Journal of Cell Science RESEARCH ARTICLE Multiple layers of regulation influence cell integrity control by the PKC ortholog Pck2 in fission yeast Marisa Madrid 1, *, Rafael Jime ´ nez 1 , Laura Sa ´ nchez-Mir 1 , Teresa Soto 1 , Alejandro Franco 1 , Jero Vicente-Soler 1 , Mariano Gacto 1 , Pilar Pe ´ rez 2 and Jose ´ Cansado 1, * ABSTRACT The fission yeast protein kinase C (PKC) ortholog Pck2 controls cell wall synthesis and is a major upstream activator of the cell integrity pathway (CIP) and its core component, the MAP kinase Pmk1 (also known as Spm1), in response to environmental stimuli. We show that in vivo phosphorylation of Pck2 at the conserved T842 activation loop during growth and in response to different stresses is mediated by the phosphoinositide-dependent kinase (PDK) ortholog Ksg1 and an autophosphorylation mechanism. However, T842 phosphorylation is not essential for Pmk1 activation, and putative phosphorylation at T846 might play an additional role in Pck2 catalytic activation and downstream signaling. These events, together with turn motif autophosphorylation at T984 and binding to small GTPases Rho1 and/or Rho2, stabilize Pck2 and render it competent to exert its biological functions. Remarkably, the target of rapamycin complex 2 (TORC2) does not participate in the catalytic activation of Pck2, but instead contributes to de novo Pck2 synthesis, which is essential to activate the CIP in response to cell wall damage or glucose exhaustion. These results unveil a novel mechanism whereby TOR regulates PKC function at a translational level, and they add a new regulatory layer to MAPK signaling cascades. KEY WORDS: MAP kinase, Pck2, Ksg1, Tor1, Pmk1, Cell integrity, S. pombe INTRODUCTION In eukaryotic organisms, the protein kinase C (PKC) family is a group of serine/threonine kinases belonging to the AGC class that play essential roles in signaling pathways controlling cell growth, morphogenesis, differentiation and cell death (Newton, 2010). PKC isoforms share a basic structure consisting of a variable N- terminal regulatory domain followed by a highly conserved C- terminal kinase domain. The core of the kinase domain contains three conserved phosphorylation sites that are crucial for catalytic activity, known as the activation loop, the turn motif and the hydrophobic motif (Freeley et al., 2011; Newton, 2010). The 3- phosphoinositide-dependent kinase-1 (PDK1), an AGC kinase family member itself, is the activation loop kinase of most PKCs and other AGC kinases such as PKC-related protein kinases (PRK1 and PRK2; also known as PKN1 and PKN2, respectively, in mammals) or protein kinase A (PKA) (Newton, 2010). Phosphorylation of the activation loop of PKCs is crucial for their activation, with the sole exception of PKCd (Liu et al., 2006). Interestingly, mammalian PKC isoforms like PKCh or PKCd become phosphorylated at the activation loop through PDK1-independent mechanisms involving autophosphorylation or unknown kinase(s) (Freeley et al., 2011). The turn and hydrophobic motifs in mammalian PKCs are phosphorylated either by mammalian target of rapamycin (mTOR) or by an autophosphorylation mechanism. However, the catalytic activity requirement for the phosphorylation of these motifs varies widely among the family members (Freeley et al., 2011), suggesting that alternative mechanisms regulate their activation and downstream signaling functions. The fission yeast Schizosaccharomyces pombe has two PKC orthologs named Pck1 and Pck2. The pck1D or pck2D single mutants are viable, but simultaneous deletion is lethal (Arellano et al., 1999). The N-terminus of both kinases contains two HR1 Rho-binding repeats resembling those present in mammalian PRK1 and PRK2, and these repeats bind to active GTPases Rho1 or Rho2 (Arellano et al., 1999; Villar-Tajadura et al., 2008). Pck1 and Pck2 are unstable, and interaction through their N-terminal PEST sequences with GTP–Rho1 or GTP–Rho2 increases their stability (Arellano et al., 1999; Villar-Tajadura et al., 2008). Rho1-mediated stabilization promotes increased phosphorylation of Pck2 at T842 within the activation loop (Sayers et al., 2000). GTP-bound Rho2 might also stabilize Pck2, because most Rho2 effects are mediated by Pck2 (Calonge et al., 2000; Ma et al., 2006), and Pck2 is stabilized in cells lacking the Rho2 GTPase- activating protein (GAP) Rga2 (Villar-Tajadura et al., 2008). Rho1 and Rho2, acting through Pck2, coordinately regulate the biosynthesis of (1,3) b-D-glucan (Rho1) and a-glucan (Rho2), the two main cell wall polymers in fission yeast (Arellano et al., 1999; Calonge et al., 2000). In addition, Rho1, Rho2 and Pck2 are key upstream activators of the cell integrity MAPK pathway (CIP), whose central element, Pmk1 (also known as Spm1), becomes activated under adverse conditions and regulates cell separation, morphogenesis, cell wall construction or ionic homeostasis (Madrid et al., 2006; Pe ´rez and Cansado, 2010; Toda et al., 1996). Rho1 and Rho2 support Pmk1 activity during vegetative growth primarily through Pck2 (Barba et al., 2008; Sa ´nchez-Mir et al., 2014b). However, whereas the Rho2–Pck2 branch is fully responsible for MAPK activation in response to hyper- and hypo-osmotic stress, both Rho1 and Rho2 target Pck2 for signaling cell wall damage to the CIP (Barba et al., 2008; Sa ´nchez-Mir et al., 2014b). In addition, Pmk1 activation under 1 Yeast Physiology Group, Department of Genetics and Microbiology, Facultad de Biologı ´a, Universidad de Murcia, 30071 Murcia, Spain. 2 Instituto de Biologı ´a Funcional y Geno ´ mica, Consejo Superior de Investigaciones Cientı ´ficas/ Departamento de Microbiologı ´a y Gene ´ tica, Universidad de Salamanca, 37007 Salamanca, Spain. *Author for correspondence (marisa@um.es; jcansado@um.es) Received 25 June 2014; Accepted 13 November 2014 ß 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 266–280 doi:10.1242/jcs.158295 266