UNIPROT:P67775 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Target Concepts:

Query: UNIPROT:P67775 (alpha isoform)
797 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although the protein kinase inhibitors (PKIs) are known to be potent and specific inhibitors of the catalytic (C) subunit of cAMP-dependent protein kinase, little is known about their physiological roles. Glutamate 203 of the C alpha isoform (C alpha E203) has been implicated in the binding of the arginine 15 residue of the skeletal isoform of PKI (PKI alpha R15) (Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N., Taylor, S.S., and Sowadski, J. M. (1991) Science 253, 414-420). To investigate the role of C alpha E203 in the binding of PKI and in vivo C-PKI interactions, in vitro mutagenesis was used to change the C alpha E203 codon of the murine C alpha cDNA to alanine and glutamine codons. Initially, the C alpha E203 mutant proteins were expressed and purified from Escherichia coli. C alpha E203 is not essential for catalysis as all of the C subunit mutants were enzymatically active. The mutation of Glu203 did increase the apparent Km for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) severalfold but did not affect the apparent Km for ATP. The Vmax(app) was not affected by the mutation of C alpha E203. The mutation of C alpha E203 compromised the ability of PKI alpha (5-24), PKI alpha, and PKI beta to inhibit phosphotransferase activity. PKI alpha was altered using in vitro mutagenesis to probe the role of Arg15 in interacting with C alpha E203. The PKI alpha R15A mutant was reduced in its inhibition of C alpha. Preliminary studies of the expression of these C alpha mutants in COS cells gave similar results. These results suggest that the C alpha E203 mutants may be useful in assessing the role of PKI in vivo.
...
PMID:Glutamic acid 203 of the cAMP-dependent protein kinase catalytic subunit participates in the inhibition by two isoforms of the protein kinase inhibitor. 790 1

The major receptor protein for cyclic GMP (cGMP) in smooth muscle is the cGMP-dependent protein kinase (cGMP kinase). The more abundant I alpha isoform (subunit M(r) congruent to 78,000) of this enzyme mediates the effects of cGMP to relax contracted vascular smooth muscle preparations. In this study, we have addressed the hypothesis that the cGMP kinase is anchored to intracellular proteins which might serve to target cGMP kinase to protein substrates. Using a gel overlay technique, immunoprecipitation, and a fluorescence binding assay for cGMP kinase, we have identified vimentin as a high-affinity and specific binding protein for cGMP kinase. Binding of cGMP kinase to vimentin is reversible and stoichiometric (one cGMP kinase dimer/vimentin dimer) with a KD of approximately 49 nM. The site of high-affinity binding between cGMP kinase and vimentin did not appear to be localized to the catalytic domain of the kinase since vimentin phosphorylated by cGMP kinase and peptide substrates for cGMP kinase did not compete for high-affinity binding. Neither the proteolytically-derived 69-kDa catalytic fragment nor the 8-kDa N-terminal fragment bound vimentin with high affinity, suggesting that the cGMP kinase dimer was necessary for the interaction. Vimentin was readily phosphorylated in vitro with the dimer, but not the monomeric 69-kDa catalytic fragment even though the monomeric 69-kDa fragment was catalytically active toward other substrates such as histone F2b and peptides. This suggests that the high-affinity interaction between cGMP kinase and vimentin occurs at the N-terminal region, thus allowing the interaction between the phosphorylation site of vimentin and the catalytic site of cGMP kinase to occur.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:High-affinity binding and localization of the cyclic GMP-dependent protein kinase with the intermediate filament protein vimentin. 802 8

The protein kinase inhibitors (PKIs) are potent inhibitors of the catalytic (C) subunit of cAMP-dependent protein kinase. In this study, the interaction between Phe10 of PKI and the C subunit residues Tyr235 and Phe239 was investigated using site-directed mutagenesis. Previous peptide studies as well as the crystal structure suggested that these residues may play a key role in C-PKI binding. The C subunit codons for Tyr235 and Phe239 were changed singly and in combination to serine codons. The mutated C alpha proteins were overexpressed in Escherichia coli. The purified C alpha Y235S, C alpha F239S, and C alpha Y235S/F239S proteins did not exhibit any differences in their Km(app) for the peptide substrate Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) or Vmax(app), with respect to wild-type C alpha. All of the C subunit mutants displayed less than 2-fold changes in their Km(app) for ATP. The PKI alpha isoform displayed increased IC50 values for C alpha Y235S (71-fold), C alpha F239S (150-fold), and C alpha Y235S/F239S (1800-fold). Similarly, the PKI beta 1 protein showed increased IC50 values against the C alpha Y235S, C alpha F239S, and C alpha Y235S/F239S proteins, 9.4-, 11-, and 44-fold, respectively. In addition, the PKI alpha F10 codon was altered to an alanine codon, and this mutation decreased its ability to inhibit C alpha kinase activity, but did not affect its ability to inhibit C alpha Y235S/F239S. The mutation of Tyr235 and Phe239 to serines, however, did not alter the ability of the type II R subunit to inhibit phosphotransferase activity. These results suggest that C alpha Y235 and C alpha F239 are important for specific inhibition by both PKI alpha and PKI beta but not the type II R subunit and that mutations at these residues would be useful for in vivo analysis of C-PKI interactions.
...
PMID:Evidence for the importance of hydrophobic residues in the interactions between the cAMP-dependent protein kinase catalytic subunit and the protein kinase inhibitors. 802 74

