A DOMINANT-NEGATIVE CYCLIN-D1 MUTANT PREVENTS NUCLEAR IMPORT OF CYCLIN-DEPENDENT KINASE-4 (CDK4) AND ITS PHOSPHORYLATION BY CDK-ACTIVATING KINASE

Authors
Citation
Ja. Diehl et Cj. Sherr, A DOMINANT-NEGATIVE CYCLIN-D1 MUTANT PREVENTS NUCLEAR IMPORT OF CYCLIN-DEPENDENT KINASE-4 (CDK4) AND ITS PHOSPHORYLATION BY CDK-ACTIVATING KINASE, Molecular and cellular biology, 17(12), 1997, pp. 7362-7374
Citations number
107
Categorie Soggetti
Biology,"Cell Biology
ISSN journal
02707306
Volume
17
Issue
12
Year of publication
1997
Pages
7362 - 7374
Database
ISI
SICI code
0270-7306(1997)17:12<7362:ADCMPN>2.0.ZU;2-D
Abstract
Cyclins contain two characteristic cyclin folds, each consisting of fi ve alpha-helical bundles, which are connected to one another by a shor t linker peptide. The first repeat makes direct contact with cyclin-de pendent kinase (CDK) subunits in assembled holoenzyme complexes, where as the second does not contribute directly to the CDK interface, Altho ugh threonine 156 in mouse cyclin III is predicted to lie at the carbo xyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1- CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156 E but not T156S) is not phosphorylated by recombinant CDK-activating k inase (CAK) in vitro, fails to undergo activating T-loop phosphorylati on in viva, and remains catalytically inactive and unable to phosphory late the retinoblastoma protein. Moreover, when it is ectopically over expressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus, CAK phosphor ylation is not required for nuclear transport of cyclin D1-CDK4 comple xes, because complexes containing wild-type cyclin D1 and a CDK4 (T172 A) mutant lacking the CAK phosphorylation site are efficiently importe d, In contrast, enforced overexpression of the CDK inhibitor p21(Cip1) together with mutant cyclin D1 (T-156A)-CDK4 complexes enhanced their nuclear localization, These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition bq CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resist ant to ubiquitination, to compete with endogenous cyclin D1 in mammali an cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inac tive complexes and dominantly inhibiting the ability of transfected NI H 3T3 fibroblasts to enter S phase.