The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RB, can inhibit S-phase entry. In PSM-RB-arrested G 1 cells, normal levels of cyclin E and cyclin E-associated kinase activity were detected, but the expression of cyclin A was inhibited. The ectopic expression of cyclin E restored cyclin A expression and drove the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB could not complete DNA replication. Microinjection of cells that have passed through the G 1 restriction point with plasmids expressing PSM-RB also led to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. These results reveal a novel role for RB in the inhibition of S-phase progression that is distinct from the inhibition of the G 1 /S transition, and suggest that continued phosphorylation of RB beyond G 1 /S is required for the completion of DNA replication.

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Murine retroviruses which encode c-myb proteins that have either complete or truncated carboxy (C) termini were used to infect haemopoietic cells from murine fetal liver. Using an agar colony assay, we could show that infection with the virus encoding the truncated protein resulted in the persistence of colony-forming cells well beyond the short period for which such cells are present in uninfected cultures. The resultant colonies failed to give rise to cell lines; however, clonal cell lines occasionally emerged from the original infected liquid cultures. The virus which encoded a myb protein with a complete C-terminus was virtually inactive in the colony assay; surprisingly, however, this virus could enhance proliferation in liquid cultures and has led to the generation of at least one cell line. In addition to demonstrating 'activation' of c-myb by C-terminal truncation, our results imply that an unaltered c-myb protein can also contribute to cellular transformation and that a second event is required to establish mybtransformed cells as a permanent cell line.

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Rasdependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serumstimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.Cross talk between different signaling systems plays an important role in signal cascades initiated by a variety of growth factors. Thyrotropin, or thyroid-stimulating hormone (TSH), is one such growth factor. The receptor for TSH is a sevenmembrane-spanning receptor which couples to the heterotrimeric G protein G S (58). Injection of an affinity-purified antibody to G S abolished TSH-induced DNA synthesis in Wistar rat thyroid (WRT) cells (41). Binding of TSH to its receptor results in activation of G S and adenylate cyclase and generation of the intracellular second messenger cyclic AMP (cAMP), activating the cAMP-dependent protein kinase (PKA). PKA appears to be required for TSH-induced mitogenesis, since microinjection of the heat-stable protein kinase inhibitor (PKI) of PKA significantly reduces TSH-induced DNA synthesis in WRT cells (31). Many of the effects of TSH on thyroid cells can be mimicked by cAMP agonists, further substantiating the role of PKA in TSH signaling (15). In addition, microinjection of a dominant-interfering Ras protein (N17 Ras) reduces TSH-stimulated DNA synthesis in WRT cells, as does injection of the Ras GTPase-activating protein GAP and the Ras effector-specific antibody Y13-259, indicating that activation of Ras is required for TSH-induced mitogenesis (2, 30, 31). Microinjection of both PKI and dominantnegative Ras together into WRT cells r...