The Type I cGMP-dependent protein kinase catalytic domain (residues 336-671 from the I alpha isoform) has been expressed as a cGMP independent kinase in a baculovirus system. Using peptide substrates, the protein retains similar substrate specificity as the native holoenzyme. The recombinant catalytic domain catalyzes the phosphorylation of histone, but does not display the inhibition using non-substrate histones which has been described for the holoenzyme. The catalytic domain is an active kinase in mammalian cells also since vascular smooth muscle cells transfected with the cDNA encoding the catalytic domain display altered morphology. The catalytic domain of G-kinase may be a useful tool for delineating the role of cGMP-mediated protein phosphorylation in cell systems.
...
PMID:Expression of the catalytic domain of cyclic GMP-dependent protein kinase in a baculovirus system. 815 80

A partial cDNA sequence indicated that the T lymphocyte early-activation gene (Tea) encodes a protein related to the dual-function ecotropic retrovirus receptor/cationic amino acid transporter (ecoR/CAT1), and RNA blots suggested highest Tea expression in T lymphocytes and liver (MacLeod, C.L., Finley, K., Kakuda, D. Kozad, C.A., and Wilkinson, M.F. (1990) Mol. Cell. Biol. 7, 3663-3674). The sequence of full-length Tea cDNA from liver (3683 bases) predicts a 657-amino-acid protein (CAT2 alpha) with 12-14 transmembrane domains. A long (515 base) region with six initiation codons and termination codons precedes the translation start codon. The liver Tea cDNA is identical to Tea cDNA from T lymphocytes (encoding CAT2 beta) with the exception of an apparent alternatively spliced sequence encoding a hydrophilic loop of 43 amino acids. The liver-specific sequence contains unique consensus sites for phosphorylation by cyclic AMP-dependent protein kinase and by protein kinase C. Injection of Xenopus oocytes with CAT2 alpha or CAT2 beta messenger RNA resulted in expression of Na(+)-independent cationic amino acid transport that was detected by current measurements under voltage-clamp. Although the amino acid sequences of the isoforms differ in only 21 of 43 residues with the majority of substitutions being conservative, the apparent affinity of CAT2 beta for arginine uptake was 70-fold higher than the CAT2 alpha isoform (Km 38 microM versus 2.7 mM). Neither isoform functioned as a receptor for ecotropic or amphotropic murine retroviruses. However, CAT1-CAT2 chimeric proteins that contain the first three putative extracellular loops of ecoR/CAT1 functioned as ecotropic receptors despite a diminished capacity to bind the viral envelope glycoprotein. The chimeric proteins also functioned as basic amino acid transporters with substrate affinities corresponding to the CAT2 isoform constituting the carboxyl-terminal portion. These results demonstrate that domains of these transporters can function in chimeric combinations to control viral receptor and transport functions.
...
PMID:Control of cationic amino acid transport and retroviral receptor functions in a membrane protein family. 819 86

Okadaic acid (2 nM) inhibited by 80-90% the protein phosphatase activities in diluted extracts of rat liver, human fibroblasts, and Xenopus eggs acting on three substrates (high mobility group protein-I(Y), caldesmon and histone H1) phosphorylated by a cyclin-dependent protein kinase (CDK) suggesting that a type-2A phosphatase was responsible for dephosphorylating each protein. This result was confirmed by anion exchange chromatography of rat liver and Xenopus extracts, which demonstrated that the phosphatases acting on these substrates coeluted with the two major species of protein phosphatase 2A, termed PP2A1 and PP2A2. When matched for activity toward glycogen phosphorylase, PP2A1 was five- to sevenfold more active than PP2A2 and 35-fold to 70-fold more active than the free catalytic subunit (PP2Ac) toward the three CDK-labeled substrates. Protein phosphatases 1, 2B, and 2C accounted for a negligible proportion of the activity toward each substrate under the assay conditions examined. The results suggest that PP2A1 is the phosphatase that dephosphorylates a number of CDK substrates in vivo and indicate that the A and B subunits that are associated with PP2Ac in PP2A1 accelerate the dephosphorylation of CDK substrates, while suppressing the dephosphorylation of most other proteins. The possibility that PP2A1 activity is regulated during the cell cycle is discussed.
...
PMID:Protein phosphatase 2A1 is the major enzyme in vertebrate cell extracts that dephosphorylates several physiological substrates for cyclin-dependent protein kinases. 840 Apr 54

