Phosphorylation-induced Conformational Changes in Rap1b: ALLOSTERIC EFFECTS ON SWITCH DOMAINS AND EFFECTOR LOOP*

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 Ser¹⁷⁹ 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 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.Rap1b, a member of the Ras superfamily of small G proteins, is a GTPase that acts as a molecular on/off switch for the transduction of several external stimuli by alternating from an inactive GDP-bound to an active GTP-bound state (1, 2). Rap1 activation is mediated by several second messengers, growth factors, cytokines, and cell adhesion molecules. The steady-state level of Rap1-GTP is tightly regulated by a family of guanine nucleotide exchange factors that catalyze the otherwise slow dissociation of GDP (i.e. activation) and GTPase-activating proteins, which stimulate the rather slow intrinsic GTPase catalytic activity (i.e. inactivation) (3). GTP binding is coupled to conformational changes in two well defined regions, the switch I (residues 30–40) and switch II (residues 60–76) domains, responsible for high affinity interaction with effector molecules (4, 5) and thus downstream signal transduction.cAMP is one among several pathways leading to Rap1 activation (6). cAMP exerts both mitogenic and anti-mitogenic responses in different cell types, and Rap1 activation is required downstream of cAMP in both scenarios (7, 8). Elevation of intracellular cAMP levels activates cAMP-dependent protein kinase (PKA)⁴ and Epac (exchange protein activated by cAMP), a Rap guanine nucleotide exchange factor (9). Expression of Rap1b in cells where cAMP is mitogenic is associated with an increase in cAMP-mediated G₁/S phase entry (7, 10), and both biochemical events, Rap activation and phosphorylation at Ser¹⁷⁹, are synergistically required for this action (11).PKA substrates able to modulate Rap1 activity (i.e. Src/C3G recruitment and GTPase-activating protein) were recently reported (12, 13). However, the role of PKA-dependent Rap1 phosphorylation at Ser¹⁷⁹ is still unknown. Rap1 phosphorylation does not affect its overall intracellular localization, its basal GTP/GDP exchange reaction, its intrinsic rate of GTP hydrolysis, or its ability to be stimulated by a cytosolic Rap GTPase-activating protein (10); however, several reports suggest that Rap1 phosphorylation is able to modulate its association with some binding partners, namely cytochrome b₅₅₈ (14) and Raf1 (15). The mechanism by which a modification of Ser¹⁷⁹ at the C-terminal end of the molecule affects the regions involved with effector interaction at its N terminus is for the moment unclear.In this study, we report a global assessment of the effects of Ser¹⁷⁹ phosphorylation on conformational change/mobility analyzed by hydrogen/deuterium exchange mass spectrometry (DXMS). The results are consistent with an allosteric effect of the C terminus (containing Ser¹⁷⁹) to the switch loops/effector domain.