Protein Kinase A-Dependent and -Independent Signaling Pathways Contribute to Cyclic AMP-Stimulated Proliferation

Abstract

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

“…Wortmannin had no e ect on TSH-stimulated S6 phosphorylation (Figure 3c), unlike its e ects on basal levels of S6 phosphorylation in RasC40 cells (Figure 1b). In contrast, the PKA-speci®c inhibitor H89 abolished TSH-stimulated S6 phosphorylation (Figure 3c), as reported for parental cells (Cass et al, 1999). These ®ndings indicate that TSH and RasC40 regulate p70s6k activity through distinct signaling pathways.…”

“…To elucidate the mechanism behind this e ect, the e ects of TSH on second messenger activation in RasC40 cells were examined. In parental cells, TSH signaling diverges to include a PI3K-dependent pathway to Akt and a PI3K-independent pathway to p70s6k (Cass et al, 1999). Consistent with these observations, TSH ( Figure 3b) or 8BrcAMP (data not shown) failed to further stimulate Akt phosphorylation in RasC40 cells.…”

“…These results demonstrate that expression of RasC40 is su cient to confer TSH-independent proliferation, and that mitogenic signaling in RasC40 cells requires PI3K and p70s6k, but not MEK activity. This inhibitor pro®le is similar to that of TSH in parental WRT cells where PI3K (Cass et al, 1999) and p70s6k (Cass and Meinkoth, 1998), but not Raf-1 or Mek (Al-Alawi et al, 1995), are required for hormone-stimulated proliferation.…”

“…TSH-stimulated proliferation is Ras- (Kupperman et al, 1993) and PI3K (Cass et al, 1999) dependent. Based on this, we examined whether Ras-mediated activation of PI3K was su cient to stimulate TSHindependent proliferation.…”

“…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). The ability of TSH to in¯uence the e ector pathways activated by Ras may be crucial for its ability to stimulate proliferation and di erentiation.…”