Protocol

Protein Phosphatase Protocols

Volume 365 of the series Methods in Molecular Biology pp 323-334

Phosphatase Targets in TOR Signaling

  • Estela JacintoAffiliated withDepartment of Physiology and Biophysics UMDNJ, Robert Wood Johnson Medical School

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Abstract

Cells undergo growth or increase in mass in the presence of nutrients. A key signaling molecule that responds to the presence of nutrients is the target of rapamycin (TOR). TOR is a highly conserved protein kinase and is the target of the growth inhibitor rapamycin. In response to nutrients, TOR promotes the phosphorylation of its downstream targets, leading to increased protein synthesis and decreased protein turnover. In yeast, a major mechanism for the downstream regulation of TOR effectors is by inhibition of the type 2A-related phosphatase SIT4. TOR negatively regulates SIT4 by promoting the association of SIT4 with TAP42. When TOR is inactivated by rapamycin treatment or nitrogen starvation, downstream effectors of TOR such as the serine/threonine protein kinase NPR1 and the TAP42 interacting protein TIP41 are dephosphorylated in a SIT4-dependent manner. The phosphorylation state of NPR1 and TIP41 provides a convenient readout in yeast to assay for TOR and SIT4 activities under growth-promoting or growth-inhibitory conditions.

Key Words

Growth TOR rapamycin SIT4 NPR1 TIP41 dephosphorylation nitrogen starvation SDS-PAGE isoelectric focusing two-dimensional gel electrophoresis