Novel Responsibilities for PKA Regulatory Subunits: Regulating Cell Migration through the Assembly of a P‐Rex1‐based Signaling Platform

Cyclic Adenosine Monophosphate (cAMP) is a ubiquitous second messenger that directly activates ion channels and Rap‐guanine nucleotide exchange factors (Epacs). Interestingly, the catalytic activity of PKA (cAMP‐dependent Protein Kinase), the most prominent effector of cAMP, is activated via an indirect mechanism, consisting on the release of inhibitory regulatory subunits (R), which in the absence of cAMP are tightly bound to their catalytic (C) partners. PKA‐R subunits have two CBD domains that in response to cAMP reduce their affinity for PKA‐C subunits, thus controlling the phosphorylation of specific substrates localized in the surroundings where the holoenzyme is recruited by interactors of the PKA‐R subunits. Then, what else do PKA‐R subunits in the cAMP‐bound active conformation? Recent findings suggest that they might control their own effectors, delineating an emerging PKA signaling landscape well beyond the established paradigm in which their attributed role is restricted to their control of the kinase activity of the C subunits. Previously, we described that PKA regulates endothelial cell migration via phosphorylation of P‐Rex1, a Rac guanine nucleotide exchange factor with which the PKA‐regulatory subunits directly interact via their CBD domains, that recognize P‐Rex1 PDZ domains, facilitating the phosphorylation of this RacGEF at its first DEP domain, leading to phosphorylation‐dependent intramolecular interactions that inhibit its catalytic DH‐PH module (Chavez‐Vargas, L., et al., J Biol Chem , 2016.) We then hypothesized that P‐Rex1 is regulated in a dual manner, activated by direct interaction with PKA‐R subunits and inhibited as a consequence of its phosphorylation by C subunits. In the current work we describe the molecular mechanism by which PKA R subunits interacts with and activates P‐Rex1/Rac signaling pathway promoting endothelial cell migration. Prostanglandin E2 or Butaprost‐dependent stimulation of angiogenic Gs‐coupled EP2 receptor promotes P‐Rex1/Rac activation and endothelial chemotaxis. Specific stimulation of type I PKA R subunits (RI) with cAMP analogues 6Bnz/8AHA‐cAMP promotes P‐Rex1/RI interaction and activates P‐Rex1; whereas H89, an inhibitor of PKA kinase activity, had no effect on Rac activation occurring in response to these cAMP analogues. Moreover, we detected RI subunits associated with active P‐Rex1 isolated by pulldown with a GTP‐free Rac GTPase construct fused to GST. Knockdown of P‐Rex1 in endothelial cells prevented Rac activation in response to specific stimulation of RI subunits. Ongoing experiments are addressing the structural basis of this pathway by reconstituting this signaling system in vitro . Our aim is to address the in vivo consequences of disturbing this interaction based on specifically designed inhibitors. Together, our results support the idea of PKA‐RI subunits are novel signaling hubs that go beyond their traditional role controlling the activity and localization of PKA kinase complexes. Furthermore, we demonstrate that P‐Rex1 is an effector of PKA regulatory subunits, activated in response to increasing concentrations of cAMP. Mechanistically, this is explained as a dual regulatory interaction based on opposite effects on P‐Rex1 of PKA subunits and a reciprocal regulation based on the mobilization of PKA associated to P‐Rex1. This dynamic signaling complex putatively defines a spatiotemporal pattern of Rac activity relevant in endothelial cell migration. Support or Funding Information Work supported by CONACYT grants 152434 (JV‐P) and 240119 (GR‐C) and Fundación Miguel Alemán (GR‐C). SAG, DC‐V and AC‐K are supported by CONACYT fellowships.