Phosphorylation of Mg(2+)-dependent protein phosphatase alpha (type 2C alpha) by casein kinase II

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Mg(2+)-dependent protein phosphatase alpha (MPP alpha) is a key enzyme involved in cellular regulation.
• Understanding the post-translational modifications of MPP alpha, such as phosphorylation, is crucial for elucidating its function.
• Investigating the phosphorylation of rat MPP alpha in a heterologous expression system provides insights into enzyme regulation.

Purpose of the Study:

• To investigate the in vivo phosphorylation of rat Mg(2+)-dependent protein phosphatase alpha (MPP alpha) expressed in Saccharomyces cerevisiae.
• To identify the specific residues and kinases responsible for MPP alpha phosphorylation.
• To compare in vivo and in vitro phosphorylation patterns of rat MPP alpha.

Main Methods:

• Expression of recombinant rat MPP alpha in Saccharomyces cerevisiae and Escherichia coli.
• In vitro phosphorylation assays using purified recombinant MPP alpha and various casein kinases (casein kinase I and II).
• Phosphopeptide mapping and amino acid analysis to identify phosphorylation sites.

Main Results:

• Rat MPP alpha expressed in yeast cells undergoes phosphorylation on serine residues in vivo.
• Recombinant rat MPP alpha is phosphorylated by casein kinase II, but not casein kinase I, in vitro.
• In vivo and in vitro phosphorylation sites are identical, involving only serine residues.
• The phosphorylation level in vitro reached 1.5 mol phosphate per mol enzyme protein.

Conclusions:

• Yeast casein kinase II likely phosphorylates rat MPP alpha in vivo when expressed in Saccharomyces cerevisiae.
• The phosphorylation sites are located in the carboxyl-terminal region of the rat MPP alpha enzyme.
• These findings contribute to understanding the regulation of Mg(2+)-dependent protein phosphatase alpha through phosphorylation.