Binding of select forms of pRB to protein phosphatase type 1 independent of catalytic activity.
The product of the retinoblastoma susceptibility gene, pRB, is a demonstrated substrate for the type 1 serine/threonine protein phosphatases (PP1). Curiously, there has been a paucity of data supporting the idea that phosphorylated pRB can be found in a complex with PP1. To more fully characterize the association between these two proteins, we utilized a PP1-affinity chromatography approach to increase our ability to capture from mammalian cell lysate populations of pRB capable of binding to PP1. Western blot analysis of the bound proteins indicates that both faster migrating, hypophosphorylated pRB, as well as slower migrating, hyperphosphorylated pRB can bind. Phosphorylated pRB binding was confirmed by immunoprecipitation of eluted 32P-labeled pRB. In addition, Western blotting of eluted proteins with pRB phosphorylated-site-specific antibodies revealed select phosphorylated forms of pRB binding to PP1. Similar binding studies performed with toxin-inhibited PP1 indicate that catalytic activity of PP1 is not required for pRB binding. The significance of this finding with respect to the functional importance of this interaction is discussed.
Tamrakar
S
S
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York, NY 14642, USA.
Mittnacht
S
S
Ludlow
J W
JW
D000818:Animals; D002384:Catalysis; D002460:Cell Line; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D052998:Microcystins; D019319:Okadaic Acid / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D010769:Phosphothreonine / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000737:chemistry / Q000378:metabolism*
D004791:Enzyme Inhibitors; D052998:Microcystins; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; D010769:Phosphothreonine; D010768:Phosphoserine; D019319:Okadaic Acid; C078588:microcystin; D010749:Phosphoprotein Phosphatases