Mck1, a member of the glycogen synthase kinase 3 family of protein kinases, is a negative regulator of pyruvate kinase in the yeast Saccharomyces cerevisiae

Abstract

An interaction between the Saccharomyces cerevisiae protein kinase Mck1 and pyruvate kinase (Pyk1) was detected by using the two-hybrid method. Purified Mck1 was able to phosphorylate purified Pyk1 on Ser in vitro. Pyruvate kinase activity was elevated in mck1⌬ cells. Several of the phenotypes of mck1⌬ mutants are similar to those observed in cells overexpressing PYK1. Co-overexpression of MCK1 suppressed all of the phenotypes associated with PYK1 overexpression. These results indicate that Mck1 negatively regulates pyruvate kinase activity, possibly by direct phosphorylation.The yeast protein kinase Mck1, a member of the glycogen synthase kinase 3 family of protein kinases (23), fulfills a number of seemingly disparate functions. MCK1 is required during meiosis and is also needed later during sporulation for ascus maturation (15). MCK1 seems to play a role in mitotic chromosome segregation (10, 21). Moreover, mck1⌬ mutants are heat sensitive (unable to grow at 37°C) and are unable to accumulate normal amounts of glycogen, which is a major storage carbohydrate in Saccharomyces cerevisiae (4, 16). However, physiological substrates of Mck1 have not been definitively identified.To identify potential targets of Mck1, we conducted a twohybrid screen to isolate genes encoding proteins that interact with Mck1. One of the interacting gene products identified was the glycolytic enzyme pyruvate kinase (Pyk1). Here we present evidence indicating that Pyk1 is negatively regulated by Mck1.Mck1 interacts with Pyk1 in vivo. For the two-hybrid screen, the MCK1 gene was fused in frame to the sequence encoding the Gal4 DNA-binding domain, generating plasmid pGBD-MCK1; to construct this plasmid, MCK1 was excised from pDD4 (12) by digestion with NdeI and BamHI and inserted into the vector pAS1-CYH (7), which had been digested with NdeI and BamHI. Strain Y190 (9) was transformed with pGBD-MCK1 and a library of S. cerevisiae cDNAs expressed as fusions to the Gal4 transcriptional activation domain (7). Library plasmids that specifically reconstitute Gal4 activity were identified, recovered, and characterized as previously described (1). Nucleotide sequencing revealed that one of the inserts recovered from this screen represented residues 14 to 464 of the PYK1 pyruvate kinase gene (5, 14), indicating that this segment of Pyk1 associates, directly or indirectly, with Mck1.To determine whether Pyk1 and Mck1 can be coimmunoprecipitated, cleared extracts were prepared from both wildtype cells (strain YPH500 [22]) and an mck1⌬ mutant (strain DBY1; Table 1), each overexpressing PYK1 from a multicopy plasmid (pPK1; Table 2). The extracts (3 mg of protein) were then subjected to immunoprecipitation with 30 l of anti-Pyk1 antibody (gift of T. Nowak); the procedures used for immunoprecipitation were described previously (24). The immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-Mck1 antibody (6) ( a DBY1 is a ura3 derivative of DDY1 (12) which carries an mck1::U...

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

“…Like mck1⌬ mutants, strains overproducing pyruvate kinase fail to grow at 37°C and do not accumulate normal amounts of glycogen. Cells overexpressing CDC19, like mck1⌬, adapt poorly to growth on non-fermentable carbon sources (11,18,19). Finally, diploid cells overexpressing CDC19 show markedly diminished sporulation efficiency (11), similar to that observed in mck1⌬/mck1⌬ diploids.…”

“…Cells overexpressing CDC19, like mck1⌬, adapt poorly to growth on non-fermentable carbon sources (11,18,19). Finally, diploid cells overexpressing CDC19 show markedly diminished sporulation efficiency (11), similar to that observed in mck1⌬/mck1⌬ diploids. Deletion of MCK1 was found to exacerbate each of these CDC19 overexpression phenotypes (11).…”

“…The findings of Brazill et al (11) suggested that Mck1p might directly phosphorylate pyruvate kinase, thereby down-regulating the activity of this enzyme. In this report we investigate this possibility.…”

“…Previous work has demonstrated that Mck1p down-regulates pyruvate kinase (EC 2.7.1.40), encoded by the CDC19/PYK1 gene, in vivo (11). Like mck1⌬ mutants, strains overproducing pyruvate kinase fail to grow at 37°C and do not accumulate normal amounts of glycogen.…”

“…Deletion of the MCK1 locus results in various defects in carbon metabolism, including reduced glycogen accumulation and poor growth on non-fermentable carbon sources (10,11). Other phenotypes resulting from deletion of MCK1 include heat and cold sensitivity (10,12), delayed sporulation (13) and sensitivity to the microtubule-destabilizing drug benomyl (12).…”