Differential Effects of Protein Kinase A on Ras Effector Pathways

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. Wistar rat thyroid cells are dependent upon thyrotropin (TSH) for growth. Although TSH-stimulated mitogenesis requires Ras, TSH activates protein kinase A (PKA) and downregulates signaling through Raf and the mitogen-activated protein kinase (MAPK) cascade. Cells expressing Ras(S35), a mutant which binds Raf, or Ras(G37), a mutant which binds RalGDS, exhibited TSH-independent proliferation. Ras(S35) stimulated morphological transformation and anchorage-independent growth. Ras(G37) stimulated proliferation but not transformation as measured by these indices. TSH exerted markedly different effects on the Ras mutants and transiently repressed MAPK phosphorylation in Ras(S35)-expressing cells. In contrast, TSH stimulated MAPK phosphorylation and growth in cells expressing Ras(G37). The Ras mutants, in turn, exerted differential effects on TSH signaling. Ras(S35) 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.