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

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We constructed deletion mutations which removed N-terminal coding sequences of various lengths from a cloned polyoma middle-size T antigen (MT antigen) gene. We introduced the MT antigen genes into a simian virus 40 expression vector so that they were expressed at high levels under the control of the simian virus 40 late promoter in COS-1 cells. The deletion mutant genes synthesized truncated MT antigens whose size was consistent with the deletion of either 70 or 106 amino acids from N termini, owing to initiation of translation at internal methionine codons in the MT antigen-coding region. The truncated MT antigens were found in cell membrane fractions but failed to show MT antigen-associated protein kinase activity. The cloned deletion mutant DNAs failed to transform rat F2408 or mouse NIH 3T3 cells. Therefore, N-terminal amino acid sequences of the polyoma MT antigen, as well as C-terminal sequences, are important for protein kinase activity and cell transformation.
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PMID:N-terminal amino acid sequences of the polyoma middle-size T antigen are important for protein kinase activity and cell transformation. 632 68

The phosphorylation of retinoic acid receptor-alpha 1 (RAR alpha 1) by PKA was investigated both in vitro and in vivo. We show that bacterially expressed RAR alpha 1 is phosphorylated in vitro by protein kinase A (PKA) at the unique serine residue 369 located in the C-terminal end of the E region. We also show that RAR alpha 1 overexpressed in COS-1 cells is phosphorylated on multiple serine residues and that phosphorylation at serine 369 occurs only when COS-1 cells are cotransfected with PKA or treated with forskolin. RAR alpha 1 mutants were constructed in which serine 369 was replaced by an alanine (S369A) or a glutamic acid (S369E) residue. Comparison of the tryptic phosphopeptide patterns of wild type and mutated RAR alpha 1 overexpressed in COS-1 cells allowed us to confirm that serine 369 is the unique phosphorylation site for PKA in cultured cells. The DNA-binding efficiency of RAR alpha/retinoid X receptor-alpha (RXR alpha) heterodimers was enhanced in vitro by the S369E mutation. However, in transfected RAC65 cells, the same S369E mutation did not affect the ligand-dependent transcriptional activation by RAR alpha 1 of reporter genes containing a retinoic acid (RA)-response element. In contrast, the S369A mutation slightly decreased both DNA binding and the efficiency of PKA to enhance RA-induced transactivation by RAR alpha 1. Finally, we show that endogenous RAR alpha is also phosphorylated in vivo at serine 369 in forskolin-treated F9 cells, supporting the idea that phosphorylation of RARs at this site is involved in the modulation of the RA-induced differentiation of F9 cells by (Bu)2cAMP.
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PMID:Phosphorylation of the retinoic acid receptor-alpha by protein kinase A. 747 69

cDNA clones encoding the third member of the RAC protein kinase family, termed RAC-PK gamma, were isolated from a rat brain cDNA library. The deduced amino acid sequence of RAC-PK gamma was highly related to those of previously identified family members, RAC-PK alpha and beta, that have a pleckstrin homology domain and a protein-serine/threonine kinase catalytic domain at the amino- and carboxyl-terminal regions, respectively. Northern blot analysis indicated that RAC-PK gamma was expressed abundantly in brain and testis. Specific activities of RAC-PK alpha, beta, and gamma purified from transfected COS-7 cells were similar when measured by using myelin basic protein as a phosphate acceptor. Analysis using fusion proteins of glutathione S-transferase revealed that the pleckstrin homology domain of the three subtypes of RAC-PK associate with both protein kinase C subspecies and beta gamma subunits of G proteins. These results suggest that the pleckstrin homology domains of RAC protein kinase family could associate more than one protein to regulate the activity and/or intracellular distribution of this enzyme family by different ways.
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PMID:Molecular cloning and characterization of a new member of the RAC protein kinase family: association of the pleckstrin homology domain of three types of RAC protein kinase with protein kinase C subspecies and beta gamma subunits of G proteins. 748 43

