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PubMed2000

A novel adapter protein employs a phosphotyrosine binding domain and exceptionally basic N-terminal domains to capture and localize an atypical protein kinase C: characterization of Caenorhabditis elegans C kinase adapter 1, a protein that avidly binds protein kinase C3.

Atypical protein kinase C isoforms (aPKCs) transmit regulatory signals to effector proteins located in the cytoplasm, nucleus, cytoskeleton, and membranes. Mechanisms by which aPKCs encounter and control effector proteins in various microenvironments are poorly understood. By using a protein interaction screen, we discovered two novel proteins that adapt a Caenorhabditis elegans aPKC (PKC3) for specialized (localized) functions; protein kinase C adapter 1 (CKA1, 593 amino acids) and CKA1S (549 amino acids) are derived from a unique mRNA by alternative utilization of two translation initiation codons. CKA1S and CKA1 are routed to the cell periphery by exceptionally basic N-terminal regions that include classical phosphorylation site domains (PSDs). Tethering of PKC3 is mediated by a segment of CKA1 that constitutes a phosphotyrosine binding (PTB) domain. Two aromatic amino acids (Phe(175) and Phe(221)) are indispensable for creation of a PKC3-binding surface and/or stabilization of CKA1.aPKC complexes. Patterns of CKA1 gene promoter activity and CKA1/CKA1S protein localization in vivo overlap with patterns established for PKC3 expression and distribution. Transfection experiments demonstrated that CKA1/CKA1S sequesters PKC3 in intact cells. Structural information in CKA1/CKA1S enables delivery of adapters to the lateral plasma membrane surface (near tight junctions) in polarized epithelial cells. Thus, a PTB domain and PSDs collaborate in a novel fashion in CKA1/CKA1S to enable tethering and targeting of PKC3. Avid ligation of a PKC isoform is a previously unappreciated function for a PTB module.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Efficient interaction of the vesicular stomatitis virus P protein with the L protein or the N protein in cells expressing the recombinant proteins.

Specific in vivo interaction between the phosphoprotein (P) and the large polymerase protein (L) from the Indiana serotype of vesicular stomatitis virus was studied using a two-hybrid system. Transfection of CHO cells with plasmids encoding GALPIND and VPLIND fusion proteins resulted in an easily detectable level of CAT activity, indicating that PIND and LIND associate in vivo in the absence of other viral proteins. Mutational studies of PIND demonstrated that both domains I and II of PIND are important for PIND-LIND association. In addition, casein kinase II (CKII)-mediated phosphorylation within domain I of PIND was necessary for efficient association with LIND. We have also used the two-hybrid system to show PIND interaction with NIND in vivo. PIND and NIND associated more strongly than PIND and LIND. A similar strong association was observed in heterologous interaction studies between Indiana and New Jersey serotype P and N proteins. Mutational studies of PIND demonstrated that, unlike what was found for PNJ-NNJ association, only the C-terminal region of the P protein was important for efficient association with NIND. Like PNJ, CKII-mediated phosphorylation within domain I of PIND was not required for P-N association and, like NNJ, the C-terminal five amino acids of the NIND protein were critical for P association with N. These results demonstrate the importance of phosphorylation and specific domains of the P protein in its interaction with the L and N proteins, which are necessary for viral transcription and replication, respectively.

Amino Acid Sequence↗
PubMed1951

Conversion of plasma protein to tissue protein without evidence of protein breakdown; results of giving plasma protein labeled with carbon 14 parenterally to dogs.

Labeled plasma proteins obtained from donor dogs, previously fed epsilon-C(14)-dl-lysine, have been given intravenously to recipient dogs. The disappearance of labeled globulin from the plasma at a rate considerably faster than albumin has been confirmed. Evidence suggesting that the mass of protein in solution in the extravascular, extracellular fluid is approximately equal to the plasma proteins in circulation has been derived from a study of the dilution of labeled plasma protein by repeated injections of non-labeled plasma protein. In a period of 7 days the transfer of C(14) from plasma to tissue proteins amounted to between 30 and 40 per cent of the activity in the labeled plasma protein injected intravenously. The conversion was accompanied by a very small loss of activity in the urine and expired air and the activity remained in the lysine residue of the liver and probably of other tissues. The data presented favor the view that plasma proteins are utilized in the body economy after partial catabolism within the cell area and provide no evidence of complete breakdown to the amino acid level.

