Parallel purification of three catalytic subunits of the protein serine/threonine phosphatase 2A family (PP2A(C), PP4(C), and PP6(C)) and analysis of the interaction of PP2A(C) with alpha4 protein

Protein Expr Purif. 2003 Sep;31(1):19-33. doi: 10.1016/s1046-5928(03)00141-4.

Abstract

The protein serine/threonine phosphatase (PP) type 2A family consists of three members: PP2A, PP4, and PP6. Specific rabbit and sheep antibodies corresponding to each catalytic subunit, as well as a rabbit antibody recognizing all three subunits, were utilized to examine the expression of these enzymes in select rat tissue extracts. PP2A, PP4, and PP6 catalytic subunits (PP2A(C), PP4(C), and PP6(C), respectively) were detected in all rat tissue extracts examined and exhibited some differences in their levels of expression. The expression of alpha4, an interacting protein for PP2A family members that may function downstream of the target of rapamycin (Tor), was also examined using specific alpha4 sheep antibodies. Like the phosphatase catalytic subunits, alpha4 was ubiquitously expressed with particularly high levels in the brain and thymus. All three PP2A family members, but not alpha4, bound to the phosphatase affinity resin microcystin-Sepharose. The phosphatase catalytic subunits were purified to apparent homogeneity (PP2A(C) and PP4(C)) or near homogeneity (PP6(C)) from bovine testes soluble extracts following ethanol precipitation and protein extraction. In contrast to PP2A(C), PP4(C) and PP6(C) exhibited relatively low phosphatase activity towards several substrates. Purified PP2A(C) and native PP2A in cellular extracts bound to GST-alpha4, and co-immunoprecipitated with endogenous alpha4 and ectopically expressed myc-tagged alpha4. The interaction of PP2A(C) with alpha4 was unaffected by rapamycin treatment of mammalian cells; however, protein serine/threonine phosphatase inhibitors such as okadaic acid and microcystin-LR disrupted the alpha4/PP2A complex. Together, these findings increase our understanding of the biochemistry of alpha4/phosphatase complexes and suggest that the alpha4 binding site within PP2A may include the phosphatase catalytic domain.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Anion Exchange Resins / chemistry
  • Antibodies / immunology
  • Antibodies / isolation & purification
  • Antibody Specificity
  • Blotting, Western
  • Brain / enzymology
  • Brain Chemistry
  • COS Cells
  • Caseins / metabolism
  • Catalytic Domain*
  • Cattle
  • Chlorocebus aethiops
  • Chromatography, Affinity
  • Cloning, Molecular
  • Ethers, Cyclic / pharmacology
  • Gene Expression
  • Glutathione Transferase / genetics
  • Glutathione Transferase / metabolism
  • Histones / metabolism
  • Intercellular Signaling Peptides and Proteins
  • Liver / chemistry
  • Liver / enzymology
  • Male
  • Mice
  • Microcystins
  • Molecular Sequence Data
  • Myocardium / chemistry
  • Myocardium / enzymology
  • Okadaic Acid / pharmacology
  • Oxazoles / pharmacology
  • Peptides, Cyclic / chemistry
  • Peptides, Cyclic / metabolism
  • Phosphoprotein Phosphatases / genetics
  • Phosphoprotein Phosphatases / isolation & purification*
  • Phosphoprotein Phosphatases / metabolism
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Phosphorylase a / metabolism
  • Phosphorylation
  • Precipitin Tests
  • Protein Binding / physiology
  • Protein Kinases / metabolism
  • Protein Phosphatase 2
  • Rats
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Resins, Synthetic
  • Ribosomal Protein S6 Kinases, 70-kDa / metabolism
  • Sequence Alignment
  • Sequence Analysis, Protein
  • Sirolimus / pharmacology
  • Testis / chemistry
  • Testis / enzymology
  • Thymus Gland / chemistry
  • Thymus Gland / enzymology

Substances

  • 1-norokadaone
  • Anion Exchange Resins
  • Antibodies
  • Caseins
  • Ethers, Cyclic
  • Histones
  • Igbp1 protein, mouse
  • Intercellular Signaling Peptides and Proteins
  • Microcystins
  • Oxazoles
  • Peptides, Cyclic
  • Phosphoproteins
  • Recombinant Fusion Proteins
  • Resins, Synthetic
  • Okadaic Acid
  • microcystin
  • Mono Q
  • Phosphorylase a
  • Glutathione Transferase
  • Protein Kinases
  • Ribosomal Protein S6 Kinases, 70-kDa
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 2
  • Sirolimus