3,4)-bisphosphate binding of the C-terminal PH domain of human pleckstrin

Pleckstrin is the major target of protein kinase C (PKC) in blood platelets. Its phosphorylation triggers responses that ultimately lead to platelet activation and blood clot formation. Pleckstrin consists of three domains: a pleckstrin homology (PH) domain at both termini and a central DEP (Dishevelled, Egl-1, Pleckstrin) domain. Here, we report the solution nuclear magnetic resonance (NMR) structure of the C-terminal PH domain (C-PH) of human pleckstrin-1. We show that this PH domain binds phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) with high specificity in protein lipid overlay assays. Using NMR titration experiments and mutational analysis, residues involved in binding to PtdIns(3,4)P2 are identified. The binding site is formed by a patch of basic residues from the beta1 and beta2 strands and the beta1-beta2 loop. Since PtdIns(3,4)P2 is an important signaling molecule in platelets, our data suggest a C-PH dependent regulation of pleckstrin function in response to PtdIns(3,4)P2.     

Expression of the protein kinase C substrate pleckstrin in macrophages: association with phagosomal membranes.

Despite evidence suggesting that protein kinase C (PKC) isoforms are important in phagocytosis by Fcgamma receptors, the mechanisms by which the substrates of these kinases act are largely unknown. We have investigated the role of one PKC substrate, pleckstrin, in cells of the monocyte/macrophage lineage. Pleckstrin expression in mouse macrophages was induced severalfold in response to bacterial LPS and IFN-gamma. In unstimulated cells, the protein was largely confined to the cytosol. Upon ingestion of IgG-opsonized zymosan particles (OPZ), however, pleckstrin accumulated on the phagosomal membrane. This association was transient, being maximal after 15 min and declining thereafter. Similar kinetics of association was also seen for both filamentous actin and the delta isoform of PKC. Ingestion of OPZ was found to induce phosphorylation of pleckstrin. To examine whether phosphorylation was required for phagosomal association, pleckstrin was expressed in CHO-IIA cells that stably express the FcgammaRIIA receptor and are competent for phagocytosis of OPZ. In these cells, both wild-type pleckstrin and mutants in which the phosphoacceptor sites had been mutated to either alanine (nonphosphorylatable) or glutamine (pseudophosphorylated) were found to accumulate on OPZ phagosomes. Thus, association of pleckstrin with phagosomes is independent of its phosphorylation. Our findings suggest that pleckstrin may serve as an intracellular adaptor/targeting protein in response to particulate stimuli. By targeting interacting ligands to the phagosomal compartment, pleckstrin may serve to regulate phagocytosis and/or early steps during maturation of the phagosome.     

Phosphorylation of pleckstrin increases proinflammatory cytokine secretion by mononuclear phagocytes in diabetes mellitus

The protein kinase C (PKC) family of intracellular enzymes plays a crucial role in signal transduction for a variety of cellular responses of mononuclear phagocytes including phagocytosis, oxidative burst, and secretion. Alterations in the activation pathways of PKC in a variety of cell types have been implicated in the pathogenesis of the complications of diabetes. In this study, we investigated the consequences of PKC activation by evaluating endogenous phosphorylation of PKC substrates with a phosphospecific PKC substrate Ab (pPKC(s)). Phosphorylation of a 40-kDa protein was significantly increased in mononuclear phagocytes from diabetics. Phosphorylation of this protein is downstream of PKC activation and its phosphorylated form was found to be associated with the membrane. Mass spectrometry analysis, immunoprecipitation, and immunoblotting experiments revealed that this 40-kDa protein is pleckstrin. We then investigated the phosphorylation and translocation of pleckstrin in response to the activation of receptor for advanced glycation end products (RAGE). The results suggest that pleckstrin is involved in RAGE signaling and advanced glycation end product (AGE)-elicited mononuclear phagocyte dysfunction. Suppression of pleckstrin expression with RNA interference silencing revealed that phosphorylation of pleckstrin is an important intermediate in the secretion and activation pathways of proinflammatory cytokines (TNF-alpha and IL-1beta) induced by RAGE activation. In summary, this study demonstrates that phosphorylation of pleckstrin is up-regulated in diabetic mononuclear phagocytes. The phosphorylation is in part due to the activation of PKC through RAGE binding, and pleckstrin is a critical molecule for proinflammatory cytokine secretion in response to elevated AGE in diabetes.     

