Identification of a novel TORC2 phosphorylation motif in Gad8
2Department of Natural and Life Science, The Open University of Israel, Israel
The target of rapamycin (TOR) is a serine/threonine kinase that forms two complexes: TOR complex 1 (TORC1) and TOR complex 2 (TORC2). In mammals, mTORC2 phosphorylates two kinases, AKT and PKC, within two sites: the turn motif and hydrophobic motif, leading to their activation via the phosphorylation of another site by the kinase PDK1.
It was shown recently that an additional site is phosphorylated by TORC2, named the TIM motif (F-X3-F-T), while the hydrophobic motif has been suggested to be an autophosphorylation site.
In Schizosaccharomyces pombe, Gad8 is the ortholog of AKT while Ksg1 is the ortholog of PKD1. We found that the TIM motif is conserved in Gad8.
Mutating the threonine of the TIM motif in Gad8 into an alanine (T520A) or a glutamate (T520E) caused a decrease in the level of phosphorylation in the Ksg1-dependent activation motif (T387), but no change was observed in the phosphorylation of the hydrophobic motif.
The T520A and T520E mutations conferred a moderate sensitivity to DNA damage and DNA replication stress, induced by camptothecin (CPT) or hydroxyurea (HU), respectively.
Surprisingly, the gad8-T520E mutation partially suppressed the strong sensitivity of TORC2 mutant cells to CPT and HU, suggesting that T520E may mimic phosphorylation and confers a TORC2-independent phenotype.
To summarize, we have characterized a novel phosphorylation site in Gad8, which is conserved from yeast to mammals. Our results suggest that mutations inhibiting or mimicking constant phosphorylation of the TIM motif decrease the fitness of S. pombe to DNA damage or DNA replication stress.