Direct and Novel Regulation of cAMP-dependent Protein Kinase by Mck1p, a Yeast Glycogen Synthase Kinase-3

Timothy F Rayner,Joseph V Gray,Jeremy W Thorner

doi:10.1074/jbc.m112349200

Abstract

The MCK1 gene of Saccharomyces cerevisiae encodes a protein kinase homologous to metazoan glycogen synthase kinase-3. Previous studies implicated Mck1p in negative regulation of pyruvate kinase. In this study we find that purified Mck1p does not phosphorylate pyruvate kinase, suggesting that the link is indirect. We find that purified Tpk1p, a cAMP-dependent protein kinase catalytic subunit, phosphorylates purified pyruvate kinase in vitro, and that loss of the cAMP-dependent protein kinase regulatory subunit, Bcy1p, increases pyruvate kinase activity in vivo. We find that purified Mck1p inhibits purified Tpk1p in vitro, in the presence or absence of Bcy1p. Mck1p must be catalytically active to inhibit Tpk1p, but Mck1p does not phosphorylate this target. We find that abolition of Mck1p autophosphorylation on tyrosine prevents the kinase from efficiently phosphorylating exogenous substrates, but does not block its ability to inhibit Tpk1p in vitro. We find that this mutant form of Mck1p appears to retain the ability to negatively regulate cAMP-dependent protein kinase in vivo. We propose that Mck1p, in addition to phosphorylating some target proteins, also acts by a separate, novel mechanism: autophosphorylated Mck1p binds to and directly inhibits, but does not phosphorylate, the catalytic subunits of cAMP-dependent protein kinase.

Highlights

The genome of Saccharomyces cerevisiae encodes four different protein kinases highly similar to mammalian glycogen synthase kinase-3 (GSK-3)1 [1]
We examined the effect of a specific inhibitor of PKA, mammalian protein kinase inhibitor (PKI), on the phosphorylation of pyruvate kinase by the purified preparation of PKA
We have found that purified, catalytically active, Mck1p cannot phosphorylate purified pyruvate kinase in vitro

Summary

Introduction

The genome of Saccharomyces cerevisiae encodes four different protein kinases highly similar (greater than 40% identity) to mammalian glycogen synthase kinase-3 (GSK-3)1 [1]. This was accomplished by using cAMPconjugated agarose, which binds to the Bcy1p subunit, to dissociate the purified Tpk1p1⁄7Bcy1p complex as described under “Experimental Procedures.” This step efficiently yielded a homogeneous preparation of Tpk1p, confirmed by silver staining of SDS-PAGE gels (Fig. 1) and Western blotting with anti-His6 tag monoclonal antibodies.2 Subsequent experiments using only the purified Tpk1p subunit to phosphorylate pyruvate kinase demonstrated that Tpk1p on its own is sufficient to phosphorylate pyruvate kinase in vitro (Fig. 2B).

Results

Conclusion