The interferon-induced P1/eIF-2 alpha protein kinase cDNA, designated PKR, was expressed both in Escherichia coli and in transfected monkey COS cells. TrpE-PKR fusion proteins and PKR nonfusion proteins were examined for their RNA-binding activity by Northwestern blot analysis. PKR is a RNA-binding protein that possesses two copies of a highly conserved motif, RI and RII, within the N-terminal region of the protein. Amino acid residues between 11 and 243 of PKR, which includes both copies of the R motif, displayed RNA-binding activity comparable to that of the full-length 551-amino-acid PKR protein. Analysis of substitution and deletion mutant PKR proteins revealed that motif RI was both necessary and sufficient for RNA-binding activity, whereas motif RII was not. Substitution of the highly conserved lysine at position 64 within the RI motif abolished RNA-binding activity, both of full-length PKR and the N-terminal 243-amino-acid truncated PKR. Finally, PKR substitution and deletion mutant cDNAs deficient for kinase function were expressed to much higher levels in transfected monkey cells than was the full-length wild-type PKR cDNA.
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PMID:Mechanism of interferon action motif I of the interferon-induced, RNA-dependent protein kinase (PKR) is sufficient to mediate RNA-binding activity. 750 74

Phosphorylation of purified Na+,K(+)-ATPase by cAMP-dependent protein kinase (protein kinase A) decreases the activity of this enzyme. We have now shown, using several experimental approaches, that a highly conserved seryl residue on the catalytic (alpha) subunit of Na+,K(+)-ATPase, corresponding to Ser943 of the rat alpha 1 isoform, is the phosphorylation site for protein kinase A. cDNAs corresponding to wild-type Na+,K(+)-ATPase and Na+,K(+)-ATPase in which Ser943 was mutated to Ala were transfected into COS cells. Treatment of the transfected cells with forskolin plus 3-isobutyl-1-methylxanthine resulted in a decrease in the activity of the wild-type enzyme but not in that of the mutated enzyme. The results suggest that, in intact cells, the activity of the Na+,K(+)-ATPase is regulated in part by signal transduction pathways that use protein kinase A-dependent phosphorylation of the Na+,K(+)-ATPase alpha subunit.
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PMID:Identification of the phosphorylation site for cAMP-dependent protein kinase on Na+,K(+)-ATPase and effects of site-directed mutagenesis. 751 Jul 9

Protein kinase C (PKC)-related cDNA clones isolated from cDNA libraries of mouse P19 embryonal carcinoma cells and mouse brain encoded a 67-kDa protein, PKC lambda. PKC lambda shows the highest amino acid sequence identity with PKC zeta (72%), the third class of the PKC family. Northern blot analysis showed that the mRNA for PKC lambda is expressed in a wide variety of cells and tissues, including P19 and NIH 3T3 cells, as well as brain, kidney, testis, and ovary. In undifferentiated P19 cells, the mRNA for PKC lambda is the most abundant among all the PKC family members. The differentiation of P19 cells results in an increase in PKC alpha and epsilon, and a decrease in PKC lambda. Antiserum raised against a peptide of PKC lambda identified a 74-kDa protein in P19 cell extracts as well as in extracts from COS cells transfected with the PKC lambda expression plasmid. Autophosphorylation of the PKC lambda that immunoprecipitated with the specific antiserum was observed, indicating that PKC lambda possesses protein kinase activity. A phorbol ester binding assay using intact COS cells expressing PKC lambda failed to detect binding activity specific to PKC lambda at phorbol dibutylate concentrations up to 300 nM, suggesting that PKC lambda does not possess phorbol ester binding activity. These results, in conjunction with the results obtained in parallel experiments with PKC zeta and other PKC members, suggest a biochemical similarity between PKC lambda and zeta and their clear difference from other PKC members.
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PMID:A new member of the third class in the protein kinase C family, PKC lambda, expressed dominantly in an undifferentiated mouse embryonal carcinoma cell line and also in many tissues and cells. 751 93

