Diversity in the regulatory B-subunits of protein phosphatase 2A: identification of a novel isoform highly expressed in brain.

The physiological role of type 2A protein phosphatases (PP2A) is dependent upon the association of the catalytic subunit with a variety of regulatory subunits. In order to understand the function of PP2A, we have undertaken purification of the holoenzymes and molecular cloning of the regulatory subunits. Two trimeric forms containing distinct B-subunits, PP2A0 and PP2A1, have been purified from rabbit skeletal muscle. The B-subunits associated with PP2A0 and PP2A1 migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with slightly different mobility, approximately 52.5 and approximately 51.5 kDa, respectively and showed distinct immunological properties. The B' form of B-subunit associated with PP2A0 was recognized by antibodies against the B-subunit present in bovine heart PP2A but not by antibodies specific to the B subunit isoforms of rabbit PP2A1. Cloning of cDNAs encoding the B subunit of PP2A1 resulted in the isolation of a cDNA highly homologous to, but distinct from, the B alpha subunit isoform. The deduced amino acid sequence of this novel isoform, which was designated B gamma, encoded a protein which was 81% and 87% identical to the B alpha and B beta isoforms, respectively. Northern blot analysis indicated that the B gamma isoform is highly expressed in rabbit brain as a transcript of 3.9 kb. Analysis of B-subunit expression by Western blot indicated a general parallel with the message levels. In conclusion, our data reveal even greater complexity of PP2A trimeric holoenzymes due to the identification of a novel B regulatory subunit isoform of PP2A1 and a distinct B' subunit associated with PP2A0.

Molecular mechanism of the synergistic phosphorylation of phosphatase inhibitor-2. Cloning, expression, and site-directed mutagenesis of inhibitor-2.

Inhibitor-2 (I-2) is the regulatory subunit of the ATP-Mg-dependent phosphatase, a cytosolic form of type 1 protein phosphatase. Phosphorylation of I-2 at Thr-72 by the protein kinase glycogen synthase kinase-3 (GSK-3) leads to activation of the enzyme. Casein kinase II action was shown to synergistically enhance phosphorylation and activation by GSK-3 (DePaoli-Roach, A.A. (1984) J. Biol. Chem. 259, 12144-12152). Rabbit skeletal muscle and liver I-2 cDNA clones have been isolated. Rabbit skeletal muscle cDNAs could be placed in two subtypes, differing in the length of the 3'-untranslated region. The coding sequence of 612 nucleotides was identical in the two skeletal muscle and the liver cDNAs and predicted a protein of 204 amino acids, consistent with analysis of the purified protein. Northern hybridization analysis indicated that the two mRNAs of 1.7 and 2.7 kilobase pairs were present in all rabbit tissues examined, except in liver, where only the larger transcript was detected, and in testis, where additional transcripts were present. Expression in Escherichia coli of wild-type and phosphorylation site mutants resulted in the production of I-2 polypeptides with apparent M(r) values of approximately 31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitory activity of the recombinant proteins was similar to that of native rabbit skeletal muscle I-2 and was unaffected by the substitution of alanine for the GSK-3 site (Thr-72) and for the casein kinase II sites (Ser-86 and Ser-120/121) or by substitution of glutamic acid and aspartic acid for Thr-72 and Ser-86. Recombinant wild-type I-2 and the Ala-120/121 mutant were phosphorylated synergistically by GSK-3 and casein kinase II. The Thr-72 and Ser-86 mutants, however, did not undergo this synergistic phosphorylation. Our studies indicate that Thr-72 is the only GSK-3 site and that Ser-86 is the casein kinase II site required for the potentiation of GSK-3 action. Furthermore, acidic residues cannot substitute for the phosphate group either in enhancing GSK-3 phosphorylation or in activating the phosphatase.

Molecular cloning and expression of the regulatory (RG1) subunit of the glycogen-associated protein phosphatase.

DNA clones encoding the glycogen-binding (RG1) subunit of glycogen-associated protein phosphatase were isolated from rabbit skeletal muscle lambda gt11 cDNA libraries. Overlapping clones provided an open reading frame of 3327 nucleotides that predicts a polypeptide of 1109 amino acids with a molecular weight of 124,257. Northern hybridization of rabbit RNA identified a major mRNA transcript of 7.5 kilobases present in skeletal, diaphragm, and cardiac muscle, but not in brain, kidney, liver, and lung. Southern analysis of rabbit genomic DNA digested with various restriction endonucleases gave rise to a single hybridizing fragment, suggesting that a single gene is present. Expression of the complete RG1 subunit coding sequence in Escherichia coli generated a protein of apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 160,000, similar to the size of the polypeptide detected by Western immunoblot in rabbit skeletal muscle extracts. The RG1 subunit shares significant homology with the Saccharomyces cerevisiae GAC1 gene product which is involved in activation of glycogen synthase and glycogen accumulation. The homology with GAC1 substantiates the role of this enzyme in control of glycogen metabolism. Hydropathy analysis of the RG1 subunit amino acid sequence revealed the presence of a hydrophobic region in the COOH terminus, suggesting a potential association with membrane. This result suggests that the same phosphatase regulatory component may be involved in targeting the enzyme both to membranes and to glycogen.