Deregulation of poly(A) polymerase interferes with cell growth.
Vertebrate poly(A) polymerase (PAP) contains a catalytic domain and a C-terminal Ser-Thr-rich regulatory region. Consensus and nonconsensus cyclin-dependent kinase (cdk) sites are conserved in the Ser-Thr-rich region in vertebrate PAPs. PAP is phosphorylated by cdc2-cyclin B on these sites in vitro and in vivo and is inactivated by hyperphosphorylation in M-phase cells, when cdc2-cyclin B is active. In the experiments described here, we undertook a genetic approach in chicken DT40 cells to study the function of PAP phosphorylation. We found that PAP is highly conserved in chicken and is essential in DT40 cells. While cells could tolerate reduced levels of PAP, even modest overexpression of either wild-type PAP or a mutant PAP with two consensus cdk sites mutated (cdk- PAP) was highly deleterious and at a minimum resulted in reduced growth rates. Importantly, cells that expressed cdk- PAP had a significantly lower growth rate than did cells that expressed similar levels of wild-type PAP, which was reflected in increased accumulation of cells in the G0-G1 phase of the cell cycle. We propose that the lower growth rate is due to the failure of hyperphosphorylation and thus M-phase inactivation of cdk- PAP.
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
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Zhao
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Manley
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002417:Cattle; D002455:Cell Division; D002460:Cell Line; D002645:Chickens; D017124:Conserved Sequence; D019926:Cyclin B / Q000378:metabolism; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D005091:Exons; D006579:Heterozygote; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011062:Polynucleotide Adenylyltransferase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011898:Ranidae; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection; D014714:Vertebrates
D019926:Cyclin B; D011994:Recombinant Proteins; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; C495905:CDK5 protein, human; C495906:Cdk5 protein, mouse; D018844:Cyclin-Dependent Kinases; D011062:Polynucleotide Adenylyltransferase