Abstract
The conserved Ser/Thr kinase target of rapamycin (TOR) serves as a central regulator in controlling cell growth-related functions. There exist two distinct TOR complexes, TORC1 and TORC2, each coupling to specific downstream effectors and signaling pathways. In Saccharomyces cerevisiae, TORC2 is involved in regulating actin organization and maintaining cell wall integrity. Ypk2 (yeast protein kinase 2), a member of the cAMP-dependent, cGMP-dependent, and PKC (AGC) kinase family, is a TORC2 substrate known to participate in actin and cell wall regulation. Employing avo3(ts) mutants with defects in TORC2 functions that are suppressible by active Ypk2, we investigated the molecular interactions involved in mediating TORC2 signaling to Ypk2. GST pulldown assays in yeast lysates demonstrated physical interactions between Ypk2 and components of TORC2. In vitro binding assays revealed that Avo1 directly binds to Ypk2. In avo3(ts) mutants, the TORC2-Ypk2 interaction was reduced and could be restored by AVO1 overexpression, highlighting the important role of Avo1 in coupling TORC2 to Ypk2. The interaction was mapped to an internal region (amino acids 600-840) of Avo1 and a C-terminal region of Ypk2. Ypk2(334-677), a truncated form of Ypk2 containing the Avo1-interacting region, was able to interfere with Avo1-Ypk2 interaction in vitro. Overexpressing Ypk2(334-677) in yeast cells resulted in a perturbation of TORC2 functions, causing defective cell wall integrity, aberrant actin organization, and diminished TORC2-dependent Ypk2 phosphorylation evidenced by the loss of an electrophoretic mobility shift. Together, our data support the conclusion that the direct Avo1-Ypk2 interaction is crucial for TORC2 signaling to the downstream Ypk2 pathway.
Highlights
Molecular mechanisms underlying target of rapamycin complex 2 (TORC2) signaling are poorly understood
Active Ypk2 Rescues Multiple Phenotypes of avo3ts Mutants—We have previously obtained two temperature-sensitive mutants of the TORC2-specific component Avo3; the growth of these avo3ts mutants is sensitive to high temperatures, and they display defects in cell wall integrity and actin organization if grown at their nonpermissive temperatures such as 37 °C [18]
The results demonstrated that GST-Ypk2, but not GST, was able to pull down major TORC2 components (Fig. 2), indicating that Ypk2 physically interacts with TORC2 in yeast cells
Summary
Molecular mechanisms underlying target of rapamycin complex 2 (TORC2) signaling are poorly understood. Results: The TORC2 component Avo directly interacts with a downstream substrate Ypk. Conclusion: Avo1-Ypk interaction is essential for TORC2-Ypk coupling and activation of the downstream effector pathway(s) regulating cell integrity and actin organization. Ypk (yeast protein kinase 2), a member of the cAMP-dependent, cGMP-dependent, and PKC (AGC) kinase family, is a TORC2 substrate known to participate in actin and cell wall regulation. Other TORC2 substrates found in yeast are Slm and Slm; these two very similar proteins can bind to phosphatidylinositol 4,5-bisphosphate and are known to be involved in the regulation of actin organization [30]. To further understand the functions of individual TORC2 components in mediating downstream signaling, we investigated the molecular interactions involved in the TORC2-mediated Ypk regulation. Our data suggest that the direct interaction between Avo and Ypk mediates the coupling of TORC2 to the downstream Ypk signaling pathway
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