Caracterización de las rutas de MAP Quinasas de respuesta a estrés e integridad celular, y su interacción funcional con otras vías de señalización intracelular en Schizosaccharomyces pombe

Abstract

Protein kinase C (PKC) family of enzymes play an essential role in the regulation of numerous physiological processes in eukaryotic organisms. Fission yeast Schizosaccharomyces pombe has two PKC orthologs, Pck1 and Pck2, which act upstream of the cell integrity MAP kinase pathway (CIP) to regulate morphogenesis and cell wall biosynthesis. We found that, despite their strong structural similarity and functional redundancy, the mechanisms regulating maturation, activation and stabilization of both kinases have a remarkably distinct biological impact. Thus, in vivo phosphorylation of Pck1 within activation loop, turn and hydrophobic motifs is essential for its stability and biological functions. In contrast, Pck2 is partially functional independent of its catalytic activity. Remarkably, constitutive activation promotes dephosphorylation and destabilization of Pck2, while it enhanced Pck1 levels, which interferes with proper downstream signalling to the CIP triggered by Pck2. These results suggest that early duplication from a common ancestor involved significant changes in the mechanisms regulating catalytic activation and stability of the PKC family members, which would allow a flexible control of their biological functions, including MAP kinase signalling. TOR plays a key role in controlling cell growth and aging, and its dysfunction causes diseases such as cancer, obesity, and diabetes. We used a multidisciplinary approach combining genetic, biochemical and cell biology techniques to study the functional interactions between the CIP and TOR pathways during cell growth and in response to stress. Our results revealed that Rhy1, the GTPase activator of the TORC2 complex in S. pombe, activates the CIP by two distinct mechanisms. The first one includes the TORC2-Gad8 and TORC1-Psk1 complexes, which promote the synthesis of Pck2 in response to stress induced by cell wall damage or glucose starvation. In the second mechanism, Ryh1 activates the MAPK Pmk1 independently of TORC2, regulating the localization and/or traffic at the plasma membrane of upstream activators of the MAPK cascade including PDK-1 ortholog Ksg1 and GEF Rgf1. Finally, activated Pmk1 acts upstream of Ryh1 to inhibit TORC2 signalling in response to stress. Coordinated regulation of TOR and CIP pathways through cross-activation and inhibitory mechanisms would allow to accurately modulate cell adaptation and survival against different environmental insults. Unlike the other protein kinases, catalytic activation of the MAPKs depends on dual phosphorylation at two tyrosine and threonine residues within the conserved -TXY- motif. We have employed S. pombe as a model organism to analyze the possible biological relevance of monophosphorylated forms of MAP kinases Pmk1 and Sty1 (stress response pathway). Our results suggest that both kinases are phosphorylated by a "processive" sequential mechanism in which tyrosine phosphorylation is a pre-requisite to further phosphorylation at the threonine residue during growth and stress. In fact, the inability of phosphorylation of both Sty1 and Pmk1 at the threonine residue within the activation loop elicited the constitutive binding between MAPKK and MAPK and subsequent hyperphosphorylation at the tyrosine residue. Threonine monophosphorylated forms of the MAP kinases Sty1 and Pmk1 are partially functional in vivo and promote a moderate adaptive response to stress.