Differential Effects of Protein Kinase A on Ras Effector Pathways

Abstract

Ras mutants with the ability to interact with different effectors have played a critical role in the identification of Ras-dependent signaling pathways. We used two mutants, Ras S35 and Ras G37 , which differ in their ability to bind Raf-1, to examine Ras-dependent signaling in thyroid epithelial cells. abolished TSH-stimulated changes in cell morphology and thyroglobulin expression, while Ras G37 had no effect on these activities. Together, the data indicate that cross talk between Ras and PKA discriminates between distinct Ras effector pathways.Ras proteins are important signaling intermediates that convey signals initiated at the cell surface to effector pathways in the cytoplasm. Ras exerts effects on cell transformation and proliferation, the actin cytoskeleton, differentiation, and apoptosis. It is likely that these effects are mediated by multiple effectors, including Raf-1, RalGDS (2), phosphatidylinositol 3-kinase (PI3K) (48), and other Ras-binding proteins (reviewed in reference 38), as well as members of the Rho family (26,44,45).The elucidation of multiple Ras effector pathways was greatly facilitated by the isolation of Ras mutants which interact with single downstream effectors (60). Ras S35 binds preferentially to Raf, while Ras G37 binds to RalGDS (49,53,60 While the differential effects of Ras S35 and Ras G37 on Raf and MAPK activity are consistently observed (23,25,27,46,49,60), other effects of these mutants vary. Both Ras S35 and Ras G37 transform some NIH 3T3 strains, inducing growth in medium with low serum levels, anchorage-independent proliferation, and tumor formation in nude mice (27). The activity of Ras G37 in these assays suggests that Ras stimulates some of the same biological effects through Raf-independent pathways and that the effector pathways used by Ras vary in different cell types. Consistently, Ras V12 stimulates transformation in most cells, while activated forms of Raf transform fibroblasts but not epithelial cells (41). If Ras signals through multiple effectors, the selection of a particular effector pathway should be regulated. One potential way to achieve such regulation is through cross talk between Ras and other signaling pathways.One of the best-studied examples of cross talk is that between Ras and protein kinase A (PKA). In many cells, cyclic AMP (cAMP) inhibits Ras signaling through Raf and the MAPK cascade (reviewed in reference 6). cAMP activates PKA, which phosphorylates Raf-1 at multiple serine residues. PKA-mediated phosphorylation reduces the affinity of Raf for Ras (17,63) and decreases Raf kinase activity (10,17,57), effects which may inhibit Ras-mediated proliferation. Ras and cAMP also collaborate to produce similar effects. In PC12 cells, Ras and cAMP induce neurite extension (13, 54) and promote cell survival (64). Ras and cAMP stimulate proliferation in thyroid cells (7,36,39), and Ras activity is required for the mitogenic effects of thyrotropin (TSH) (32). Despite the requirement for Ras, TSH downregulates signaling through Raf and the MAPK cascade...

Ras signaling through PI3K confers hormone-independent proliferation that is compatible with differentiation

“…Similarly, Tg expression was comparable in exponentially growing RasC40 and parental cells (data not shown). In contrast, as previously reported (Miller et al, 1998), TSH failed to stimulate Tg expression in RasS35 cells (Figure 5b). The absence of Tg expression in these cells was not due to alterations in the kinetics of Tg expression, as treatment with TSH for times ranging from 24 ± 72 h failed to induce Tg expression (data not shown).…”

“…Similar results were obtained in three experiments in RasC40 B3 cells, and in a total of eight experiments in ®ve independent RasC40 cell lines. MAPK phosphorylation was observed in three independent RasS35 cell lines 3a), e ects very di erent from those observed in RasS35 cells (Miller et al, 1998). Indeed, cAMP elevating agents modestly, but consistently enhanced DNA synthesis in RasC40 cells.…”

“…The growth rate of thyroid cells expressing RasV12S35 is reduced in the presence of TSH, an e ect that appears to be mediated by inhibitory e ects of PKA on RasS35-stimulated MAPK activity (Miller et al, 1998). Given that the mitogenic e ects of TSH require PI3K activity, we speculated that TSH would not impair RasC40-stimulated proliferation.…”

“…To address the possibility that overexpression of RasC40 abolished the speci®city of its e ector interactions, MAPK activity was examined. Although MAPK was constitutively activated in cells expressing RasV12S35 (referred to as RasS35 cells (Miller et al, 1998), a mutant that selectively binds to Raf-1, phosphorylated MAPK was not detected in RasC40 cells (Figure 1c). These results indicate that the speci®city of RasC40 is maintained despite its threefold overexpression relative to cellular Ras.…”

“…Multiple Ras-dependent pathways converge in the regulation of di erentiation and cell cycle arrest in PC12 cells, where the e ects of Ras on neurite extension and growth arrest are mimicked by Raf-1 and PI3K, while RalGDS inhibits withdrawal from the cell cycle (Goi et al, 1999). In thyroid cells, cAMP impairs Ras signaling through Raf-1 (Al-Alawi et al, 1995) and augments Ras signaling through RalGDS (Miller et al, 1998). In addition, cAMP activates and requires PI3K-dependent signals for its mitogenic e ects (Cass et al, 1999).…”