Amino Acids↗
PubMed1997

The role of CCAAT/enhancer-binding protein alpha and a protein that binds to the activator-protein-1 site in the regulation of liver-specific expression of the winter flounder antifreeze protein gene.

Winter flounder, Pleuronectes americanus, produces antifreeze proteins (AFPs) to avoid freezing during winter. The AFP in the blood is synthesized by the liver and its annual level is regulated by environmental factors and somatotropin. The only intron (+106 to +602) of the AFP gene contains putative binding sites for liver-enriched transcription activators. Due to the lack of an appropriate fish liver cell line, a mammalian model system was used to demonstrate the liver-specific enhancer activity of the intron. Deletion analysis of the intron indicated nucleotide sequences between +192 bp and +334 bp were important for the enhancer activity. Further refinement showed that the element B (+303 to +322 bp) resided in this region interacted with proteins from rat nuclear extracts by DNase I footprinting and mobility shift analysis. Gel retardation with competition and supershift experiments defined element B to include binding sites for CCAAT/enhancer binding protein a (C/EBP alpha), a liver-enriched transcription factor, and a novel activator protein-1 (AP-1) site binding protein, designated as antifreeze enhancer binding protein (AEP). Residues important to DNA-protein interaction in element B were mapped by methylation interference. Mutations in element B by site-directed mutagenesis destroyed the enhancer activity of the intron. Ultraviolet crosslinking experiments using element B revealed the specific binding of two proteins of approximately 43 and 80 kDa. The present findings provide a mechanism(s) by which the tissue specificity of AFP gene expression can be achieved. Integration of the present information with the somatotropin-mediated signal transduction pathways has generated a working model for the hormonal regulation and seasonal expression of the AFP gene.

Animals↗
PubMed2005

Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induced syncytium formation.

Amphotropic murine leukemia virus (A-MuLV) utilizes the PiT2 sodium-dependent phosphate transporter as its cell surface receptor to infect mammalian cells. The process of A-MuLV infection requires cleavage of the R peptide from the envelope protein. This occurs within virions thereby rendering them competent to fuse with target cells. Envelope proteins lacking the inhibitory R peptide (e.g. envelope (R-) proteins) induce viral envelope-mediated cell-cell fusion (syncytium). Here we have performed studies to determine if cell signaling through protein kinases is involved in the regulation of PiT2-mediated A-MuLV envelope (R-)-induced syncytium formation. Truncated A-MuLV retroviral envelope protein lacking the inhibitory R peptide (R-) was used to induce viral envelope-mediated cell-cell fusion. Signaling through cyclic AMP to activate PKA was found to inhibit envelope-induced cell-cell fusion, whereas treatment of cells with PKA inhibitors H89, KT5720, and PKA Catalpha siRNA all enhanced this cell fusion process. It was noted that activation of PKC, as well as overexpression of PKCepsilon, up-regulated A-MuLV envelope protein-induced cell-cell fusion, whereas exposure to PKC inhibitors and expression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) inhibited syncytium formation. v-ras transformed NIH3T3 cells were highly susceptible to A-MuLV envelope-induced cell-cell fusion, whereas expression of a dominant-negative mutant of Ras (N17Ras) inhibited this cell fusion process. Importantly, activation of Raf-1 protein kinase also is required for A-MuLV envelope-induced syncytium formation. Expression of constitutively active BXB Raf supported, whereas expression of a dominant-negative mutant of Raf-1 (Raf301) blocked, A-MuLV-induced cell-cell fusion. These results indicate that specific cell signaling components are involved in regulating PiT2-mediated A-MuLV-induced cell-cell fusion. Selective pharmacological modulation of these signaling components may be an effective means of altering cell susceptibility to viral-mediated cytopathic effects.

Animals↗
PubMed1994

Escherichia coli signal peptides direct inefficient secretion of an outer membrane protein (OmpA) and periplasmic proteins (maltose-binding protein, ribose-binding protein, and alkaline phosphatase) in Bacillus subtilis.

