The carboxyl-terminal region of the retinoblastoma protein binds non-competitively to protein phosphatase type 1alpha and inhibits catalytic activity.
pRB, a negative-growth regulatory protein, is a demonstrated substrate for type 1 serine/threonine protein phosphatases (PP1). In a recent report from this laboratory, we demonstrated that select forms of phosphorylated as well as hypophosphorylated pRB can be found complexed with the alpha-isotype of PP1 (PP1alpha). This complex can also be observed when PP1 is rendered catalytically dead by toxin inhibition. These data suggested to us that pRB may bind to PP1 at one or more sites other than the catalytically active one on the enzyme and that such binding may play a role other than bringing the substrate into contact with the enzyme to facilitate catalysis. To address this possibility we utilized a series of pRB deletion mutants and coprecipitation studies to map the pRB domain involved in binding to PP1. Together with competition assays using in vivo expression of SV40 T-antigen, we show here that the carboxyl-terminal region of pRB is both necessary and sufficient for physical interaction with PP1. Subsequent biochemical analyses demonstrated inhibition of PP1 catalytic activity toward the standard substrate phosphorylase a when this enzyme is bound to pRB containing this region. K(m) and V(max) calculations revealed that pRB binds to PP1 in a non-competitive manner. These data support the notion that pRB, in addition to being a substrate for PP1, also functions as a PP1 inhibitor. The significance of this finding with respect to the functional importance of this interaction is discussed.
Tamrakar
S
S
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry and University of Rochester Cancer Center, Rochester, New York 14642, USA.
Ludlow
J W
JW
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D007700:Kinetics; D010762:Phosphorylase a / Q000378:metabolism; D010766:Phosphorylation; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D010762:Phosphorylase a; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases