The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormoneindependent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k-and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression.Cyclic AMP (cAMP) exerts differential effects on cell proliferation. In many cells, including CHO cells, aortic smooth muscle cells, and Rat-1 fibroblasts, cAMP inhibits the mitogenic response to growth factors (8). Growth-inhibitory effects of cAMP are mediated partly through activation of cAMPdependent protein kinase A (PKA), which interferes with Raf activation and signaling (23). Less is known regarding how cAMP stimulates growth, although accumulating evidence has dissociated the mitogenic effects of cAMP from effects on mitogen-activated protein kinase (MAPK) (37,42,76). In contrast, the effects of cAMP on p70s6k correlate with effects on proliferation, and inhibition of p70s6k activation abolishes cAMP-stimulated DNA synthesis (10). These results prompted us to examine the role of phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways in cAMP-stimulated proliferation.Multiple isoforms of PI3K that vary in lipid substrate specificity and subunit structure have been identified (reviewed in reference 71). Typically, mitogens that activate receptor tyrosine kinases stimulate PI3K␣/␤ whereas those that activate G-protein-coupled receptors stimulate PI3K␥, although exceptions have been noted (36,52,66,67). PI3K is required for the mitogenic activity of many growth factors, including platel...

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...

Although abundant in well-differentiated rat thyroid cells, Rap1GAP expression was extinguished in a subset of human thyroid tumor-derived cell lines. Intriguingly, Rap1GAP was downregulated selectively in tumor cell lines that had acquired a mesenchymal morphology. Restoring Rap1GAP expression to these cells inhibited cell migration and invasion, effects that were correlated with the inhibition of Rap1 and Rac1 activity. The reexpression of Rap1GAP also inhibited DNA synthesis and anchorage-independent proliferation. Conversely, eliminating Rap1GAP expression in rat thyroid cells induced a transient increase in cell number. Strikingly, Rap1GAP expression was abolished by Ras transformation. The downregulation of Rap1GAP by Ras required the activation of the Raf/MEK/extracellular signal-regulated kinase cascade and was correlated with the induction of mesenchymal morphology and migratory behavior. Remarkably, the acute expression of oncogenic Ras was sufficient to downregulate Rap1GAP expression in rat thyroid cells, identifying Rap1GAP as a novel target of oncogenic Ras. Collectively, these data implicate Rap1GAP as a putative tumor/invasion suppressor in the thyroid. In support of that notion, Rap1GAP was highly expressed in normal human thyroid cells and downregulated in primary thyroid tumors. Rap1GAP (30,33) is a member of a family of GTPaseactivating proteins (GAPs) for Rap1/2 GTPases that includes the splice variant Rap1GAPII, SPA-1, and E6TP1. Rap1GAP shares structural similarities with the RhebGAP tuberin. Tuberin is subject to mutational inactivation and loss in tuberous sclerosis, a disease syndrome associated with the formation of multiple benign tumors (15,19,22,37). The downregulation of E6TP1 by human papillomavirus E6 protein is believed to contribute to cervical cancer (10, 11), and an SPA-1 deficiency in mice results in a spectrum of myelodysplastic disorders similar to chronic myelogenous leukemia (13). The rap1GAP gene has been mapped to 1p35-36, a chromosomal region subject to deletion in a variety of human tumors including breast (28) and endocrine (41) neoplasia. Recently, decreased expression and loss of heterozygosity for Rap1GAP were reported for human oropharyngeal squamous cell (43) and pancreatic (21, 42) carcinomas.Rap1GAP is abundant in rat thyroid epithelial cells, where thyroid-stimulating hormone (TSH) regulates Rap1GAP protein stability. The stable overexpression of Rap1GAP in thyroid cells impaired DNA synthesis and the growth rate, and based on this, we suggested that Rap1GAP might function as a tumor suppressor (34). We now provide further support for this idea. Eliminating Rap1GAP expression in differentiated rat thyroid cells induced a transient increase in cell proliferation. Moreover, while highly expressed in normal thyroid follicular cells, Rap1GAP expression was downregulated in primary thyroid tumors and in thyroid carcinoma cell lines. In vitro, decreased expression of Rap1GAP was observed selectively in thyroid carcinoma cell lines that exhibited migratory and inva...

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Thyrotropin (TSH) regulates thyroid cell proliferation and function through cAMP-mediated signaling pathways that activate protein kinase A (PKA) and Epac/Rap1. The respective roles of PKA versus Epac/Rap1 in TSH signaling remain unclear. We set out to determine whether PKA and/or Rap1 mediate extracellular signal-regulated kinase (ERK) activation by TSH. Neither blocking Rap1 activity nor silencing the expression of Rap1 impaired TSH or forskolin-induced ERK activation in Wistar rat thyroid cells. Direct activation of Epac1 failed to stimulate ERK activity in starved cells, suggesting that Epac-induced Rap1 activity is not coupled to ERK activation in rat thyroid cells. By contrast, PKA activity was required for cAMP-stimulated ERK phosphorylation and was sufficient to increase ERK phosphorylation in starved cells. Expression of dominant-negative Ras inhibited ERK activation by TSH, forskolin, and N 6 -monobutyryl (6MB)-cAMP, a selective activator of PKA. Silencing the expression of B-Raf also inhibited ERK activation by TSH, forskolin, and 6MB-cAMP, but not that stimulated by insulin or serum. Depletion of B-Raf impaired TSH-induced DNA synthesis, indicating a functional role for B-Raf in TSH-regulated proliferation. Collectively, these results position PKA, Ras, and B-Raf as upstream regulators of ERK activation and identify B-Raf as a selective target of cAMP-elevating agents in thyroid cells. These data provide the first evidence for a functional role for B-Raf in TSH signaling.