Activation loop of PKA catalytic isoforms is differentially phosphorylated by Pkh protein kinases

HAESENDONCKX S., TUDISCA V, VOORDECKERS K., MORENO S., THEVELEIN J. AND PORTELA, P.

BIOCHEMICAL JOURNAL

PORTLAND PRESS LTD

Lugar: Londres; Año: 2012 vol. 448 p. 307 - 320

0264-6021

Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates PKA in vitro. Docking of the hydrophobic motif (HM) in their C-tail 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 site of each Tpk in vivo with different efficiency. Pkh inactivation reduces the interaction of each catalytic subunit with 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, because of the weak interaction of Tpk3 with Pkh1. We show here that this is due to the atypical HM found in Tpk3. In conclusion, our data show that each Tpk catalytic subunit is differentially phosphorylated by the Pkh protein kinases.