Signal peptides of gram-positive exoproteins generally carry a higher net positive charge at their amino termini (N regions) and have longer hydrophobic cores (h regions) and carboxy termini (C regions) than do signal peptides of Escherichia coli envelope proteins. To determine if these differences are functionally significant, the ability of Bacillus subtilis to secrete four different E. coli envelope proteins was tested. A pulse-chase analysis demonstrated that the periplasmic maltose-binding protein (MBP), ribose-binding protein (RBP), alkaline phosphatase (PhoA), and outer membrane protein OmpA were only inefficiently secreted. Inefficient secretion could be ascribed largely to properties of the homologous signal peptides, since replacing them with the B. amyloliquefaciens alkaline protease signal peptide resulted in significant increases in both the rate and extent of export. The relative efficiency with which the native precursors were secreted (OmpA >> RBP > MBP > PhoA) was most closely correlated with the overall hydrophobicity of their h regions. This correlation was strengthened by the observation that the B. amyloliquefaciens levansucrase signal peptide, whose h region has an overall hydrophobicity similar to that of E. coli signal peptides, was able to direct secretion of only modest levels of MBP and OmpA. These results imply that there are differences between the secretion machineries of B. subtilis and E. coli and demonstrate that the outer membrane protein OmpA can be translocated across the cytoplasmic membrane of B. subtilis.

ATP-Binding Cassette Transporters↗
PubMed1992

Specificities of protein-protein and protein-DNA interaction of GABP alpha and two newly defined ets-related proteins.

The ets-related protein GABP alpha interacts with the four ankyrin-type (ANK) repeats of GABP beta to form a high-affinity DNA-binding complex that recognizes a site important for herpes simplex virus type I immediate early gene activation. To investigate the selectivity and specificity of the GABP complex, we have isolated two new ETS family members, termed ER81 and ER71. ER81 and GABP alpha were present in most tissues of adult mice, whereas ER71 was restricted to testis. We have compared the DNA-binding specificities of these proteins by binding site selection. GABP alpha, ER71, and ER81 recognized the common pentanucleotide DNA sequence 5'-CGGAA/T-3'. Although subtle differences were observed for nucleotide preferences flanking this pentanucleotide core, the overall similarity of the selected sequences was most striking. Given the observation that GABP alpha interaction with GABP beta requires its intact ETS domain, we further compared the ability of GABP beta to interact with other ETS proteins. GABP beta did not augment the DNA-binding activity of the highly similar ETS domains of ER81, ER71, or Ets-1. Moreover, probing of total tissue extracts with radiolabeled GABP beta demonstrated its exceedingly stringent specificity for GABP alpha. Given that the DNA-binding specificities of these ETS proteins are similar and that the protein-protein interactions between GABP beta and GABP alpha are highly specific, we conclude that the protein interactions determine the target site selection by GABP alpha.

Amino Acid Sequence↗
PubMed2004

A system based on specific protein-RNA interactions for analysis of target protein-protein interactions in vitro: successful selection of membrane-bound Bak-Bcl-xL proteins in vitro.

Ribosome display systems are very effective and powerful tools for in vitro screening of transcribed mRNAs that encode proteins (or peptides) with specific (known or unknown) functions. We have modified such a system by exploiting the interaction between a tandemly fused MS2 coat-protein (MSp) dimer and the RNA sequence of the corresponding specific binding motif, C-variant (or Cv). We placed the MSp dimer at the N-terminus of a nascent protein and the Cv binding motif was attached to the 5' end of the protein's mRNA. This configuration enhanced the stability of the ribosome-mRNA complex. We demonstrate here that this improved ribosome display system provides an effective method for identifying the gene for a protein that binds to a protein of interest. We visualized the formation of polysome complexes in this advanced polysome display by atomic force microscopy (AFM) and found that the AFM images of polysomes in our system were different from those observed in the case of conventional ribosome display systems. Our results suggest that our technology might usefully complement yeast two-hybrid assays.

Capsid Proteins↗
PubMed1989

Phosphorylation of P1, a high mobility group-like protein, catalyzed by casein kinase II, protein kinase C, cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II.

P1, a high mobility group-like nuclear protein, phosphorylated by casein kinase II on multiple sites in situ, has been found to be phosphorylated in vitro by protein kinase C, cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II on multiple and mostly distinct thermolytic peptides. All these enzymes phosphorylated predominantly serine residues, with casein kinase II and protein kinase C also labeling threonine residues. Both casein kinase II and second messenger-regulated protein kinases, particularly protein kinase C, might therefore be involved in the physiological regulation of multisite phosphorylation of P1.

Adenosine Triphosphate↗
PubMed1990

Phosphorylation of connexin 32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II.

