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mRNA expression of the immediate-early gene NGFI-B was investigated in T cells during the G0/G1 transition as well as throughout the G1 phase. After stimulation of T lymphocytes with Con A or phorbol 12,13 dibutyrate (PDBu), NGFI-B gene expression showed an induction of at least sevenfold within 3 h of stimulation. Twenty-four h later, however, the level of NGFI-B transcripts had fallen to almost basal levels. Activation of the Ca2+ signaling pathway also produced an induction of this gene, although to a lesser extent than the one obtained after protein kinase C activation. Similar transient kinetics of NGFI-B mRNA were also observed after PDBu stimulation of G1 lymphoblasts. However, the induction of NGFI-B by IL-2 is dependent on the presence of cycloheximide. Con A-induced activation of NGFI-B gene expression was not overcome by cyclosporin A or by 8Br-cAMP, but was partially prevented by dexamethasone. In lymphoblasts, okadaic acid caused the induction of NGFI-B gene expression, indicating a role for the serine/threonine protein phosphatases PP1 and PP2A in the regulation of this gene in resting cells. Okadaic acid-induced NGFI-B transcripts were significantly more stable than PDBu-induced NGFI-B mRNA. Thus, the level of NGFI-B transcripts in T cells might be determined by the balance between the activities of several serine/threonine protein kinases and phosphatases. Together, these findings indicate that the transient induction of NGFI-B transcripts is associated with normal lymphocyte activation. Because the mRNA for NGFI-B codes for a zinc-finger DNA-binding protein, these results suggest that NGFI-B participates in transcriptional regulation during T cell activation.

Antibody prepared against the catalytic subunit of protein phosphatase-2A from rabbit skeletal muscle, could completely inhibit this enzyme, but did not significantly affect the activities of protein phosphatases-1, 2B and 2C. The antibody was used to establish the following points. The three forms of protein phosphatase-2A that can be resolved by ion-exchange chromatography, termed 2A0, 2A1, and 2A2, share the same catalytic subunit. The antigenic sites on the catalytic subunit of protein phosphatase-2A remain accessible to the antibody, when the catalytic subunit is complexed with the other subunits of protein phosphatases-2A0, 2A1 and 2A2. The catalytic subunits of protein phosphatase-2A from rabbit skeletal muscle and rabbit liver are very similar, as judged by immunotitration experiments. Protein phosphatase-1 and protein phosphatase-2A account for virtually all the phosphorylase phosphatase activity in dilute tissue extracts prepared from skeletal muscle, liver, heart, brain and kidney, and for essentially all the glycogen synthase phosphatase activity in dilute skeletal muscle and liver extracts. Protein phosphatase-2A is almost absent from the protein-glycogen complex prepared from skeletal muscle or liver extracts. Protein phosphatase-2A accounts for a major proportion of the phosphatase activity in dilute liver extracts towards 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, 6-phosphofructo-1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase and phenylalanine hydroxylase, the major phosphorylated enzymes involved in the hormonal control of hepatic glycolysis and gluconeogenesis.

The dephosphorylation of glycogen synthase by protein phosphatase-1 in hepatic glycogen and microsomes was inhibited by nanomolar concentrations of phosphorylase a. The I50 for phosphorylase a was 1000-fold lower than its Km as a substrate, while tryptic digestion increased the I50 1000-fold without affecting Km. Protein phosphatase-1 from skeletal muscle and protein phosphatase-2A from liver were only inhibited at 1000-fold higher concentrations. Protein phosphatase-1 became desensitized to phosphorylase a when released from hepatic microsomes, but sensitivity was partially restored by readdition of the solubilized enzyme to the microsomes. The results demonstrate that phosphorylase a is a potent allosteric inhibitor of hepatic protein phosphatase-1 and suggest that inhibition may be conferred by a novel phosphorylase a-binding subunit.

A 2.5 kb clone containing the full-length coding sequence of a type-2A protein phosphatase catalytic subunit has been isolated from a rabbit skeletal muscle cDNA library constructed in lambda gt10. The sequence of the protein deduced from the cDNA contains 309 residues (35.58 kDa). A major mRNA species at 2.0 kb and a minor component at 2.8 kb were visualized by Northern blotting in both skeletal muscle and liver. The type-2A enzyme showed weak homology with mammalian alkaline phosphatases between residues 55 and 95. The protein sequence of the type-2A phosphatase from rabbit skeletal muscle differs from that reported for the bovine adrenal enzyme in three regions.

