PHOSPHORYLATION-INDUCED CONFORMATIONAL CHANGES IN Rap1b: ALLOSTERIC EFFECTS ON EFFECTOR DOMAIN

EDREIRA, MARTÍN M.; SHENG, LI; RIBEIRO-NETO, FERNANDO; YEH, JOANNE; BUCK, MATTHIA; WOODS, JR, VIRGIL L.; ALTSCHULER, DANIEL L.

Congreso; The First South American Spring Symposium in Signal Transduction and Molecular Medicine; 2010

SISTAM

  In models where cAMP exerts mitogenic responses, elevation of intracellular cAMP levels activates PKA and Epac, which synergistically stimulates Rap activation and phosphorylation at S179. It has been shown that the binding of GTP to Rap1b induces conformational changes at the switch I (residues 30-40) and the switch II (residues 60-76) domains, which are responsible for interaction with effectors. On the other hand, the role of phosphorylation is still unknown. Reports suggest that phosphorylation is able to modulate Rap1 association with some binding partners. The mechanism by which a modification at the carboxy-end of the molecule affects the regions involved with effector interaction at its N-terminus is for the moment unclear. We used amide hydrogen/deuterium exchange coupled with mass spectroscopy (DXMS), crystallography and NMR, to assess potentional conformational changes induced by phosphorylation. Our results indicate that phosphorylation had no major impact on the secondary structure, but are consistent with an allosterically effect of the C-terminus to the switch loops/effector domain, that might act as an allosteric discriminator of conformational states.