Regulation of the RGS14/Gia1‐GDP signaling complex by phosphorylation

Abstract

RGS14 is a poorly understood brain protein that we show acts as a scaffold to bind inactive Gia1/3‐GDP, active H‐Ras and Rap2, and Raf kinases to modulate MAPkinase signaling. Our previous work showed that RGS14 is phosphorylated by PKA to regulate its interactions with Gia1. Here we report that RGS14 is phosphorylated in cells by a different kinase when in complex with Gia1. Recombinant RGS14 localizes to the cytosol in cells, but is recruited to the plasma membrane when co‐expressed with Gia1 to form a stable RGS14/Gia1‐GDP complex. RGS14 post complex with Gia1‐GDP appears as a prominent second higher molecular weight (HMW) immunoreactive band by SGS‐PAGE. We hypothesize this ‘upper’ band is due to addition of a novel phosphorylated residue on RGS14. Consistent with RGS14 being phosphorylated, we find that treatment of samples with alkyline phosphatase eliminates the HMW form of RGS14. Mutation of known PKA and Erk phosphorylation sites on RGS14 fail to eliminate the phosphorylated form of RGS14, suggesting the involvement of different kinase(s). Ongoing studies are designed to identify the specific phosphorylation site(s), involved kinase(s), and the functional consequences of phosphorylation. A deeper understanding of the regulation of RGS14 by phosphorylation will be important to understand its role(s) as a novel integrator of G protein and MAPkinase signaling in brain.