Phosphorylation-induced conformational changes in rap1b: Allosteric effects on switch domains and effector loop

MARTIN M. EDREIRA, SHENG LI, DANIEL HOCHBAUM, SERGIO WONG, ALEMAYEHU A. GORFE, FERNANDO RIBEIRO-NETO, VIRGIL L. WOODS, JR, AND DANIEL L. ALTSCHULER

JOURNAL OF BIOLOGICAL CHEMISTRY

Año: 2009 vol. 284 p. 27480 - 27486

0021-9258

Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b onSer179 at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation.Wereport here DXMS data comparing exchange rates for PKAphosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactlythe same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser179 phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.