Saccharomyces cerevisiae pyruvate kinase Pyk1 is PKA phosphorylation substrate in vitro

Area of Science:

• Biochemistry
• Molecular Biology
• Yeast Genetics

Background:

• cAMP-dependent protein kinase (PKA) regulates numerous cellular processes in Saccharomyces cerevisiae.
• Yeast pyruvate kinase (Pyk1) is a key enzyme in glycolysis, but its regulation by PKA is not fully understood.

Purpose of the Study:

• To investigate the interaction between PKA and Pyk1 in yeast.
• To determine if PKA can phosphorylate Pyk1 and identify the specific PKA subunits involved.

Main Methods:

• Fractionation of Saccharomyces cerevisiae postribosomal extract using DEAE-cellulose chromatography.
• Immunoblotting with anti-Bcy1 and anti-Pyk1 antibodies to confirm protein presence and identify copurifying proteins.
• In vitro phosphorylation assays using purified PKA and Pyk1 in the presence of cAMP and cGMP.
• Two-dimensional gel electrophoresis to analyze phosphorylated Pyk1 forms.

Main Results:

• Two fractions of PKA (PKA-1 and PKA-2) were isolated from yeast extract.
• Yeast pyruvate kinase (Pyk1) copurified with the PKA-1 fraction.
• Pyk1 was phosphorylated in vitro by yeast PKA, generating four distinct phosphorylated forms.
• The Tpk2 catalytic subunit of PKA was primarily involved in Pyk1 phosphorylation.

Conclusions:

• Pyk1 interacts with a specific fraction of yeast PKA (PKA-1).
• Yeast PKA, particularly the Tpk2 subunit, directly phosphorylates Pyk1 in vitro, suggesting a regulatory role.
• This phosphorylation modifies Pyk1, potentially impacting its enzymatic activity and glycolytic flux in Saccharomyces cerevisiae.