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)

Murine promonocytic leukemias involving insertional mutagenesis of the c-myb locus can be induced by replication-competent retroviruses. In previously studied promonocytic leukemic cells induced by Moloney murine leukemia virus (called MML), the provirus has been invariably integrated upstream of exons 3 or 4 and the leukemic cells expressed aberrant RNAs with fused virus-myb sequences. Furthermore, Myb expressed by these cells has been shown to be truncated by 47 or 71 amino acids. The present report examines the mechanisms of myb activation in leukemias induced by two other retroviruses, amphotropic virus 4070A and Friend strain FB29 (the leukemias are called AMPH-ML and FB-ML, respectively). This study revealed two additional c-myb proviral insertion sites in these promonocytic leukemias. One FB-ML had a proviral integration in exon 9, and expressed a C-terminally truncated Myb protein of 47 kDa similar to that previously demonstrated to be expressed in the myelomonocytic cell lines NFS60 and VFL-2. However, a sequence of reverse-transcribed and amplified RNA from this leukemia demonstrated that the truncation involved a loss of 248 amino acids compared with a loss of 240 amino acids in the myelomonocytic cell lines. Another leukemia had a provirus integrated in the 5' end of c-myb upstream of exon 2 (in the first intron) and produced a Myb protein that was indistinguishable on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from normal Myb. This latter leukemia (FB-ML R1-4-10) expressed Myb with the smallest N-terminal truncation observed so far in promonocytic leukemias; translation begins at an ATG within c-myb exon 2, leading to loss of only 20 amino acids from the N terminus. Unlike the proteins produced in Moloney murine leukemia virus-induced promonocytic leukemias (MML) that have larger truncations, this protein has an intact DNA binding region and does not contain N-terminal amino acids encoded by gag. However, this protein is similar to all N-terminally truncated Mybs so far studied, in that the truncation resulted in deletion of a casein kinase II phosphorylation site which has been proposed to be involved in regulation of DNA binding.
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PMID:New sites of proviral integration associated with murine promonocytic leukemias and evidence for alternate modes of c-myb activation. 152 51

The v-erbA oncogene of avian erythroblastosis virus (AEV) encodes a ligand-independent mutated version of the chicken c-erbA alpha-encoded thyroid hormone receptor. The v-erbA gene product, a 75-kD gag/v-erbA fusion protein, is phosphorylated on Ser-16/17 of its v-erbA-encoded domain, and phosphorylation at this site is increased in vivo after activation of either the PKA or PKC signal transduction pathways. To test the hypothesis that phosphorylation of Ser-16/17 regulates gag/v-erbA protein function, mutant proteins in which Ser-16/17 had been changed to alanine or threonine residues were analyzed for their ability to inhibit erythroid differentiation of ts v-erbB or ts v-sea-transformed erythroblasts at nonpermissive temperature. Conversion of Ser-16/17 into alanine, although not affecting nuclear localization or DNA binding of the gag/erbA protein, prevented phosphorylation of the v-erbA-encoded domain of the protein both in unstimulated cells or after stimulation by PKA and PKC activators. The nonphosphorylatable AA-gag/v-erbA protein proved unable to inhibit temperature-induced differentiation of ts v-erbB and ts v-sea-transformed erythroblasts and to block expression of the erythrocyte-specific genes band 3 and carbonic anhydrase II. Back mutation of these alanine residues to serine resulted in the recovery of both normal phosphorylation levels and wild-type biological activity. In contrast, substitution of Ser-16/17 for threonine, which preserved phosphorylation in unstimulated cells but not PKA- and PKC-enhanced phosphorylation, resulted in a partially active gag/v-erbA protein. These results, together with the fact that the protein kinase inhibitor H7 resulted in both a dose-dependent inhibition of gag/v-erbA protein phosphorylation and the induction of terminal differentiation of AEV-transformed erythroblasts show that phosphorylation of gag/v-erbA protein is required for full biological activity. These results support the hypothesis that phosphorylation of the gag/v-erbA protein is important for transcriptional repression of at least some of its target genes in erythroid cells.
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PMID:Phosphorylation of the v-erbA protein is required for its function as an oncogene. 197 40

We investigated cell cycle-dependent regulation of protein kinase activity encoded by the viral mos gene by using a normal rat kidney cell line (NRK-6m2) chronically infected with a temperature-sensitive mutant (ts110) of Moloney murine sarcoma virus, which produces the P85gag-mos transforming protein. In elutriation experiments, in which cells in various phases of the cell cycle are separated based upon size, a twofold increase in the specific activity of the P85gag-mos protein kinase was observed as cells progressed from G0/G1 through S and G2/M. A three- to fourfold increase in gas-mos protein kinase specific activity relative to unsynchronized cells was observed in mitotic NRK-6m2 cells synchronized by treatment with thymidine followed by colcemid or with nocodazole alone. Interestingly, the gag-mos protein was structurally altered in mitotic cells generating a protein species moving slower than P85gag-mos in SDS-polyacrylamide gels. Our findings indicate that viral mos protein kinase activity is regulated during the cell cycle via phosphorylation. We propose that the mos transforming protein functions in a pleiotropic manner, affecting both cytoplasmic and nuclear targets.
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PMID:Cell cycle-mediated structural and functional alteration of P85gag-mos protein kinase activity. 213 25