Phosphorylation of connexin 32, the major liver gap-junction protein, was studied in purified liver gap junctions and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK), by protein kinase C (PKC) and by Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-PK II). Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 32 demonstrated that cAMP-PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1. PKC also phosphorylated seryl residues in additional peptides. CA2+/CaM-PK II phosphorylated serine and to a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL-amine (residues 228-239 based on the deduced amino acid sequence of rat connexin 32) was phosphorylated by cAMP-PK and by PKC, with kinetic properties being similar to those for other physiological substrates phosphorylated by these enzymes. Ca2+/CaM-PK II did not phosphorylate the peptide. Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated that Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by cAMP-PK or by PKC. In hepatocytes labeled with [32P]orthophosphoric acid, treatment with forskolin or 20-deoxy-20-oxophorbol 12,13-dibutyrate (PDBt) resulted in increased 32P-incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the cAMP-PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced by treatment with PDBt. Thus, activation of PKC may have differential effects on junctional permeability in different cell types; one source of this variability may be differences in the sites of phosphorylation in different gap-junction proteins.

Amino Acid Sequence↗
PubMed2001

Cytoplasmic retention of HIV-1 regulatory protein Vpr by protein-protein interaction with a novel human cytoplasmic protein VprBP.

Vpr is an HIV-1 auxiliary regulatory protein packaged in the virion. It has been shown to enhance the nuclear transport of the HIV-1 pre-integration complex, activate transcription of cellular and viral promoters, and arrest the cell cycle at the G2/M check-point. We previously identified a cellular protein of 180 kDa (RIP) that interacted with HIV-1 Vpr specifically. We now rename this cellular protein as Vpr-binding protein, or VprBP. In this report, we describe the cloning of the VprBP cDNA that encodes 1507 aa residues and is identical to the previously cloned cDNA KIAA0800. We demonstrate that Vpr specifically interacts with recombinantly expressed VprBP in vitro as well as in vivo. Furthermore, Vpr interacts with the cellular endogenous VprBP in the context of the HIV-1 life cycle. Mutational analysis of VprBP suggests that the Vpr binding domain is located within the C-terminal half of VprBP, which has a Pro-rich domain and several Phe-x-x-Phe repeats. Subcellular fractionation studies show that both the endogenous VprBP and the adenovirus-expressed VprBP are distributed predominantly in the cytoplasmic fraction. Consistent with previous reports, the adenovirus-expressed Vpr is distributed in both the cytoplasmic and the nuclear fractions. However, when VprBP and Vpr are expressed together, Vpr is found almost exclusively in the cytoplasm. Expression of VprBP does not affect the nuclear transport of the adenoviral nuclear protein, pTP. VprBP expressed in insect cells also blocks the nuclear transport of a Vpr-GFP fusion protein, and VprBP mutants incapable of interacting with Vpr fail to block Vpr-GFP nuclear transport. We hypothesize that Vpr interaction with VprBP may cause changes in the host cell cytoplasm that affect HIV-1 pathogenesis as well as HIV-1 replication.

Amino Acid Sequence↗
PubMed1989

Diagnosis and treatment of homozygous protein C deficiency. Report of the Working Party on Homozygous Protein C Deficiency of the Subcommittee on Protein C and Protein S, International Committee on Thrombosis and Haemostasis.

This report summarizes the documented cases of homozygous protein C deficiency in the United States and Europe. Procedures for diagnosing and treating this disorder (both initially and over the long term) have been compiled by a working party on homozygous protein C deficiency of the Subcommittee on Protein C of the International Committee on Thrombosis and Haemostasis. Homozygous protein C deficiency is an autosomal recessive disorder that usually manifests itself by purpura fulminans and, less commonly, by massive large vein thrombosis; severe diffuse intravascular coagulation also develops in these infants, and there is evidence of intrauterine thrombosis. For confirmation of homozygous protein C deficiency in a neonate with purpura fulminans or massive venous thrombosis, the infant should have undetectable protein C activity and both parents should be heterozygous for protein C deficiency. At the onset of symptoms, the initial treatment should be plasma (8 to 12 ml/kg every 12 hours) until all lesions have healed. Two modalities for long-term treatment are accepted as useful in these children: oral anticoagulant therapy or protein C replacement (fresh frozen plasma or prothrombin complex concentrate). Liver transplantation has been performed in only one child, with success. Oral anticoagulation (vitamin K antagonists, maintaining the prothrombin time from one and one-half to two times control values or at the International Normalized Ratio of 2.5 to 4.4) is our recommendation of choice for long-term treatment. With appropriate care, these children are able to be free of coagulopathy and live relatively normal lives.

