Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb.
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.
Ezhevsky
S A
SA
Howard Hughes Medical Institute and Division of Molecular Oncology, Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Nagahara
H
H
Vocero-Akbani
A M
AM
Gius
D R
DR
Wei
M C
MC
Dowdy
S F
SF
D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D002460:Cell Line; D056741:Cyclin D; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D016193:G1 Phase; D006801:Humans; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D015398:Signal Transduction; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
C493966:CDKN2B protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056741:Cyclin D; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D017346:Protein Serine-Threonine Kinases; C495900:CDK4 protein, human; C495911:CDK6 protein, human; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases