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

PDK1 (phosphoinositide-dependent protein kinase 1) phosphorylates and activates (cAMP-dependent protein kinase) in vitro. Docking of the HM (hydrophobic motif) in the C-terminal tail of the duanyu1529 catalytic subunits on to the PIF (PDK1-interacting fragment) pocket of PDK1 is a critical step in this activation process. However, PDK1 regulation of duanyu1529 in vivo remains controversial. Saccharomyces cerevisiae contains three duanyu1529 catalytic subunits, TPK1, TPK2 and TPK3. We demonstrate that Pkh [PKB (protein kinase B)-activating kinase homologue] protein kinases phosphorylate the activation loop of each Tpk in vivo with various efficiencies. Pkh inactivation reduces the interaction of each catalytic subunit with the regulatory subunit Bcy1 without affecting the specific kinase activity of Comparative analysis of the in vitro interaction and phosphorylation of Tpks by Pkh1 shows that Tpk1 and Tpk2 interact with Pkh1 through an 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, the results of the present study show that Pkh protein kinases contribute to the divergent regulation of the Tpk catalytic subunits.

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