Anticoagulants↗
PubMed1983

Use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: the replication initiator protein of plasmid R6K.

The initiation of DNA replication of plasmid R6K is triggered by a 35-kilodalton initiator protein. The initiator protein had been elusive because of its lability and the lack of a convenient assay procedure to aid its purification. Using recombinant DNA techniques, we have fused the cistron of the initiator near its COOH-terminal end, in the correct reading frame, to the lacZ cistron of Escherichia coli at the ninth codon from the NH2 terminus. The fused cistron yielded a protein that was not only stable in vivo but also had dual activities: initiation of DNA replication in vivo and in vitro and hydrolysis of beta-galactoside. Using an affinity column that is specific for beta-galactosidase, we have demonstrated the rapid purification of the hybrid protein to near homogeneity. Exploiting the polymeric structure of the initiator, we have also isolated the nonfused form of the initiator protein, associated through subunit interaction with the beta-galactosidase-fused protein, which permits its purification by affinity chromatography. NH2-terminal amino acid sequence analysis of the heteropolymer has not only shown that the fused and nonfused initiators have the same sequence but also confirmed the protein sequence of the initiator as predicted from its nucleotide sequence. The techniques described here should be generally useful for the isolation of other proteins that are difficult to purify by conventional procedures.

Bacterial Proteins↗
PubMed1993

Identification of AML-1 and the (8;21) translocation protein (AML-1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions.

The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) translocation associated with acute myelogenous leukemia and encodes a protein with a central 118-amino-acid domain with 69% homology to the Drosophila pair-rule gene, runt. We demonstrate that AML-1 is a DNA-binding protein which specifically interacts with a sequence belonging to the group of enhancer core motifs, TGT/cGGT. Electrophoretic mobility shift analysis of cell extracts identified two AML-1-containing protein-DNA complexes whose electrophoretic mobilities were slower than those of complexes formed with AML-1 produced in vitro. Mixing of in vitro-produced AML-1 with cell extracts prior to gel mobility shift analysis resulted in the formation of higher-order complexes. Deletion mutagenesis of AML-1 revealed that the runt homology domain mediates both sequence-specific DNA binding and protein-protein interactions. The hybrid product, AML-1/ETO, which results from the (8;21) translocation and retains the runt homology domain, both recognizes the AML-1 consensus sequence and interacts with other cellular proteins.

Animals↗
PubMed1983

Acute phase proteins with special reference to C-reactive protein and related proteins (pentaxins) and serum amyloid A protein.

The acute phase response among plasma proteins is a normal response to tissue injury and is therefore a fundamental aspect of many diverse disease processes. It probably usually has a beneficial net function in limiting damage and promoting repair but in some circumstances it may have pathological consequences. Sustained high levels of acute phase proteins and especially SAA are associated with the development of amyloidosis in some individuals. Increased concentrations of CRP may, by activating the complement system, contribute to inflammation and enhance tissue damage. Failure of the normal or appropriate CRP response may also possibly have deleterious effects. SAA is a polymorphic protein which is normally present only in trace amounts but which, during the acute phase response, becomes one of the major apolipoproteins associated with high-density lipoprotein particles. The function of apoSAA is not known but it must have considerable physiological significance apart from its role as the putative precursor of amyloid A protein fibrils. CRP and SAP have been very stably conserved throughout vertebrate evolution and homologous proteins are apparently present even in vertebrates. This strongly suggests that they have important functions although these have not yet been precisely delineated. The main role of CRP may be to provide for enhanced clearance of inappropriate materials from the plasma whether these are of extrinsic origin, such as microorganisms and their products, or the autologous products of cell damage and death. The interaction between aggregated CRP and plasma low-density lipoprotein may play a significant part in the normal function of CRP and may also have a role in lipoprotein metabolism, clearance, and deposition. SAP is a normal tissue protein as well as being a plasma protein. Aggregated SAP selectively binds fibronectin and this may represent an aspect of the normal function of SAP. The deposition of SAP in amyloid is evidently not a normal function but it is not known whether this deposition is involved in the pathogenesis of amyloid or whether it is merely an epiphenomenon. In any case immunohistochemical staining for SAP is useful in the diagnosis of amyloid, in investigation of glomerulonephritis, and in studying disorders of elastic tissue. Regardless of its physiological or pathophysiological functions, the assay of serum CRP is a valuable aid to clinical management in a number of different situations and in different diseases provided results are interpreted in the light of full clinical information.(ABSTRACT TRUNCATED AT 400 WORDS)

Amyloid↗
PubMedDate not supplied

Intracellular signaling in arthropod blood cells: involvement of protein kinase C and protein tyrosine phosphorylation in the response to the 76-kDa protein or the beta-1,3-glucan-binding protein in crayfish.

