Biochemical characterization of recombinant yeast PPZ1, a protein phosphatase involved in salt tolerance

FEBS Letters

Bollen, Mathieu²  Ariño, Joaquín¹  Posas, Francesc¹  Stalmans, Willy² 

[1] Departament de Bioquímica i Biologia Molecular, Facultat de Veterinària Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain;Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium

关键词: Bacterial expression;    Fusion protein;    Protein phosphatase;    Saccharomyces cerevisiae;    CK-2;    casein kinase-2;    GST;    glutathione-S-transferase;    PK-A;    CAMP-dependent protein kinase;    PK-C;    protein kinase C;    PP;    protein phosphatase;   

DOI  :  10.1016/0014-5793(95)00593-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The Saccharomyces cerevisiae gene PPZ1 codes for a 692-residues protein that shows in its carboxyl-terminal half about 60% identity with the catalytic subunit of mammalian and yeast protein phosphatase-1 and that is involved in salt homeostasis. T The complete PPZ1 protein has been succesfully expressed as a soluble glutathione-S-transferase fusion protein. The recombinant protein, after purification by a single affinity chromatography step, displayed phosphatase activity towards a number of substrates, including myelin basic protein, histone 2A and casein, but was ineffective in dephosphorylating glycogen phosphorylase. It was also active towards p-nitrophenylphosphate. The activity was severalfold increased by the presence of Mn²⁺ ions and by limited trypsinolysis. The enzyme was inhibited by okadaic acid and microcystin-LR at concentrations comparable to what is found for type 1 protein phosphatase although it was much less sensitive to inhibitor-2. The recombinant protein was phosphorylated in vitro by cAMP-dependent protein kinase, protein kinase C and casein kinase-2. Phosphorylation affected preferentially sites located in the amino-terminal half of the protein and did not alter the activity of the phosphatase.

【 授权许可】

Unknown