Multiple regulatory levels influence cell integrity control by PKC ortholog Pck2 in fission yeast

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.

Multiple crosstalk between TOR and the cell integrity MAPK signaling pathway in fission yeast

“…Nevertheless, this complex contributes to de novo Pck2 synthesis, which is essential to activate the CIP in response to cell wall damage or glucose exhaustion, but not to other stimuli like saline stress29. To identify the main components of this novel regulatory mechanism, we analyzed Pck2 levels and Pmk1 activation in a series of mutants lacking upstream regulators or downstream effectors of the TORC2 signaling cascade.…”

“…Rho GTPases Rho1 and Rho2 are the main positive regulators of the CIP through Pck2, one of the two orthologs of protein kinase C (PKC) in this organism28. Similar to Gad8 and Psk1, Pck2 activation requires activation loop phosphorylation by Ksg1 (PDK1), and undergoes subsequent autophosphorylation of the turn motif29. These events, together with binding to Rho1 and/or Rho2, stabilize and render Pck2 catalytically competent to modulate cell integrity through activation of Pmk129.…”

“…Similar to Gad8 and Psk1, Pck2 activation requires activation loop phosphorylation by Ksg1 (PDK1), and undergoes subsequent autophosphorylation of the turn motif29. These events, together with binding to Rho1 and/or Rho2, stabilize and render Pck2 catalytically competent to modulate cell integrity through activation of Pmk129. We recently found that increased de novo Pck2 synthesis is essential to trigger Pmk1 activation in response to cell wall damage or glucose starvation, and that TORC2 is involved in such control2729.…”

“…These events, together with binding to Rho1 and/or Rho2, stabilize and render Pck2 catalytically competent to modulate cell integrity through activation of Pmk129. We recently found that increased de novo Pck2 synthesis is essential to trigger Pmk1 activation in response to cell wall damage or glucose starvation, and that TORC2 is involved in such control2729. Also, Pmk1 might negatively regulate the TORC2-Gad8 signaling30, suggesting that the CIP and TORC2 pathways extensively crosstalk in vivo , although the mechanisms involved are currently unknown.…”

Eisosomes Regulate Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Cortical Clusters and Mitogen-activated Protein (MAP) Kinase Signaling upon Osmotic Stress

“…In contrast, the fission yeast pathway is strongly activated by osmotic stress, whereas heat and cell wall stress lead to a less pronounced activation with slower kinetics (18,20). We have recently shown that delayed Pmk1 activation under cell wall stress or glucose deprivation, but not during osmotic stress, is linked to enhanced Pck2 translation mediated by the TORC2 complex (52). Thus, different activation mechanisms may have evolved to use this conserved response pathway for distinct environmental conditions in different species.…”