Degranulation (regulated exocytosis) of crayfish granular blood cells and release of the prophenoloxidase activating system can be triggered by two endogenous ligands, a 76-kDa cell adhesion protein or a beta-1,3-glucan-binding protein when reacted with beta-1,3-glucan (beta GBP-L). These ligands bind a recently described membrane receptor. Degranulation triggered by these ligands was inhibited by the protein kinase C (pkC) inhibitor staurosporine or by the tyrosine kinase inhibitor herbimycin A. Incubation with the 76-kDa protein increased tyrosine phosphorylation of a granular cell protein of approximately 80 kDa. In addition, the pkC activating phorbol esters PMA or beta PDD degranulated the cells in a dose-dependent manner, whereas the control isomeric phorbol ester alpha PDD that does not activate pkC did not have any effect on the cells. Thus, we propose that binding of the 76-kDa protein or beta GBP-L to the receptor triggers cellular responses via a pathway that includes pkC activation and protein tyrosine phosphorylation.

Alkaloids↗
PubMed2002

Rabconnectin-3, a novel protein that binds both GDP/GTP exchange protein and GTPase-activating protein for Rab3 small G protein family.

Rab3A, a member of the Rab3 small G protein family, regulates Ca(2+)-dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP), and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). It remains unknown how or in which step of the multiple exocytosis steps these regulators are activated and inactivated. We isolated here a novel protein that was co-immunoprecipitated with Rab3 GEP and GAP by their respective antibodies from the crude synaptic vesicle fraction of rat brain. The protein, named rabconnectin-3, bound both Rab3 GEP and GAP. The cDNA of rabconnectin-3 was cloned from a human cDNA library and its primary structure was determined. Human rabconnectin-3 consisted of 3,036 amino acids and showed a calculated M(r) of 339,753. It had 12 WD domains. Tissue and subcellular distribution analyses in rat indicated that rabconnectin-3 was abundantly expressed in the brain where it was enriched in the synaptic vesicle fraction. Immunofluorescence and immunoelectron microscopy revealed that rabconnectin-3 was concentrated on synaptic vesicles at synapses. These results indicate that rabconnectin-3 serves as a scaffold molecule for both Rab3 GEP and GAP on synaptic vesicles.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Expression and intracellular localization of protein phosphatases 2A and 2B, protein kinase a, A-Kinase anchoring protein (AKAP79), and binding of the regulatory (RII) subunit of protein kinase a to AKAP79 in human myometrium.

OBJECTIVE: To determine the expression and intracellular localization of protein phosphatases 2A (PP2A) and 2B (PP2B), protein kinase A (PKA), and A-kinase anchoring protein (AKAP79), and expression of PKA (RII subunit) binding to AKAP79 in human postmenopausal and pregnant myometrium and to correlate their expressions to blood levels of estradiol, progesterone, and oxytocin. METHODS: Myometrial samples were taken from postmenopausal hysterectomy specimens (group 1, n = 5), from pregnant nonlaboring women (group 2, n = 7) and pregnant laboring women (group 3, n = 5) at cesarean. Western immunoblotting, immunohistochemical, and RII overlay assays were performed. Blood samples were assayed for estradiol, progesterone, and oxytocin levels. RESULTS: There were no significant differences in expression of PP2A, PKA, AKAP79, or PKA(RII) binding to AKAP79 between the three groups. Expression of PP2B was significantly greater in the nonlabor group (group 2) compared with groups 1 and 3. Protein phosphatase 2B, PKA, and AKAP79 expressions were localized in myometrial cytoplasm, but PP2A was localized in blood vessel endothelium. There was no significant correlation between the protein expression and the hormone level in the three groups. CONCLUSION: Human postmenopausal and pregnant (nonlabor and labor) myometrium expressed PP2A, PP2B, PKA, AKAP79, and PKA (RII)-AKAP79 binding. Levels of PP2A, PKA, and AKAP79 expression did not appear to be determinants of human myometrial contractility at parturition. Expression of PP2B may play a role in uterine quiescence. No association was found between protein expression and hormone level.

A Kinase Anchor Proteins↗