A novel auto-phosphorylation mechanism of PKA activity regulation in Saccharomyces cerevisiae.

SOLARI, CLARA; CAMARANO ADRIANA; MORENO SILVIA; PORTELA PAULA

Puerto Varas

Congreso; XII PABMB Congress; 2013

PABMB, SAIB, soc bioq y biol mol chile

In the AGC family of serine/threonine kinases, the activities of catalytic subunits are regulated by multiple phosphorylation. In Saccharomyces cerevisiae, the catalytic subunit of PKA is encoded by three genes: TPK1, TPK2 and TPK3; while the regulatory subunit is encoded by the gene BCY1. Pkh1 (homolog of mammalian PDK1) phosphorylates the activation loop of Tpk1 (Thr241) in newly synthesized Tpk1 independently of the carbon source. Pkh inactivation reduces the interaction Tpk1-Bcy1 without affecting the specific kinase activity of Tpk1. We have described that Tpk1 increases its phosphorylation status during glucose stimulus by an autophosphorylation mechanism. Here we demonstrate that Ser179 is a residue target of glucose induced phosphorylation only on Tpk1 active molecules. Studies evaluating the relationship between Thr241 and Ser179 phosphorylation indicated that the negative charge on Ser179 did not affect Thr241 phosphorylation status, while low phosphorylation on Thr241 increased the phosphorylation status of Ser179 in vivo. The kinetic effects of adding a negative charge on Ser179 were measured using purified  Tpk1S179D and Tpk1S179A. Phosphomimetic Tpk1S179D mutant showed a 20-fold increase in Vmax compared with Tpk1S179A; however its Km for a peptide substrate and the A0,5 for cAMP activation of the holoenzyme were not significantly affected . Therefore, we demonstrate that an autophosphorylation mechanism controls PKA activity in response to glucose availability.