scholarly journals Regulation of the protein kinase PKR by the vaccinia virus pseudosubstrate inhibitor K3L is dependent on residues conserved between the K3L protein and the PKR substrate eIF2alpha.

1997 ◽  
Vol 17 (7) ◽  
pp. 4146-4158 ◽  
Author(s):  
M Kawagishi-Kobayashi ◽  
J B Silverman ◽  
T L Ung ◽  
T E Dever
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Vaccinia Virus ◽  
Defense Mechanism ◽  
Mutational Analysis ◽  
Substrate Recognition ◽  
Phosphorylation Site ◽  
Antiviral Defense ◽  
Loss Of Function ◽  
Yeast Saccharomyces Cerevisiae

The mammalian double-stranded RNA-activated protein kinase PKR is a component of the cellular antiviral defense mechanism and phosphorylates Ser-51 on the alpha subunit of the translation factor eIF2 to inhibit protein synthesis. To identify the molecular determinants that specify substrate recognition by PKR, we performed a mutational analysis on the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR. High-level expression of PKR is lethal in the yeast Saccharomyces cerevisiae because PKR phosphorylates eIF2alpha and inhibits protein synthesis. We show that coexpression of vaccinia virus K3L can suppress the growth-inhibitory effects of PKR in yeast, and using this system, we identified both loss-of-function and hyperactivating mutations in K3L. Truncation of, or point mutations within, the C-terminal portion of the K3L protein, homologous to residues 79 to 83 in eIF2alpha, abolished PKR inhibitory activity, whereas the hyperactivating mutation, K3L-H47R, increased the homology between the K3L protein and eIF2alpha adjacent to the phosphorylation site at Ser-51. Biochemical and yeast two-hybrid analyses revealed that the suppressor phenotype of the K3L mutations correlated with the affinity of the K3L protein for PKR and was inversely related to the level of eIF2alpha phosphorylation in the cell. These results support the idea that residues conserved between the pseudosubstrate K3L protein and the authentic substrate eIF2alpha play an important role in substrate recognition, and they suggest that PKR utilizes sequences both near and over 30 residues from the site of phosphorylation for substrate recognition. Finally, by reconstituting part of the mammalian antiviral defense mechanism in yeast, we have established a genetically useful system to study viral regulators of PKR.

scholarly journals Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis.

1993 ◽  
Vol 13 (5) ◽  
pp. 2870-2881 ◽  
Author(s):  
L C Robinson ◽  
M M Menold ◽  
S Garrett ◽  
M R Culbertson
Keyword(s):  
Protein Kinase ◽  
Eukaryotic Cell ◽  
Casein Kinase ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Restrictive Temperature ◽  
Loss Of Function ◽  
Casein Kinase I ◽  
Yeast Saccharomyces Cerevisiae

Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.

scholarly journals Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A

2011 ◽  
Vol 300 (5) ◽  
pp. C989-C997 ◽  
Author(s):  
Pimthanya Wanichawan ◽  
William E. Louch ◽  
Kristin H. Hortemo ◽  
Bjørg Austbø ◽  
Per Kristian Lunde ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Phosphorylation Site ◽  
Full Length ◽  
Kinase A

The cardiac Na+/Ca2+ exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-32P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.

scholarly journals Dual requirement for a newly identified phosphorylation site in p70s6k.

1997 ◽  
Vol 17 (9) ◽  
pp. 5648-5655 ◽  
Author(s):  
B A Moser ◽  
P B Dennis ◽  
N Pullen ◽  
R B Pearson ◽  
N A Williamson ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Mutational Analysis ◽  
Phosphorylation Site ◽  
Serum Stimulation ◽  
Kinase C ◽  
Homologous Site ◽  
Phosphopeptide Analysis

The activation of p70s6k is associated with multiple phosphorylations at two sets of sites. The first set, S411, S418, T421, and S424, reside within the autoinhibitory domain, and each contains a hydrophobic residue at -2 and a proline at +1. The second set of sites, T229 (in the catalytic domain) and T389 and S404 (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues. Here we describe the identification and mutational analysis of three new phosphorylation sites, T367, S371, and T447, all of which have a recognition motif similar to that of the first set of sites. A mutation of T367 or T447 to either alanine or glutamic acid had no apparent effect on p70s6k activity, whereas similar mutations of S371 abolished kinase activity. Of these three sites and their surrounding motifs, only S371 is conserved in p70s6k homologs from Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family. Serum stimulation increased S371 phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S371 phosphorylation is regulated by an external mechanism. Phosphopeptide analysis of S371 mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T389 phosphorylation, a phosphorylation site previously shown to be essential for kinase activity. Nevertheless, the substitution of an acidic residue at T389, which mimics phosphorylation at this site, did not rescue mutant p70s6k activity, indicating that S371 phosphorylation plays an independent role in regulating intrinsic kinase activity.

Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis

1993 ◽  
Vol 13 (5) ◽  
pp. 2870-2881
Author(s):  
L C Robinson ◽  
M M Menold ◽  
S Garrett ◽  
M R Culbertson
Keyword(s):  
Protein Kinase ◽  
Eukaryotic Cell ◽  
Casein Kinase ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Restrictive Temperature ◽  
Loss Of Function ◽  
Casein Kinase I ◽  
Yeast Saccharomyces Cerevisiae

Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.

scholarly journals Regulation of chromosome segregation by Glc8p, a structural homolog of mammalian inhibitor 2 that functions as both an activator and an inhibitor of yeast protein phosphatase 1.

1995 ◽  
Vol 15 (11) ◽  
pp. 6064-6074 ◽  
Author(s):  
H Y Tung ◽  
W Wang ◽  
C S Chan
Keyword(s):  
Protein Kinase ◽  
Chromosome Segregation ◽  
Protein Phosphatase ◽  
Site Directed Mutagenesis ◽  
Protein Phosphatase 1 ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Yeast Saccharomyces Cerevisiae ◽  
Growth Phenotype

The Ipl1 protein kinase is essential for proper chromosome segregation and cell viability in the budding yeast Saccharomyces cerevisiae. We have previously shown that the temperature-sensitive growth phenotype of conditional ipl1-1ts mutants can be suppressed by a partial loss-of-function mutation in the GLC7 gene, which encodes the catalytic subunit (PP1C) of protein phosphatase 1, thus suggesting that this enzyme acts in opposition to the Ipl1 protein kinase in regulating yeast chromosome segregation. We report here that the Glc8 protein, which is related in primary sequence to mammalian inhibitor 2, also participates in this regulation. Like inhibitor 2, the Glc8 protein is heat stable, exhibits anomalous electrophoretic mobility, and functions in vitro as an inhibitor of yeast as well as rabbit skeletal muscle PP1C. Interestingly, overexpression as well as deletion of the GLC8 gene results in a partial suppression of the temperature-sensitive growth phenotype of ipl1ts mutants and also moderately reduces the amount of protein phosphatase 1 activity which is assayable in crude yeast lysates. In addition, the chromosome missegregation phenotype caused by an increase in the dosage of GLC7 is totally suppressed by the glc8-delta 101::LEU2 deletion mutation. These findings together suggest that the Glc8 protein is involved in vivo in the activation of PP1C and that when the Glc8 protein is overproduced, it may also inhibit PP1C function. Furthermore, site-directed mutagenesis studies of GLC8 suggest that Thr-118 of the Glc8 protein, which is equivalent to Thr-72 of inhibitor 2, may play a central role in the ability of this protein to activate and/or inhibit PP1C in vivo.

scholarly journals Loss of Protein Kinase PKR Expression in Human HeLa Cells Complements the Vaccinia Virus E3L Deletion Mutant Phenotype by Restoration of Viral Protein Synthesis

2007 ◽  
Vol 82 (2) ◽  
pp. 840-848 ◽  
Author(s):  
Ping Zhang ◽  
Bertram L. Jacobs ◽  
Charles E. Samuel
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Vaccinia Virus ◽  
Hela Cells ◽  
Viral Protein ◽  
Deletion Mutant ◽  
Mutant Phenotype ◽  
Viral Protein Synthesis ◽  
Virus Growth ◽  
Α Subunit

ABSTRACT The E3L proteins encoded by vaccinia virus bind double-stranded RNA and mediate interferon resistance, promote virus growth, and impair virus-mediated apoptosis. Among the cellular proteins implicated as targets of E3L is the protein kinase regulated by RNA (PKR). To test in human cells the role of PKR in conferring the E3L mutant phenotype, HeLa cells stably deficient in PKR generated by an RNA interference-silencing strategy were compared to parental and control knockdown cells following infection with either an E3L deletion mutant (ΔE3L) or wild-type (WT) virus. The growth yields of WT virus were comparable in PKR-sufficient and -deficient cells. By contrast, the single-cycle yield of ΔE3L virus was increased by nearly 2 log10 in PKR-deficient cells over the impaired growth in PKR-sufficient cells. Furthermore, virus-induced apoptosis characteristic of the ΔE3L mutant in PKR-sufficient cells was effectively abolished in PKR-deficient HeLa cells. The viral protein synthesis pattern was altered in ΔE3L-infected PKR-sufficient cells, characterized by an inhibition of late viral protein expression, whereas in PKR-deficient cells, late protein accumulation was restored. Phosphorylation of both PKR and the α subunit of protein synthesis initiation factor 2 (eIF-2α) was elevated severalfold in ΔE3L-infected PKR-sufficient, but not PKR-deficient, cells. WT virus did not significantly increase PKR or eIF-2α phosphorylation in either PKR-sufficient or -deficient cells, both of which supported efficient WT viral protein production. Finally, apoptosis induced by infection of PKR-sufficient HeLa cells with ΔE3L virus was blocked by a caspase antagonist, but mutant virus growth was not rescued, suggesting that translation inhibition rather than apoptosis activation is a principal factor limiting virus growth.

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