ABSTRACT
We identified a species relevant to polo-like kinase family, a human homologue of mouse serum-inducible kinase, hSNK gene, whose mRNA expression was rapidly increased in cultured human thyroid cells after X-ray irradiation. The cDNA cloning and genomic analysis of the hSNK gene showed the presence of 14 exons spanning over 6 kb of genomic DNA that encodes a 2.9-kb mRNA product. Promoter analysis demonstrated possible existence of a radiation-responsive element in the p53 binding homology element (p53RE) localized to near upstream of basal promoter of the hSNK gene. Nuclear protein extracts from HeLa and various human thyroid carcinoma cell lines bound selectively to p53RE. Anti-p53 or anti-p73 antibodies, however, failed to recognize the p53RE-protein complex formed in the presence of such nuclear extracts. These results suggest that radiation-responsive transcription factor(s) directly participates in the regulation of hSNK gene expression via the binding to p53RE in promoter region.
Subject(s)
Protein Kinases/biosynthesis , Protein Kinases/genetics , Thyroid Gland/metabolism , Thyroid Gland/radiation effects , Transcription, Genetic , Tumor Suppressor Protein p53/metabolism , Base Sequence , Blotting, Northern , Cell Nucleus/metabolism , Cells, Cultured , Cloning, Molecular , DNA/metabolism , DNA, Complementary/metabolism , DNA, Complementary/radiation effects , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Radiation , Exons , Gene Deletion , Gene Library , Genes, Reporter , Genes, Tumor Suppressor , HeLa Cells , Humans , Luciferases/metabolism , Models, Genetic , Molecular Sequence Data , Nuclear Proteins/metabolism , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Kinases/chemistry , Protein Serine-Threonine Kinases , RNA/radiation effects , RNA, Messenger/metabolism , Sequence Analysis, DNA , Thyroid Neoplasms/metabolism , Transfection , Tumor Cells, Cultured , Tumor Protein p73 , Tumor Suppressor ProteinsABSTRACT
POP2 protein of Saccharomyces cerevisiae is a component of a protein complex that regulates the transcription of many genes. We found that the 97th threonine residue (Thr 97) of Pop2p was phosphorylated upon glucose limitation. The Thr 97 phosphorylation occurred within 2 min after removing glucose and was reversed within 1 min after the readdition of glucose. The effects of hexokinase mutations and glucose analogs indicate that this phosphorylation is dependent on glucose phosphorylating activity. We purified a protein kinase that phosphorylates a peptide containing Thr 97 of Pop2p and identified it as Yak1p, a DYRK family kinase. Phosphorylation of Pop2p was barely detectable in a yak1Delta strain. We found that Yak1p interacted with Bmh1p and Bmh2p only in the presence of glucose. A GFP-Yak1p fusion protein shuttled rapidly between the nucleus and the cytoplasm in response to glucose. A strain with alanine substituted for Thr 97 in Pop2p showed overgrowth in the postdiauxic transition and failed to stop the cell cycle at G(1) phase in response to glucose deprivation. Thus, Yak1p and Pop2p are part of a novel glucose-sensing system in yeast that is involved in growth control in response to glucose availability.
Subject(s)
Carrier Proteins/metabolism , Cell Nucleus/metabolism , Glucose/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Ribonucleases , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Cell Division , Electrophoresis, Polyacrylamide Gel , Intracellular Signaling Peptides and Proteins , Phosphorylation , Protein Serine-Threonine Kinases/isolation & purification , Protein-Tyrosine Kinases/isolation & purification , Signal Transduction , Dyrk KinasesABSTRACT
The yeast POP2 protein (Pop2p) is a component of a global transcription regulatory complex and is required for gene expression of many genes in Saccharomyces cerevisiae. We constructed POP2 deletion plasmids encoding various Pop2p regions under the native POP2 promoter and found that the minimum functional region was located in two-thirds of the carboxyl terminal region. A mouse homologue of the POP2 gene (mCAF1), which corresponds to the Pop2p minimum region, partially rescued the growth defect of pop2 null mutant cells. Addition of the Pop2p amino terminal region to mCAF1 strengthened the suppression. mCAF1 also weakly suppressed the relatively high expression of the SUC2 gene of pop2 cells under glucose-repressing conditions; however, it failed to suppress the defect of full expression of the SUC2 gene under glucose-derepressing conditions. Our findings clearly demonstrate that a mammalian homologue can substitute for the yeast POP2 gene in some aspect.
