| Summary: | 博士 === 國立陽明大學 === 生化暨分子生物研究所 === 100 === Target of rapamycin (TOR) is a serine/threonine kinase conserved from yeasts to mammals. There exist two distinct TOR complexes, TORC1 and TORC2, each coupling to specific downstream effectors and signaling pathways. In S. cerevisiae, TORC2, consisting of Tor2, Lst8, Avo1, Avo2, Avo3, and Bit61, is involved in regulating actin organization and maintaining the cell wall integrity. To investigate the molecular mechanisms underlying TORC2 signaling, we have previously generated temperature-sensitive (ts) mutants of AVO3/TSC11 which are defective in TORC2 functions. The yeast protein kinase 2 (Ypk2), a member of the cAMP-dependent, cGMP-dependent, and PKC (AGC) kinase family, is a TORC2 substrate known to participate in the regulation of actin and cell wall. We also found that the defects of avo3ts mutants can be suppressed by overexpression of active Ypk2. GST pulldown assays have demonstrated the physical interaction between Ypk2 and components of TORC2, and this interaction depends on the direct binding of Ypk2 to Avo1. In avo3ts mutants, the TORC2-Ypk2 interaction is reduced and can be restored by AVO1 overexpression, highlighting the important role of Avo1 in coupling TORC2 to Ypk2. Ypk2334–677, a truncated form of Ypk2 containing the Avo1-interacting region, is able to interfere with the Avo1-Ypk2 interaction in vitro. Overexpressing Ypk2334–677 in yeast cells can result in a perturbation of TORC2 functions, causing defective cell wall integrity, aberrant actin organization, and diminished TORC2-dependent Ypk2 phosphorylation. These results together support a conclusion that the direct Avo1-Ypk2 interaction is crucial for TORC2 signaling to the downstream Ypk2 pathway.
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