Mutations of p34cdc2 phosphorylation sites induce premature mitotic events in HeLa cells: evidence for a double block to p34cdc2 kinase activation in vertebrates

In vertebrates, entry into mitosis is accompanied by dephosphorylation of p34cdc2 kinase on threonine 14 (Thr14) and tyrosine 15 (Tyr15). To examine the role of these residues in controlling p34cdc2 kinase activation, and hence the onset of mitosis, we replaced Thr14 and/or Tyr15 by non‐phosphorylat...

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Bibliographic Details
Published in:The EMBO journal 1991-11, Vol.10 (11), p.3331-3341
Main Authors: Krek, W., Nigg, E.A.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Biological and medical sciences
CDC2 Protein Kinase - genetics
CDC2 Protein Kinase - metabolism
Cell cycle, cell proliferation
Cell physiology
Chickens
DNA - genetics
Enzyme Activation
Fluorescent Antibody Technique
Fundamental and applied biological sciences. Psychology
HeLa Cells
Humans
Mitosis
Molecular and cellular biology
Molecular Sequence Data
Mutation
Phenotype
Phosphorylation
Plasmids
Precipitin Tests
Protamine Kinase - metabolism
Threonine - genetics
Transfection
Tyrosine - genetics
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Summary:In vertebrates, entry into mitosis is accompanied by dephosphorylation of p34cdc2 kinase on threonine 14 (Thr14) and tyrosine 15 (Tyr15). To examine the role of these residues in controlling p34cdc2 kinase activation, and hence the onset of mitosis, we replaced Thr14 and/or Tyr15 by non‐phosphorylatable residues and transfected wild‐type and mutant chicken p34cdc2 cDNAs into HeLa cells. While expression of wild‐type p34cdc2 did not interfere with normal cell cycle progression, p34cdc2 carrying mutations at both Thr14 and Tyr15 displayed increased histone H1 kinase activity and rapidly induced premature mitotic events, including chromosome condensation and lamina disassembly. No phenotype was observed in response to mutation of only Thr14, and although single‐site mutation at Tyr15 did induce premature mitotic events, effects were partial and their onset was delayed. These results identify both Thr14 and Tyr15 as sites of negative regulation of vertebrate p34cdc2 kinase, and they suggest that dephosphorylation of p34cdc2 represents the rate‐limiting step controlling entry of vertebrate cells into mitosis.
ISSN:0261-4189
1460-2075
DOI:10.1002/j.1460-2075.1991.tb04897.x