Plasma membrane recruitment and activation of the AGC kinase Ypk1 is mediated by target of rapamycin complex 2 (TORC2) and its effector proteins Slm1 and Slm2

Abstract

The yeast AGC kinase orthologs Ypk1 and Ypk2 control several important cellular processes, including actin polarization, endocytosis, and sphingolipid metabolism. Activation of Ypk1/2 requires phosphorylation by kinases localized at the plasma membrane (PM), including the 3-phosphoinositide-dependent kinase 1 orthologs Pkh1/Pkh2 and the target of rapamycin complex 2 (TORC2). Unlike their mammalian counterparts SGK and Akt, Ypk1 and Ypk2 lack an identifiable lipid-targeting motif; therefore, how these proteins are recruited to the PM has remained elusive. To explore Ypk1/2 function, we constructed ATP analog-sensitive alleles of both kinases and monitored global changes in gene expression following their inhibition, where we observed increased expression of stress-responsive target genes controlled by Ca 2+ -dependent phosphatase calcineurin. TORC2 has been shown previously to negatively regulate calcineurin in part by phosphorylating two related proteins, Slm1 and Slm2, which associate with the PM via plextrin homology domains. We therefore investigated the relationship between Slm1 and Ypk1 and discovered that these proteins interact physically and that Slm1 recruits Ypk1 to the PM for phosphorylation by TORC2. We observed further that these steps facilitate subsequent phosphorylation of Ypk1 by Pkh1/2. Remarkably, a requirement for Slm1, can be bypassed by fusing the plextrin homology domain of Slm1 alone onto Ypk1, demonstrating that the essential function of Slm1 is largely attributable to its role in Ypk1 activation. These findings both extend the scope of cellular processes regulated by Ypk1/2 to include negative regulation of calcineurin and broaden the repertoire of mechanisms for membrane recruitment and activation of a protein kinase.

Modulation of TORC2 Signaling by a Conserved Lkb1 Signaling Axis in Budding Yeast

“…Full activity of Ypk1/2 also requires phosphorylation by Pkh1 and Pkh2, another pair of kinase paralogs that are the yeast homologs of vertebrate PDK1 (Casamayor et al 1999). Expression of constitutively active Ypk1 rescues lethality caused by inactivation of TORC2, which suggests that Ypk1/2 are amongst the most important targets of TORC2 (Kamada et al 2005;Niles et al 2012). Activation of SGK kinases by TORC2 and PDK1 is conserved in vertebrates (Biondi et al 2001;García-Martínez and Alessi 2008).…”

“…To test whether TORC2 signaling and Rts1 phosphorylation are correlated with growth rate, we used centrifugal elutriation to isolate small cells in early G1 phase and then assayed TORC2 activity and Rts1 phosphorylation as the cells progressed through the cell cycle. TORC2 activity was assayed with a phosphospecific antibody that recognizes a TORC2 site present on Ypk1 and Ypk2 (Niles et al 2012). The same samples were probed for the mitotic cyclin Clb2 to provide a marker for mitosis ( Figure 2A).…”

“…TORC2 directly phosphorylates and activates a pair of partially redundant kinase paralogs called Ypk1 and Ypk2, which are the budding yeast homologs of vertebrate SGK kinases (Casamayor et al 1999;Kamada et al 2005;Niles et al 2012). Full activity of Ypk1/2 also requires phosphorylation by Pkh1 and Pkh2, another pair of kinase paralogs that are the yeast homologs of vertebrate PDK1 (Casamayor et al 1999).…”

Sphingolipid Homeostasis in the Endoplasmic Reticulum and Beyond

“…Here a prominent role for Slm proteins is suggested by reports that the PH-domain-containing proteins Slm1 and Slm2 are necessary and sufficient for recruitment of Ypk1 to Torc2 and for Ypk1 activation (Fig. 4D) (Berchtold et al 2012;Niles et al 2012). Furthermore, inhibition of sphingolipid synthesis causes a rapid relocalization of Slm proteins from eisosomes to membrane domains containing Torc2, and this relocalization is required for phosphorylation of Ypk1 at Thr662 (Berchtold et al 2012).…”

“…Furthermore, the increased phosphorylation of Orm1/2 caused by inhibition of sphingolipid synthesis is paralleled by a corresponding increase in Ypk1 activity (Roelants et al 2011). This stimulation of Ypk1 activity is in turn the result of Torc2-mediated phosphorylation of Ypk1 at Thr662 (Roelants et al 2011;Niles et al 2012;Sun et al 2012).…”