Biological function of the retinoblastoma protein requires distinct domains for hyperphosphorylation and transcription factor binding.
Despite the importance of the retinoblastoma susceptibility gene to tumor growth control, the structural features of its encoded protein (pRb) and their relationship to protein function have not been well explored. We constructed a panel of deletion mutants of pRb expression vectors and used a biological assay for pRb that measures growth inhibition and morphologic changes in pRb-transfected Saos-2 cells to correlate structural alterations of the pRb coding region with function. We tested the deleted proteins for the ability to bind to viral oncoprotein E1A and to the transcription factor E2F. We also measured the ability of the mutant proteins to become hyperphosphorylated in vivo and to be recognized as substrates in vitro by a cell cycle-regulatory kinase associated with cyclin A. We identified two regions of pRb that are required for E2F binding and for hyperphosphorylation. E1A binding domains partially overlap but are distinct from both of these other two regions. Biological function of pRb is dependent on retention of the integrity of both of these biochemically defined domains. These data support the model that pRb is a transducer of afferent signals (via the kinase that phosphorylates it) and efferent signals (through transcription factor binding), using distinct structural elements. Preservation of both of these features is essential for the ability of pRb to induce growth inhibition and morphologic changes upon reintroduction into transfected cells.
Qian
Y
Y
Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106.
Luckey
C
C
Horton
L
L
Esser
M
M
Templeton
D J
DJ
D017500:Adenovirus E1A Proteins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D005091:Exons; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D017500:Adenovirus E1A Proteins; D016160:Retinoblastoma Protein; D014157:Transcription Factors; D004247:DNA