Abstract
The retinoblastoma protein (pRb) acts to constrain the G1-S transition in mammalian cells. Phosphorylation of pRb in G1 inactivates its growth-inhibitory function, allowing for cell cycle progression. Although several cyclins and associated cyclin-dependent kinases (cdks) have been implicated in pRb phosphorylation, the precise mechanism by which pRb is phosphorylated in vivo remains unclear. By inhibiting selectively either cdk4/6 or cdk2, we show that endogenous D-type cyclins, acting with cdk4/6, are able to phosphorylate pRb only partially, a process that is likely to be completed by cyclin E-cdk2 complexes. Furthermore, cyclin E-cdk2 is unable to phosphorylate pRb in the absence of prior phosphorylation by cyclin D-cdk4/6 complexes. Complete phosphorylation of pRb, inactivation of E2F binding, and activation of E2F transcription occur only after sequential action of at least two distinct G1 cyclin kinase complexes.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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CDC2-CDC28 Kinases*
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Carrier Proteins*
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Cell Cycle Proteins / metabolism
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Cell Nucleus / metabolism
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Cells, Cultured
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Cyclin D
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Cyclin E / metabolism*
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinase 4
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Cyclin-Dependent Kinase 6
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Cyclin-Dependent Kinases / metabolism*
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Cyclins / metabolism*
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DNA-Binding Proteins / metabolism
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E2F Transcription Factors
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G1 Phase
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Humans
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Phosphorylation
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Protein Serine-Threonine Kinases / metabolism*
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Proto-Oncogene Proteins*
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Retinoblastoma Protein / metabolism*
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Retinoblastoma-Binding Protein 1
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Transcription Factor DP1
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Transcription Factors / metabolism
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Transfection
Substances
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Carrier Proteins
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Cell Cycle Proteins
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Cyclin D
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Cyclin E
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Cyclins
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DNA-Binding Proteins
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E2F Transcription Factors
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Proto-Oncogene Proteins
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Retinoblastoma Protein
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Retinoblastoma-Binding Protein 1
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Transcription Factor DP1
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Transcription Factors
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Protein Serine-Threonine Kinases
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CDC2-CDC28 Kinases
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CDK2 protein, human
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CDK4 protein, human
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CDK6 protein, human
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinase 4
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Cyclin-Dependent Kinase 6
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Cyclin-Dependent Kinases