Dual mechanisms for the inhibition of E2F binding to RB by cyclin-dependent kinase-mediated RB phosphorylation.
The growth suppression function of RB is dependent on its protein binding activity. RB contains at least three distinct protein binding functions: (i) the A/B pocket, which binds proteins with the LXCXE motif; (ii) the C pocket, which binds the c-Abl tyrosine kinase; and (iii) the large A/B pocket, which binds the E2F family of transcription factors. Phosphorylation of RB, which is catalyzed by cyclin-dependent protein kinases, inhibits all three protein binding activities. We have previously shown that LXCXE binding is inactivated by the phosphorylation of two threonines (Thr821 and Thr826), while the C pocket is inhibited by the phosphorylation of two serines (Ser807 and Ser811). In this report, we show that the E2F binding activity of RB is inhibited by two sets of phosphorylation sites acting through distinct mechanisms. Phosphorylation at several of the seven C-terminal sites can inhibit E2F binding. Additionally, phosphorylation of two serine sites in the insert domain can inhibit E2F binding, but this inhibition requires the presence of the RB N-terminal region. RB mutant proteins lacking all seven C-terminal sites and two insert domain serines can block Rat-1 cells in G1. These RB mutants can bind LXCXE proteins, c-Abl, and E2F even after they become phosphorylated at the remaining nonmutated sites. Thus, multiple phosphorylation sites regulate the protein binding activities of RB through different mechanisms, and a constitutive growth suppressor can be generated through the combined mutation of the relevant phosphorylation sites in RB.
Knudsen
E S
ES
Department of Biology and Center for Molecular Genetics, University of California at San Diego, La Jolla 92093-0322, USA.
Wang
J Y
JY
D000818:Animals; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002455:Cell Division; D002478:Cells, Cultured; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D016193:G1 Phase; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D012694:Serine / Q000378:metabolism; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D012694:Serine; D011505:Protein-Tyrosine Kinases; D016315:Proto-Oncogene Proteins c-abl; D018844:Cyclin-Dependent Kinases