The Retinoblastoma Protein Is Linked to the Activation of Ras

Abstract

The inner membrane-bound protein Ras integrates various extracellular signals that are subsequently communicated from the cytoplasm to the nucleus via the Raf/MEK/MAPK cascade. Here we show that the retinoblastoma protein pRb, previously reported to be a nuclear target of this pathway, can in turn influence the activation state of Ras. Rb-deficient fibroblasts display elevated levels (up to 30-fold) of activated Ras during G₁. Expression of wild-type pRb or a number of pRb mutants defective in E2F regulation reverses this effect. We provide evidence that the mid-G₁ activation of Ras in Rb-deficient cells, which occurs at the level of guanine nucleotide binding, differs from that of epidermal growth factor-induced stimulation of Ras, being dependent on protein synthesis. The aberrant levels of Ras activity associated with loss of pRb may be responsible for the differentiation defects in Rb-deficient cells, because suppression of Ras activity in Rb^−/− fibroblasts restores the transactivation function of MyoD and the expression of a late marker of skeletal muscle differentiation. These data suggest that nuclear-cytoplasmic communication between pRb and Ras is bidirectional.

ACKNOWLEDGMENTS

We thank D. Shalloway and S. Taylor for the GST-RBD construct; S. Reeves for the retroviral vector; W. Pear and D. Baltimore for Bosc23 cells; G. Cooper for the dominant-negative Ras plasmid; A. Lassar for the MCK reporter, MyoD plasmid, and retrovirus; W. Chin for the GR plasmid and GRE reporter; W. Sellers, F. Kaye, and W. Kaelin for pRb plasmids; J. DeCaprio for TAg plasmids; C. Sherr and M. Roussel for the NIH 3T3 cells and their cyclin D1 derivative; T. Jacks, N. Dyson, and E. Harlow for p107-deficient MEFs; T. Upton for p107^−/− 3T3 cells; E. Flemington for the E2F-1 and reporter plasmid; and J. Lamb, R. Weinberg, O. Iliopoulos, W. Sellers, M. Weber, J. Griffin, D. Livingston, T. Roberts, and the members of the Ewen Laboratory for critical review of the manuscript.

This work was supported by National Cancer Institute grant CA65842 to M.E.E. M.E.E. is a Scholar for the Leukemia Society of America.