The RB/p107/p130 phosphorylation pathway is not inhibited in rapamycin-induced G₁-prolongation of NIH3T3 cells

The immunosuppressant rapamycin has previously been shown to inhibit G₁/S transition in several cell types. In Swiss-3T3 cells, rapamycin prolongs G₁ through the inhibition of the S6-kinase. In T-lymphocytes, rapamycin blocks the mitogen-induced down regulation of p27(Kip1), inhibitor of the cdk/cyclin complexes. We show here that an NIH3T3 line (N-3T3) is also sensitive to the G₁/S inhibitory effect of rapamycin. Unlike lymphocytes, rapamycin does not affect p27(Kip1) in these immortalized fibroblasts, nor does rapamycin affect the activity of cyclin D- or cyclin E-dependent kinases. As a result, rapamycin does not inhibit the phosphorylation of the retinoblastoma protein (RB) or two RE-related proteins, p107 and p130. Despite the phosphorylation of RB/pl07/p130, the expression of cyclin A and its associated kinase activity is delayed in rapamycin-treated N-3T3 cells. Ectopic expression of cyclin A, but not cyclins D and E or E2F-1 and -4, can overcome the effect of rapamycin. Taken together, these results suggest that entry into S-phase is likely to involve rapamycin-sensitive pathways other than the phosphorylation of the pocket proteins.