Functional analyses of mouse ASK, an activation subunit for Cdc7 kinase, using conditional ASK knockout ES cells.
ASK (activator of S phase kinase) is an activation subunit for mammalian Cdc7 kinase. We have generated mutant ES cell lines in which ASK can be conditionally inactivated. Upon loss of the ASK genes, the mutant ES cells rapidly cease cell growth. In keeping with its expected roles in activation of the essential S phase kinase, DNA synthesis is arrested and significant cell death is eventually induced in ASK-deficient cells, demonstrating essential roles of ASK for viability of ES cells. Using these mutant cells, we have set up a system where ASK molecules can be functionally dissected. In keeping with previous results from yeasts, conserved motif-M and motif-C were shown to be essential for in vivo functions of ASK, whereas a long C-terminal tail, found only in ASK-related molecules in higher eukaryotes, is not required. Unexpectedly, the motif-N, related to the BRCT motif and dispensable for viability in yeasts, is essential for the viability of ES cells. Further characterization reveals that motif-N is required for the maximum phosphorylation of MCM in cells, whereas the autophosphorylation activity of Cdc7 is not significantly affected by its loss. These results may suggest that motif-N of ASK may facilitate recruitment of substrates for Cdc7 kinase.
Yamashita
Nobuyuki
N
Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan.
Kim
Jung-Min
JM
Koiwai
Osamu
O
Arai
Ken-ichi
K
Masai
Hisao
H
D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D018797:Cell Cycle Proteins; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D004261:DNA Replication; D004789:Enzyme Activation; D005785:Gene Conversion; D015870:Gene Expression; D005822:Genetic Vectors; D048848:MAP Kinase Kinase Kinase 5 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008957:Models, Genetic; D009154:Mutation; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases; D016196:S Phase; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology*; D014162:Transfection; D019076:Transgenes
D018797:Cell Cycle Proteins; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases; D048848:MAP Kinase Kinase Kinase 5; C482859:Map3k5 protein, mouse