The porcine group C rotavirus (Cowden strain) NSP3 protein (the group C equivalent of the group A gene 7 product, formerly called NS34) shares homology with known double-stranded RNA-binding proteins, such as the interferon-induced, double-stranded RNA-dependent protein kinase PKR. A clone of NSP3, expressed both in vitro and in COS-1 cells, led to the synthesis of minor amounts of a product with an M(r) of 45,000 (the expected full-length M(r) of NSP3) and major amounts of products with M(r)s of 38,000 and 8,000. Restriction enzyme digestion analysis prior to expression in vitro and amino-terminal sequence analysis suggest that the products with M(r)s of 38,000 and 8,000 are cleavage products of the protein with an M(r) of 45,000. The full-length protein and the product with an M(r) of 8,000, both of which contain the motif present in double-stranded RNA-binding proteins, bound specifically to double-stranded RNA. The products with M(r)s of 45,000 and 8,000 were also detected in Cowden strain-infected MA104 cells. NSP3 products expressed in COS-1 cells were capable of inhibiting activation of the double-stranded RNA-dependent protein kinase similar to other double-stranded RNA-binding proteins, and NSP3 products expressed in HeLa cells were capable of rescuing the replication of an interferon-sensitive deletion mutant of vaccinia virus.
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PMID:Products of the porcine group C rotavirus NSP3 gene bind specifically to double-stranded RNA and inhibit activation of the interferon-induced protein kinase PKR. 751 79

B lymphocytes which reside in the germinal center region of lymphoid follicles are functionally and phenotypically distinct from the surrounding mantle zone B cells. We have isolated cDNA clones for several genes that are differentially expressed between these two populations of B lymphocytes. One such gene, BL44, is preferentially expressed in germinal center B cells. The nucleotide sequence of a 2,874-base pair BL44 cDNA was determined and a 2,451-bp open reading frame found that encodes for a 97-kDa serine/threonine protein kinase referred to as GC kinase. It has an NH2-terminal catalytic domain most similar to that of the Drosophila NinaC protein and the yeast STE20 protein. GC kinase mRNA transcripts are not unique to germinal center B cells and are found in several other tissues, including brain, lung, and placenta. The GC kinase protein was immunoprecipitated from transfected COS cells and from the Burkitt cell line RAMOS. GC kinase immunoprecipitated from transfected COS cells phosphorylated the substrates casein and myelin basic protein. In addition, a 97-kDa phosphoprotein likely to be GC kinase itself was detected. GC kinase may participate in an important signal transduction pathway in germinal center B cells.
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PMID:Differential expression of a novel protein kinase in human B lymphocytes. Preferential localization in the germinal center. 751 85

We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.
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PMID:Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides. 751 72

The interferon-inducible, RNA-dependent protein kinase (PKR) is an important regulator of viral protein synthesis. Activated PKR inhibits protein synthesis by phosphorylating initiation factor eIF-2 alpha. The reovirus S4 gene, whose 1196 nucleotide mRNA transcript does not activate the PKR kinase, is efficiently expressed in vector-transfected monkey COS cells. By contrast, the 1463 nucleotide S1 gene of reovirus, which is a potent activator of PKR, is poorly expressed in COS cells. Virus genetic engineering was therefore used to examine the effect of the PKR activator sequence from the reovirus S1 gene on the expression of chimeric genes of reovirus in transfected COS cells. Chimeric S1/S4 and S4/S1/S4 reovirus constructions that included the PKR activator sequence from S1 in the sigma 3 ORF of S4 were expressed much less efficiently than wild-type S4. However, expression of sigma 3 from S4 (3'UTR/S1), which included the PKR activator sequence from S1 within the 3'-UTR of S4, was comparable to that from wild-type S4. Treatment of COS cells with 2-aminopurine, an inhibitor of PKR, increased the expression of the reovirus S1, S1/S4, and S4/S1/S4 chimeric genes but not the S4 gene or S4 (3'UTR/S1) chimera in transfected COS cells. Likewise, coexpression of the phosphotransfer-negative mutant PKR (K296R) increased the expression of reovirus S1, S1/S4 and S4/S1/S4 chimeric genes but not the S4 gene or S4 (3'UTR/S1) chimera in cotransfected COS cells. Truncated PKR(1-243) which includes the dsRNA binding domain but not the kinase catalytic subdomains was able to enhance the expression of reovirus S1, but did not affect S4 expression. The dsRNA binding protein E3L encoded by vaccinia virus also increased S1 expression similar to PKR (1-243) and PKR(K296R). These results suggest that the translational repression in vivo mediated by PKR is selective for mRNAs that possess the kinase activator region, and that the dominant negative effect of PKR on gene expression is likely mediated by the RNA binding activity of the PKR protein.
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PMID:Mechanism of interferon action. Translational control and the RNA-dependent protein kinase (PKR): antagonists of PKR enhance the translational activity of mRNAs that include a 161 nucleotide region from reovirus S1 mRNA. 752 9


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