cAMP stimulates the proliferation of numerous cell types by mechanisms that remain largely undefined. Our work has revealed that Rap1b is critical for the transduction of the cAMP mitogenic signal. Rap1b is activated by cAMP and phosphorylated by PKA. Expression of constitutively active Rap 1b in systems where cAMP stimulates mitogenesis, leads to an increased mitogenic and tumorigenic response. In thyroid follicular cells, either TSH or cAMP stimulation of DNA synthesis require both the binding of GTP to Rap1b as well as the phosphorylation of Rap1b by PKA. Moreover, specific expression of active Rap1b in the thyroid of transgenic mice leads to the development of benign hyperplasia, adenomas and carcinomas. Because the mitogenic actions of Rap 1b in cellular models and the observed pathology in transgenic mice only occurs in the presence of cAMP signaling, we have proposed that Rap 1b is a conditional oncogene. Thus, it became necessary to identify biochemical processes involved in the mitogenic action of Rap 1b. We found that TSH, via cAMP and PKA, inhibits Ala, and Rap1b is strictly required for this inhibition. Moreover, cAMP inhibition of Akt requires GTP-bound and phosphorylated Rap1b, suggesting that phospho-Rap1b-GTP is a critical component in the transduction of the cAMP mitogenic signal. In addition, TSH stimulation switches insulin-dependent Erk activation from a transient to a sustained profile. Interestingly, the cAMP/phospho-Rap1b-GTP mediated inhibition of Akt follows a time course identical to the Erk activation. Since Akt phosphorylates and inhibits the Erk stimulator c-Raf/B-Kaf, protein kinases critical for G1/S entry, we propose that Rap 1b plays a gating function in the cross talk between cAMP-Akt and Raf-Erk: transient inhibition of AM by cAMP/phospho-Rap1b-GTP is responsible for the sustained Erk activity required for G1/S. In this proposal we put forward experiments designed to elucidate the molecular mechanisms underlying the transduction of the cAMP mitogenic signal by Rap 1b and its relatedness to Akt and Erk.
The specific aims of this proposal are: 1) To identify effector pathways that mediate the cAMP/Rap1b-dependent inhibition of Akt. This will be accomplished by identifying Rap 1b effector domain mutants that prevent cAMP from signaling to Akt and G1/S; 2) To assess the role of Rap 1b phosphorylation on its function. Our hypothesis is that the state of Rap 1b phosphorylation regulates its affinity for effector proteins. We will determine the effect of Rap 1b phosphorylation on its binding to effectors by saturation binding experiments and by real time kinetics using a newly developed FRET assay. We also intend to uncover new phosphorylation-dependent Rap1 effectors using pull-down and two hybrid assays; 3) To study the mechanism and biological significance of Akt inhibition by cAMP-Rap 1b. We plan to establish the role of cAMP and Rap 1b on the regulation of an Akt phosphatase and demonstrate the role of Rap1 and its phosphorylation in TSH/insulin sustained Erk activation.
| Hochbaum, Daniel; Barila, Guillermo; Ribeiro-Neto, Fernando et al. (2011) Radixin assembles cAMP effectors Epac and PKA into a functional cAMP compartment: role in cAMP-dependent cell proliferation. J Biol Chem 286:859-66 |
| Edreira, Martin M; Li, Sheng; Hochbaum, Daniel et al. (2009) Phosphorylation-induced conformational changes in Rap1b: allosteric effects on switch domains and effector loop. J Biol Chem 284:27480-6 |
