Hyperphosphorylation of the retinoblastoma gene product is determined by domains outside the simian virus 40 large-T-antigen-binding regions.
With the murine retinoblastoma (RB) cDNA, a series of RB mutants were expressed in COS-1 cells and the pRB products were assessed for their ability (i) to bind to large T antigen (large T), (ii) to become modified by phosphorylation, and (iii) to localize in the nucleus. All point mutations and deletions introduced into regions previously defined as contributing to binding to large T abolished pRB-large T complex formation and prevented hyperphosphorylation of the RB protein. In contrast, a series of deletions 5' to these sites did not interfere with binding to large T. While some of the 5' deletion mutants were clearly phosphorylated in a cell cycle-dependent manner, one, delta Pvu, failed to be phosphorylated depsite binding to large T. pRB with mutations created at three putative p34cdc2 phosphorylation sites in the N-terminal region behaved similarly to wild-type pRB, whereas the construct delta P5-6-7-8, mutated at four serine residues C terminal to the large T-binding site, failed to become hyperphosphorylated despite retaining the ability to bind large T. All of the mutants described were also found to localize in the nucleus. These results demonstrate that the domains in pRB responsible for binding to large T are distinct from those recognized by the relevant pRB-specific kinase(s) and/or those which contain cell cycle-dependent phosphorylation sites. Furthermore, these data are consistent with a model in which cell cycle-dependent phosphorylation of pRB requires complex formation with other cellular proteins.
Hamel
P A
PA
Division of Immunology and Cancer, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.
Cohen
B L
BL
Sorce
L M
LM
Gallie
B L
BL
Phillips
R A
RA
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002872:Chromosome Deletion; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010766:Phosphorylation; D014176:Protein Biosynthesis; D016160:Retinoblastoma Protein / Q000235:genetics* / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D015345:Oligonucleotide Probes; D016160:Retinoblastoma Protein