Bourgoint, Clelia, Rispal , Delphine, Berti, Marina, Filipuzzi, Ireos, Helliwell, Stephen, Prouteau, Manuel and Loewith, Robbie (2018) The Target of Rapamycin Complex 2-dependent phosphorylation of Pan1 by Akl1 controls endocytosis dynamics. Journal of Biological Chemistry, 293 (31). ISSN 0021-92581083-351X

Abstract

The Target of Rapamycin Complex 2 (TORC2) is a widely conserved serine/threonine protein kinase. In Saccharomyces cerevisiae, TORC2 is essential, playing a key role in plasma membrane homeostasis. To do this TORC2 regulates diverse processes including sphingolipid synthesis, glycerol production and efflux, polarization of the actin cytoskeleton and endocytosis. The major direct substrate of TORC2 is the AGC-family kinase Ypk1. Ypk1 connects TORC2 signaling to actin polarization, and to endocytosis via the flippase kinases Fpk1 and Fpk2. Here we report that Fpk1 and Fpk2 mediate TORC2 signals to actin polarization, but not endocytosis, via the amino-phospholipid flippases. To search for novel targets of the flippases kinases we exploited the fact that Fpk1 prefers to phosphorylate Ser residues found within the sequence RXS[L/Y] [D/E], which is present ~90 times in the yeast proteome. We observed that 25 of these are phosphorylated by Fpk1 in vitro. We focused on the Ark/Prk family kinase Akl1, as it has been previously implicated in endocytosis. Using a potent ATP-competitive small molecule, CMB4563, to acutely inhibit TORC2, we found that Fpk1-mediated Akl1 phosphorylation inhibits Akl1 activity, which reduces phosphorylation of Pan1 and other endocytic coat proteins and ultimately contributes to an arrest of endocytosis. These results demonstrate that the regulation of actin polarization and endocytosis downstream of TORC2 is signaled by separate pathways that bifurcate at the level of the flippase kinases.

Item Type:	Article
Date Deposited:	21 Aug 2018 00:45
Last Modified:	21 Aug 2018 00:45
URI:	https://oak.novartis.com/id/eprint/35004