A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H-7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH-SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH-SY5Y cells and studied their modifications during differentiation. The alpha, beta 1, delta, and epsilon isoforms were present in SH-SY5Y cells, as well as in rat brain. Protein kinase C-alpha and -beta 1 were the most abundant isoforms in SH-SY5Y cells, and immunoreactive protein kinase C-delta and -epsilon were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the alpha isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12-tetradecanoyl-13-acetyl-beta-phorbol or staurosporine, and that protein kinase C-epsilon is predominantly membrane-bound. Its localization did not change during differentiation. Western blots of total SH-SY5Y cell extracts and of subcellular fractions probed with isoform-specific polyclonal antibodies showed that when SH-SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C-alpha and -epsilon decreased, and protein kinase C-beta 1 did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase alpha and epsilon in neuritogenesis.
...
PMID:Differential expression and subcellular localization of protein kinase C alpha, beta, gamma, delta, and epsilon isoforms in SH-SY5Y neuroblastoma cells: modifications during differentiation. 841 48

The gene for the alpha isoform of Ca2+/calmodulin-dependent kinase II (alpha CaMKII) codes for a multifunctional protein kinase that is found exclusively in the brain. Here we show that in skeletal muscle, an alternative nonkinase product, hereafter referred to as alpha KAP (alpha CaMKII association protein), is expressed from the same gene. alpha KAP consists of a C-terminal region that is identical to the association domain of alpha CaMKII, with the exception of 11 amino acids inserted in the variable region. The N-terminal sequence of alpha KAP is highly hydrophobic and not present in any known CaMKII protein. The catalytic and regulatory domains of alpha CaMKII are missing in alpha KAP. Analysis of the exon-intron structure revealed that the alpha KAP transcript is derived from the alpha CaMKII gene by alternative promoter usage and RNA splicing. The transcriptional start site of alpha KAP mRNA is located within an intron of the alpha CaMKII gene. Therefore, the relationship between alpha KAP and alpha CaMKII is that of a gene within a gene. Immunostaining using anti-alpha KAP antibodies suggests that alpha KAP is associated with sarcomeres of skeletal muscle fibers. On the basis of its primary structure and specific location, the possible function of alpha KAP as an anchoring protein for CaMKII is discussed.
...
PMID:An alternative, nonkinase product of the brain-specifically expressed Ca2+/calmodulin-dependent kinase II alpha isoform gene in skeletal muscle. 852 7

Cyclic ADP-ribose (cADPR) is generated in pancreatic islets by glucose stimulation, serving as a second messenger for Ca2+ mobilization from the endoplasmic reticulum for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373). In the present study, we observed that the addition of calmodulin (CaM) to rat islet microsomes sensitized and activated the cADPR-mediated Ca2+ release. Inhibitors for CaM-dependent protein kinase II (CaM kinase II) completely abolished the glucose-induced insulin secretion as well as the cADPR-mediated and CaM-activated Ca2+ mobilization. Western blot analysis revealed that the microsomes contain the alpha isoform of CaM kinase II but do not contain CaM. When the active 30-kDa chymotryptic fragment of CaM kinase II was added to the microsomes, fully activated cADPR-mediated Ca2+ release was observed in the absence of CaM. These results along with available evidence strongly suggest that CaM kinase II is required to phosphorylate and activate the ryanodine-like receptor, a Ca2+ channel for cADPR as an endogenous activator, for the cADPR-mediated Ca2+ release.
...
PMID:Requirement of calmodulin-dependent protein kinase II in cyclic ADP-ribose-mediated intracellular Ca2+ mobilization. 853 Apr 41

Because of the low abundance of the two major isoforms (C alpha and C beta) of catalytic (C) subunit of cAMP-dependent protein kinase, it has been difficult to monitor their expression and virtually impossible to quantify their synthesis, phosphorylation, and turnover in intact mammalian cells. We now describe sensitive and quantitative immunochemical methods using a goat antibody raised against the recombinant C alpha isoform of murine C subunit that enable studies of the expression and metabolism of C subunit in cultured cells. The antibody reacts well with C alpha and C beta isoforms of murine C subunit and with C subunits from rat, hamster, and human cell lines, so it should have widespread utility. Immunoreactivity with bovine heart C subunit was substantially weaker. For quantitation of C subunit radioactivity in extracts of cells labeled metabolically with [35S]methionine, we developed a two-cycle immunoadsorption protocol that reduces nonspecific adsorption to negligible levels. A tritium-labeled, truncated C subunit marker protein is added to extracts as an internal marker to monitor C subunit recoveries in different samples. For analysis of expression of C subunit isoforms in different cells or tissues, we describe a nonradioactive Western immunoblot procedure that can quantitate C subunit in amounts as low as 12 pg. Using extraction conditions that either stabilize or destabilize the phosphate on Thr-197, we show how the relative expression and phosphorylation of C alpha and C beta isoforms can be estimated from SDS-gel patterns resulting from either immunoblot or immunoadsorption procedures.
...
PMID:Methods for studying synthesis, turnover, and phosphorylation of catalytic subunit of cAMP-dependent protein kinase in mammalian cells. 864 25

<< Previous 1 2 3 4 5 6 7 8 Next >>