(519.9) A phosphorylation network controls the stability of alpha-arrestins Aly1/Art6 and Aly2/Art3

Poster Board Number: A444

Eric Jordahl (University of Pittsburgh), Ray Bowman (University of Pittsburgh), Sydnie Davis (University of Pittsburgh), Nejla Ozbaki-Yagan (University of Pittsburgh), Annette Chiang (University of Pittsburgh), Stefanie Hedayati (University of Pittsburgh), Hannah Barsouk (University of Pittsburgh), Allyson ODonnell (University of Pittsburgh)

Selective protein trafficking controls the repertoire of membrane proteins at the cell surface. This contingent of surface proteins regulates nutrient/metabolite balance and response to extracellular cues. Environmental changes trigger cellular signaling that drives transitions in the plasma membrane proteome. These alterations are achieved in no small part through regulation of the α-arrestins, an emergent and powerful class of protein trafficking adaptors. The α-arrestins are largely cytosolic proteins that are transiently recruited to membranes via binding to membrane protein motifs. α-Arrestins bring with them a ubiquitin ligase, which stimulates ubiquitination and subsequent endocytosis of membrane proteins. Phosphorylation of α-arrestins is key to regulating their trafficking function; dephosphorylated α-arrestins are generally ‘active’ endocytic adaptors. While some kinases and phosphatases for α-arrestins are known, the degree of α-arrestins phosphorylation is staggering, with gt;40 phosphorylation sites identified for a single α-arrestin, and suggests we still have much to learn about α-arrestins regulation. We sought to determine what kinases and phosphates regulate paralogous α-arrestins Aly1 and Aly2 using a genetic screen in yeast. Using an array of all known non-essential kinase and phosphatase deletions, we determined which of these influenced α-arrestin-mediated resistance to rapamycin, an inhibitor of the TORC1 nutrient-sensing kinase. We identified a large cohort of kinases and phosphates that influenced Aly-dependent phenotypes, causing electrophoretic mobility changes and, in many cases, diminishing the abundance of these α-arrestins. We focused our studies on the Sit4 protein phosphatase, a key regulator downstream of TORC1 signaling able to influence other α-arrestins, identified in our screen. Strikingly, Aly1 and Aly2 were hyperphosphorylated and destabilized in the absence of Sit4, suggesting that excessive phosphorylation may promote degradation of these α-arrestins. For Aly2, but not Aly1, degradation in the sit4∆ cells could be reversed by loss of the vacuolar protease Pep4, indicating that vacuolar degradation predominates for Aly2 under these conditions. Loss of the Npr1 kinase in sit4∆ cells restored Aly protein abundances and reversed the hyperphosphorylation, demonstrating that this kinase is responsible for the excess Aly phosphorylation in sit4∆ cells. This is a remarkable finding as typically Npr1 is considered inactive in sit4∆, however, we suggest that Npr1 is selectively active in the absence of Sit4, able to modify some substrates but not others. We define new features of TORC1 signaling in regulating α-arrestin phosphorylation and stability.

NSF Career (AFO), Norman H. Horowitz Research Fellowship, Samuel D. Colella Research Fellowship