ENGINEERED MUTANTS OF PRB WITH IMPROVED GROWTH SUPPRESSION POTENTIAL

We have constructed a panel of substitution mutants which affect one o r more of the putative cdk target sites of the RB protein. We have exa mined the activity of these mutants relative to wild-type RB by both a transcriptional repression assay and by measuring growth suppression in vitro. We find that some phosphorylation site mutants of pRB can re press E2 transcription more strongly than wild-type RB. These mutants are partially resistant to phosphorylation by cdks and can arrest tumo r cells in G1 in vitro. Our results indicate a functional correlation between the ability to repress E2F-dependent transcription and the abi lity to suppress tumor cell growth in vitro. In addition, we describe two classes of RB mutants: N-terminal truncated p56(RB) and a novel mu tant of RB containing multiple substitutions near its nuclear localiza tion signal. Both classes of RB mutants have greater activity than the mild-type protein. Because RB is a key regulator of cell cycle progre ssion, expression of a more potent, phosphorylation resistant RB may h ave utility in both RB(-/-) and RB(+/+) tumors as well as in hyperprol iferative disorders.