Activation loop of PKA catalytic isoforms is differentially phosphorylated by Pkh protein kinases in Saccharomyces cerevisiae.

Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates PKA in vitro. Docking of the hydrophobic motif (HM) in the C-tail of the PKA catalytic subunits onto the PDK1-interacting fragment (PIF) pocket of PDK1 kinase is a critical step in this activation process. However, PDK1 regulation of PKA in vivo remains ...
controversial. Saccharomyces cerevisiae contains three PKA catalytic subunits, TPK1, TPK2 and TPK3. We demonstrate that Pkh protein kinases phosphorylate the activation loop of each Tpk in vivo with varying efficiency. Pkh inactivation reduces the interaction of each catalytic subunit with the regulatory subunit Bcy1 without affecting the specific kinase activity of PKA. Comparative analysis of in vitro interaction and phosphorylation of Tpks by Pkh1 shows that Tpk1 and Tpk2 interact with Pkh1 through HM-PIF pocket interaction. Unlike Tpk1, mutagenesis of the activation loop site in Tpk2 does not abolish in vitro phosphorylation, suggesting that Tpk2 contains other, as yet uncharacterized Pkh1 target sites. Tpk3 is poorly phosphorylated on its activation loop site, and this is due to the weak interaction of Tpk3 with Pkh1 because of the atypical HM found in Tpk3. In conclusion, our data show that Pkh protein kinases contribute to the divergent regulation of the Tpk catalytic subunits.
Biochem. J.
Date: Sep. 07, 2012
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