PL48: a novel gene associated with cytotrophoblast and lineage-specific HL-60 cell differentiation

The cDNA for a novel gene, PL48, isolated by subtractive hybridization between undifferentiated human term cytotrophoblast and differentiating cytotrophoblast, has been cloned and sequenced. PL48 contains an open reading frame coding for a 537-amino-acid protein, has multiple potential PKC, casein kinase II, and cAMP/cGMP-dependent kinase phosphorylation sites, and N-linked glycosylation sites. It is not present in a wide variety of proliferating cancer cells, but PL48 mRNA shows marked expression during cytotrophoblast and granulocyte lineage-specific HL-60 promyelocytic cell differentiation induced by DMSO.     

High-affinity L-arabinose transport operon. Nucleotide sequence and analysis of gene products

The nucleotide sequence of the "high-affinity" L-arabinose transport operon has been determined 3' from the regulatory region and found to contain three open reading frames designated araF, araG and araH. The first gene 3' to the regulatory region, araF, encodes the 23-residue signal peptide and the 306-residue mature form of the L-arabinose binding protein (33,200 Mr). The binding protein, which has been described elsewhere, is hydrophilic, soluble and found in the periplasm of Escherichia coli. This gene is followed by an intragenic space of 72 nucleotides, which contains a region of dyad symmetry 23 nucleotides long capable of forming an 11-member stem-loop. The second gene, designated araG, contains an open reading frame capable of encoding an equally hydrophilic protein containing 504 residues (55,000 Mr). Following a 14-nucleotide spacer, which does not appear to have any secondary structure, the third open reading frame, herein designated araH, is capable of encoding a hydrophobic protein containing 329 residues (34,000 Mr) that can only be envisioned as having an integral membrane location. 3' to araH there is a T-rich region containing a 24-nucleotide area of dyad symmetry centered 55 nucleotides from the termination codon. Analysis of the derived primary sequences of the araG and araH products indicates the nature and potential features of these components. The araG protein was found to possess internal homology between its amino and carboxyl-terminal halves, suggesting a common origin. The araG gene product has been shown to be homologous to the rbsA gene product, the hisP product, the ptsB product and the malK product, all of which presumably play similar roles in their respective transport systems. Putative ATP binding sites are observed within the regions of homology. The araH gene product has been shown to be homologous to the rbsC gene product, which is the first observed homology between two purported membrane proteins.     

The nucleotide sequence of tobacco rattle virus RNA-2 (CAM strain).

The nucleotide sequence of the smaller genomic strand (RNA-2) of the bipartite tobacco rattle virus (CAM strain) has been determined. RNA-2 is capped at the 5' terminus and contains 1799 nucleotide residues. There is a single 223 codon long open reading frame extending from nucleotide 574 to 1242 which designates a protein of Mr 23,654. The derived amino acid composition, in percent, matches that previously determined for the virus capsid protein. The long open reading frame is flanked by 5' and 3' untranslated regions of 573 and 554 nucleotides, respectively. The 5' leader sequence contains two different sets of direct repeats, one of 119 nucleotides and the other of 76. It also contains 13 apparently unused AUG codons, four of which lie in the same frame as the capsid protein cistron. The 3' terminal sequence of RNA-2 is identical to that of the larger genomic strand (RNA-1) for 459 nucleotides.     

Molecular cloning of the rat stomach (H+ + K+)-ATPase

We have isolated cDNA clones for the rat stomach (H+ + K+)-ATPase by employing a novel procedure involving the use of oligonucleotides corresponding to conserved amino acid sequences of related cation transport ATPase and a cross-hybridization with the sheep kidney (Na+ + K+)-ATPase alpha-subunit cDNA. The complete nucleotide sequence of the cDNA has been determined and the amino acid sequence of the protein deduced. The ATPase consists of 1,033 amino acids and has an Mr of 114,012. Amino acid homology and hydropathy plot comparisons between the gastric ATPase and the (Na+ + K+)-ATPase catalytic subunit demonstrate a striking similarity which suggests that their higher order structure and mechanism of action are virtually identical. The greatest homology occurs in the phosphorylation site region and in domains which may be involved in nucleotide binding and energy transduction. The most substantial differences occur in the N-terminal region and in the transmembrane domains. In addition, we report the presence of an open reading frame 5' to the translation initiation site of the gastric ATPase, which raises the possibility that the mRNA is polycistronic.