title: Phosphorylation-induced conformational changes in Rap1b: allosteric effects on switch domains and effector loop.
abstract: 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 on Ser(179) 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. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the 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 Ser(179) 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.
authors: Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
authors: Martin M
authors: MM
authors: Edreira
authors: Sheng
authors: S
authors: Li
authors: Daniel
authors: D
authors: Hochbaum
authors: Sergio
authors: S
authors: Wong
authors: Alemayehu A
authors: AA
authors: Gorfe
authors: Fernando
authors: F
authors: Ribeiro-Neto
authors: Virgil L
authors: VL
authors: Woods
authors: Daniel L
authors: DL
authors: Altschuler
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D002460:Cell Line; D041961:Deuterium Exchange Measurement; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D010766:Phosphorylation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D020668:rap GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C116753:RAP1B protein, human; D020668:rap GTP-Binding Proteins