Abstract
In cycling cells, the retinoblastoma protein (pRb) is un- and/or hypo-phosphorylated in early G1 and becomes hyper-phosphorylated in late G1. The role of hypo-phosphorylation and identity of the relevant kinase(s) remains unknown. We show here that hypo-phosphorylated pRb associates with E2F in vivo and is therefore active. Increasing the intracellular concentration of the Cdk4/6 specific inhibitor p15(INK4b) by transforming growth factor beta treatment of keratinocytes results in G1 arrest and loss of hypo-phosphorylated pRb with an increase in unphosphorylated pRb. Conversely, p15(INK4b)-independent transforming growth factor beta-mediated G1 arrest of hepatocellular carcinoma cells results in loss of Cdk2 kinase activity with continued Cdk6 kinase activity and pRb remains only hypo-phosphorylated. Introduction of the Cdk4/6 inhibitor p16(INK4a) protein into cells by fusion to a protein transduction domain also prevents pRb hypo-phosphorylation with an increase in unphosphorylated pRb. We conclude that cyclin D:Cdk4/6 complexes hypo-phosphorylate pRb in early G1 allowing continued E2F binding.
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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Carrier Proteins / metabolism
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Cell Cycle Proteins*
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Cell Line
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Cyclin D
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Cyclin-Dependent Kinase 4
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Cyclin-Dependent Kinase 6
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Cyclin-Dependent Kinase Inhibitor p15
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Cyclin-Dependent Kinase Inhibitor p16 / metabolism
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Cyclin-Dependent Kinases / metabolism*
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Cyclins / metabolism*
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DNA-Binding Proteins*
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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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Signal Transduction
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Transcription Factor DP1
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Transcription Factors / metabolism
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Transforming Growth Factor beta / pharmacology
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Tumor Cells, Cultured
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Tumor Suppressor Proteins*
Substances
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CDKN2B protein, human
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Carrier Proteins
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Cell Cycle Proteins
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Cyclin D
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Cyclin-Dependent Kinase Inhibitor p15
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Cyclin-Dependent Kinase Inhibitor p16
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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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Transforming Growth Factor beta
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Tumor Suppressor Proteins
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Protein Serine-Threonine Kinases
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CDK4 protein, human
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CDK6 protein, human
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Cyclin-Dependent Kinase 4
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Cyclin-Dependent Kinase 6
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Cyclin-Dependent Kinases