Distinct and site-specific phosphorylation of the retinoblastoma protein at serine 612 in differentiated cells.
The retinoblastoma susceptibility protein (pRB) is a phosphoprotein that regulates cell cycle progression at the G1/S transition. In quiescent and early G1 cells, pRB predominantly exists in the active hypophosphorylated form. The cyclin/cyclin-dependent protein kinase complexes phosphorylate pRB at the late G1 phase to inactivate pRB. This event leads to the dissociation and activation of E2F family transcriptional factors. At least 12 serine/threonine residues in pRB are phosphorylated in vivo. Although there have been many reports describing bulk phosphorylation of pRB, detail research describing the function of each phosphorylation site remains unknown. Besides its G1/S inhibitory function, pRB is involved in differentiation, prevention of cell death and control of tissue fate. To uncover the function of phosphorylation of pRB in various cellular conditions, we have been investigating phosphorylation of each serine/threonine residue in pRB with site-specific phospho-serine/threonine antibodies. Here we demonstrate that pRB is specifically phosphorylated at Ser612 in differentiated cells in a known kinase-independent manner. We also found that pRB phosphorylated at Ser612 still associates with E2F-1 and tightly binds to nuclear structures including chromatin. Moreover, expression of the Ser612Ala mutant pRB failed to induce differentiation. The findings suggest that phosphorylation of Ser612 provides a distinct function that differs from the function of phosphorylation of other serine/threonine residues in pRB.
Division of Biochemistry and Molecular Biology, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan ; Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore.
Takayuki
T
Hattori
Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore ; Research Equipment Center, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan.
Chiharu
C
Uchida
Division of Cell-Free Sciences, Proteo-Science Center, Ehime University, Matsuyama, Ehime, Japan ; Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Hirotaka
H
Takahashi
Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Naoki
N
Yamamoto
Division of Biochemistry and Molecular Biology, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan.
Mikihiko
M
Naito
Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore ; Department of Biochemistry, National University of Singapore, Singapore, Singapore.
Yoichi
Y
Taya
D002454:Cell Differentiation / Q000502:physiology*; D002843:Chromatin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D050687:E2F1 Transcription Factor / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology*; D016160:Retinoblastoma Protein / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D020298:U937 Cells
D002843:Chromatin; D004268:DNA-Binding Proteins; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D009687:Nuclear Proteins; D016160:Retinoblastoma Protein; D013912:Threonine; D012694:Serine