A series of wild-type and mutant raf genes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine- and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and CR2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.
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PMID:Mutational activation of c-raf-1 and definition of the minimal transforming sequence. 218 91

Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH2-terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. We have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activity relationships. Expression of the yeast NMT1 gene in Escherichia coli, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH2-terminal 39 amino acids. Each E. coli-synthesized NMT species has fatty acid and peptide substrate specificities that are indistinguishable from those of NMT recovered from Saccharomyces cerevisiae, suggesting that the NH2-terminal domain of this enzyme is not required for its catalytic activity. By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted in E. coli by simultaneous expression of the yeast NMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A). The fatty acid specificity of N-myristoylation was preserved in this system: [9,10(n)-3H]myristate but not [9,10(n)3H]palmitate was efficiently linked to Gly-1 of the C subunit. [13,14(n)-3H]10-Propoxydecanoic acid, a heteroatom-containing analog of myristic acid with reduced hydrophobicity but similar chain length, was an effective alternative substrate for NMT that also could be incorporated into the C subunit of PK-A. Such analogs have recently been shown to inhibit replication of certain retroviruses that depend upon linkage of a myristoyl group to their gag polyprotein precursors (e.g., the Pr55gag of human immunodeficiency virus type 1). A major advantage of the bacterial system over eukaryotic systems is the absence of endogenous NMT and substrates, providing a more straightforward way of preparing myristoylated, analog-substituted, and nonmyristoylated forms of a given protein for comparison of their structural and functional properties. The system should facilitate screening of enzyme inhibitors as well as alternative NMT fatty acid substrates for their ability to be incorporated into a specific target protein. Our experimental system may prove useful for recapitulating other eukaryotic protein modifications in E. coli so that structure-activity relationships of modifying enzymes and their substrates can be more readily assessed.
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PMID:Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria. 240 21

Protein myristoylation was first discovered in the catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent protein kinase. Subsequently, various cellular and viral myristoylated proteins were detected. In each case, the myristoyl moiety was found in an amide linkage with the amino terminal glycine residue of the modified proteins. The biological functions of protein myristoylation of various cellular protein, oncogene product, and viral structural proteins have been studied by many biochemists. Two of the most thoroughly studies myristoylated proteins are the transforming protein of Rous sarcoma virus, pp60v-src, and the proto-oncogene product, pp60c-src. Deletion, modification of the first 14 NH2-terminal amino acid of pp60v-src, or chemical antimyristoylation of the protein with N-myristoyl glycinal diethylacetal does not affect intrinsic tyrosine src-kinase activity, but prevents myristoylation and membrane association, and abolishes the transforming activity of the protein. Protein myristoylations of some viral structural proteins were also studied by many investigators, and X-ray crystallographic studies of poliovirus suggest that myristate moiety may play a central role in capsid assembly. Recently, human immunodeficiency virus, HIV-I, process a myristoylated p17gag protein, which is proteolytically derived from the NH2-terminus of a gag precursor protein, and its myristate moiety may be important for virus assembly. In this review, we detailed recent studies of the protein myristoylation in cellular regulation and virus proliferation.
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PMID:[Function of protein myristoylation in cellular regulation and viral proliferation]. 254 55

The c-erbA alpha progenitor of the v-erbA oncogene of avian erythroblastosis virus (AEV) encodes a nuclear receptor for the thyroid hormone triiodothyronine (T3) which acts as a ligand-dependent transcription factor. As previously reported (Goldberg et al., EMBO J., 7, 2425-2433), the 46 kd chicken c-erbA alpha-encoded T3 receptor (ck-ErbA alpha) is phosphorylated at two major sites. Only one of these sites (Ser28/Ser29) is retained in the v-erbA-encoded P75gag-v-erbA protein. We report here the identification of the second phosphorylation site of ck-ErbA alpha as a single serine residue localized at position 12. We propose that casein kinase II, a protein kinase distributed in the cytosolic and nuclear compartments of a number of different tissues, is responsible for serine 12 phosphorylation on the following grounds. First, serine 12 is part of a sequence containing multiple acidic amino-acids, a feature common to all sites phosphorylated by casein kinase II in physiological substrates. Second, ck-ErbA alpha was found to be phosphorylated by purified casein kinase II in vitro at the same site, as defined by two-dimensional mapping experiments, as that observed in vivo. Third, conversion of serine 12 into an unphosphorylatable alanine residue by site directed mutagenesis abolishes the phosphorylation of ck-ErbA alpha by casein kinase II in vitro. Phosphorylation of serine 12 is likely to play a role in the modulation of ErbA alpha function since both serine 12 and the casein kinase II phosphorylation sequence motif are phylogenetically conserved in all known members of the c-erbA alpha gene family encoding T3 binding proteins. The codon specifying serine 12 in ck-ErbA alpha being precisely the point where recombination between gag and ck-c-erbA alpha occurred to generate v-erbA, our results furthermore suggest that deletion of serine 12 could contribute to the oncogenic activation of v-erbA.
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PMID:The c-erbA alpha-encoded thyroid hormone receptor is phosphorylated in its amino terminal domain by casein kinase II. 255 74

We have developed a one-step purification procedure for proteins containing the N-terminal portion of the gag protein of avian sarcoma and leukemia viruses. In this procedure, a resin with a covalently attached monoclonal antibody to the gag protein p19 is used to bind gag-containing proteins from crude extracts. After washing of the resin, the bound proteins are eluted with 2 M MgCl2. For the transforming protein kinase encoded by Fujinami sarcoma virus p130gag-fps, this procedure gave an enrichment of several thousand-fold, a yield of over 10%, a final purity of over 20%, and no significant loss of protein kinase activity. Similar purifications were obtained with three other gag-containing proteins. The immunoaffinity purification described may be of general utility as a first step in purification of the several other avian retroviral transforming proteins that are synthesized from fusions of an oncogene with the viral gag gene.
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PMID:A simple method for immunoaffinity purification of nondenatured avian sarcoma and leukemia virus gag-containing proteins. 282 89

A new avian transforming retrovirus, NK24, was isolated from a chicken with a nephroblastoma. This transforming virus induced fibrosarcomas with osteogenic cell proliferation and nephroblastomas in vivo and transformed fibroblast cells in vitro. From extracts of NK24-transformed cells, anti-gag serum immunoprecipitated a 100-kilodalton nonglycosylated protein with no detectable protein kinase activity. An NK24 provirus present in infected quail cells was molecularly cloned and subjected to nucleotide sequence analysis. The genome of NK24 was 5.3 kilobases long and had a 1,126-base-pair sequence of cellular origin in place of a viral sequence of avian leukosis virus containing the 3' half of the gag gene and the 5' half of the pol gene. Although the entire env gene was retained, it appeared to be inactive, possibly owing to the loss of function of its splice acceptor site as a result of a second deletion of 1,598 bases in the 3' half of the pol gene that extended to the acceptor site. Nucleotide sequence analysis revealed that the NK24 virus contained the fos gene, previously identified as the oncogene of FBJ and FBR murine osteosarcoma viruses. Unlike the v-fos gene products of FBJ and FBR, which suffer a structural alteration at their carboxyl termini, the NK24 v-fos gene product seemed to have the same carboxyl-terminal structure as the chicken c-fos gene product. A comparison of the structures of the products of the NK24 v-fos and mouse c-fos genes suggested that the fos gene product consists of highly conserved regions and relatively divergent regions.
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PMID:An avian transforming retrovirus isolated from a nephroblastoma that carries the fos gene as the oncogene. 282 11

Twelve independent isolates of avian sarcoma viruses (ASVs) can be divided into four groups according to the transforming genes harbored in the viral genomes. The first group is represented by viruses containing the transforming sequence, src, inserted in the viral genome as an independent gene; the other three groups of viruses contain transforming genes fps, yes or ros fused to various length of the truncated structural gene gag. These transforming sequences have been obtained by avian retroviruses from chicken cellular DNA by recombination. The src-containing viruses code for an independent polypeptide, p60src; and the representative fps, yes and ros-containing ASVs code for P140/130gag-fps, P90gag-yes and P68gag-ros fusion polypeptides respectively. All of these transforming proteins are associated with the tyrosine-specific protein kinase activity capable of autophosphorylation and phosphorylating certain foreign substrates. p60src and P68gag-ros are integral cellular membrane proteins and P140/130gag-fps and P90gag-yes are only loosely associated with the plasma membrane. Cells transformed by ASVs contain many newly phosphorylated proteins and in most cases have an elevated level of total phosphotyrosine. However, no definitive correlation between phosphorylation of a particular substrate and transformation has been established except that a marked increase of the tyrosine phosphorylation of a 34,000 to 37,000 dalton protein is observed in most ASV transformed cells. The kinase activity of ASV transforming proteins appears to be essential, but not sufficient for transformation. The N-terminal domain of p60src required for myristylation and membrane binding is also crucial for transformation. By contrast, the gag portion of the FSV P130gag-fps is dispensable for in vitro transformation and removal of it has only an attenuating effect on in vivo tumorigenicity. The products of cellular src, fps and yes proto-oncogenes have been identified and shown to also have tyrosine-specific protein kinase activity. The transforming potential of c-src and c-fps has been studied and shown that certain structural changes are necessary to convert them into transforming genes. Among the cellular proto-oncogenes related to the four ASV transforming genes, c-ros most likely codes for a growth factor receptor-like molecule. It is possible that the oncogene products of ASVs act through certain membrane receptor(s) or enzyme(s), such as protein kinase C, in the process of cell transformation.
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PMID:Avian sarcoma viruses. 283 47


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