Phosphorylation by the stress-activated MAPK Slt2 down-regulates the yeast TOR complex 2

Abstract

Saccharomyces cerevisiae target of rapamycin (TOR) complex 2 (TORC2) is an essential regulator of plasma membrane lipid and protein homeostasis. How TORC2 activity is modulated in response to changes in the status of the cell envelope is unclear. Here we document that TORC2 subunit Avo2 is a direct target of Slt2, the mitogenactivated protein kinase (MAPK) of the cell wall integrity pathway. Activation of Slt2 by overexpression of a constitutively active allele of an upstream Slt2 activator (Pkc1) or by auxin-induced degradation of a negative Slt2 regulator (Sln1) caused hyperphosphorylation of Avo2 at its MAPK phosphoacceptor sites in a Slt2-dependent manner and diminished TORC2-mediated phosphorylation of its major downstream effector, protein kinase Ypk1. Deletion of Avo2 or expression of a phosphomimetic Avo2 allele rendered cells sensitive to two stresses (myriocin treatment and elevated exogenous acetic acid) that the cell requires Ypk1 activation by TORC2 to survive. Thus, Avo2 is necessary for optimal TORC2 activity, and Slt2-mediated phosphorylation of Avo2 down-regulates TORC2 signaling. Compared with wild-type Avo2, phosphomimetic Avo2 shows significant displacement from the plasma membrane, suggesting that Slt2 inhibits TORC2 by promoting Avo2 dissociation. Our findings are the first demonstration that TORC2 function is regulated by MAPK-mediated phosphorylation.

Fungal plasma membrane domains

“…The C-terminus of Avo1 contains a Pleckstrin homology (PH) domain that binds PI(4,5)P 2 and mediates the plasma membrane anchoring of TORC2 (Berchtold and Walther 2009). Avo2 is a relatively small subunit essential for cell survival during stress conditions (Leskoske et al 2018). Bit61 also binds to TORC2 but its role remains unknown (Loewith et al 2002).…”

“…MCT-MCW -In both fission and budding yeast, cell wall stress results in activation of CWI pathway that negatively regulates the growth-promoting functions of MCT (Cohen, Kupiec and Weisman 2014;Leskoske et al 2018). Recently, it was shown that activation of either CWI via Pkc1 or the HOG pathway resulted in the phosphorylation of the Avo2 subunit of TORC2 by the mitogen-activated protein kinase Slt2, leading to the down-regulation of TORC2 activity (Leskoske et al 2018).…”

Regulation of TORC2 function and localization by Rab5 GTPases inSaccharomyces cerevisiae

“…Basal activity and stability of Ypk1 also requires its phosphorylation at S644, which lies within a conserved sequence (dubbed the "turn motif") located downstream of its kinase homology domain within a C-terminal regulatory domain [16]. This modification is installed by TORC2, which is also largely PM-associated [17][18][19][20][21]. Under certain stressful conditions that stimulate TORC2-mediated phosphorylation of Ypk1, such as sphingolipid limitation [22], heat stress [23], hypotonic conditions [24,25], and acetic acid stress [26], TORC2 further elevates Ypk1 activity by phosphorylating four additional sites in its C-terminal regulatory domain, paramount among them is T662, which lies within another conserved sequence (dubbed the "hydrophobic motif") in the C-terminal domain [13,16].…”

“…Phosphorylation of Ypk1 at these locations further enhances both its activity and stability. Under other stressful conditions, such as hypertonic shock [27,28], treatments that damage the cell wall [19], and treatments that decrease "membrane tension" [29], TORC2-mediated phosphorylation of Ypk1 is dramatically reduced.…”

“…Where? Localization of TORC2 in S. cerevisiae to discrete puncta residing at the cell cortex has been well documented, based on analysis of fixed cells by indirect immunofluorescence [20] or immunogold EM [6], or examination of live cells expressing fluorescently-tagged derivatives of Tor2 [18,19,21] or TORC2-specific subunits that associate tightly with Tor2 [17,19,21]. In addition, the effector protein kinases (Ypk1 and Ypk2) directly phosphorylated by TORC2 are, as mentioned earlier, also obligatorily phosphorylated by other protein kinases (Pkh1 and Pkh2) that are tightly associated with the PM-anchored eisosomes.…”