Differential regulation of retinoblastoma protein function by specific Cdk phosphorylation sites.
The retinoblastoma tumor suppressor protein, RB, contains at least three distinct protein binding domains. The A/B pocket binds proteins with the LXCXE motif, the C pocket binds the nuclear c-Abl tyrosine kinase, and the large A/B pocket binds the transcription factor E2F. Dissociation of RB from its targets is observed as RB becomes phosphorylated during G1/S progression. There are 16 Cdk consensus phosphorylation sites in RB. It was previously unknown whether the many phosphorylation sites had redundant or distinct functions in the regulation of RB. Using RB mutant proteins lacking specific phosphorylation sites, we show that each of the binding domains is inhibited by different sites. Thr-821/826 phosphorylation is required to inhibit the binding to LXCXE containing proteins. Mutation of these two sites does not interfere with the hyperphosphorylation of RB. However, this phosphorylated mutant retains the ability to bind T-Ag, E7, and Elf-1, all of which contain the LXCXE motif. In contrast, Ser-807/811 phosphorylation is required to disrupt c-Abl binding. Mutation of Ser-807/811 and Thr-821/826 does not abolish the regulation of E2F binding. Taken together, these results show that the protein binding domains of RB are each regulated by distinct Cdk phosphorylation sites.
Knudsen
E S
ES
Department of Biology, University of California at San Diego, La Jolla, California 92093-0347, USA.
Wang
J Y
JY
D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002455:Cell Division; D016384:Consensus Sequence; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D016213:Cyclins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D010750:Phosphoproteins; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D014157:Transcription Factors; D016315:Proto-Oncogene Proteins c-abl; D018844:Cyclin-Dependent Kinases