Protein phosphatases-2A0, 2A1 and 2A2 have been purified to homogeneity from rabbit skeletal muscle. Approximately 1 mg of phosphatase-2A0 and 2A1, and 0.5 mg of phosphatase-2A2, was isolated from 4000 g muscle within 10 days. Protein phosphatases-2A0 and 2A1 each comprised three subunits, termed A, B' and C (2A0) or A, B and C (2A1), while phosphatase-2A2 contained only two subunits, A and C. The A and C components of phosphatases-2A0, 2A1 and 2A2 had indistinguishable mobilities on sodium dodecyl sulphate/polyacrylamide gels and identical peptide maps. By these criteria, the C component was also identical to the catalytic subunit of phosphatase-2A purified from ethanol-treated muscle extracts. The electrophoretic mobilities of the B and B' subunits were slightly different, and their peptide maps were distinct. The molecular masses of the native enzymes determined by sedimentation equilibrium centrifugation were 181 +/- 6 kDa (2A0), 202 +/- 6 kDa (2A1) and 107 +/- 5 kDa (2A2), while those of the subunits estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis were 60 kDa (A), 55 kDa (B), 54 kDa (B') and 36 kDa (C). These values, in conjunction with molar ratios estimated by densitometric analyses of the gels, suggest that the subunit structures of the enzymes are AB'C2 (2A0), ABC2 (2A1) and AC (2A2). Protein phosphatase-2A2 appears to be derived from 2A0 and/or 2A1 during purification through degradation or dissociation of the B' and/or B subunits. Protein phosphatases-2A0, 2A1 and 2A2 were the only phosphorylase phosphatases in rabbit skeletal muscle that were activated by the basic proteins, protamine (A0.5 = 0.25 microM), histone H1 (A0.5 = 0.3 microM) and polylysine (A0.5 = 0.04 microM). Activation by protamine varied over 5-20-fold for phosphatase-2A0 and 5-7-fold for phosphatases-2A1 and 2A2. The dephosphorylation of glycogen synthase was activated by basic proteins in a similar manner to the phosphorylase phosphatase activity. The isolated C subunit was also stimulated by histone H1 and protamine, but 5-10-fold higher concentrations were required, and with phosphorylase as substrate, maximum activation was only about 2-fold. Activation by basic proteins appears to involve their interaction with the A and/or C subunits, but not with the B or B' subunits, or substrates phosphorylase and glycogen synthase.

Glycogen synthase (labelled in sites-3) and glycogen phosphorylase from rabbit skeletal muscle were used as substrates to investigate the nature of the protein phosphatases that act on these proteins in the glycogen and microsomal fractions of rat liver. Under the assay conditions employed, glycogen synthase phosphatase and phosphorylase phosphatase activities in both subcellular fractions could be inhibited 80-90% by inhibitor-1 or inhibitor-2, and the concentrations required for half-maximal inhibition were similar. Glycogen synthase phosphatase and phosphorylase phosphatase activities coeluted from Sephadex G-100 as broad peaks, stretching from the void volume to an apparent molecular mass of about 50 kDa. Incubation with trypsin decreased the apparent molecular mass of both activities to about 35 kDa, and decreased their I50 for inhibitors-1 and -2 in an identical manner. After tryptic digestion, the I50 values for inhibitors-1 and -2 were very similar to those of the catalytic subunit of protein phosphatase-1 from rabbit skeletal muscle. The glycogen and microsomal fractions of rat liver dephosphorylated the beta-subunit of phosphorylase kinase much faster than the alpha-subunit and dephosphorylation of the beta-subunit was prevented by the same concentrations of inhibitor-1 and inhibitor-2 that were required to inhibit the dephosphorylation of phosphorylase. The same experiments performed with the glycogen plus microsomal fraction from rabbit skeletal muscle revealed that the properties of glycogen synthase phosphatase and phosphorylase phosphatase were very similar to the corresponding activities in the hepatic glycogen fraction, except that the two activities coeluted as sharp peaks near the void volume of Sephadex G-100 (before tryptic digestion). Tryptic digestion of the hepatic glycogen and microsomal fractions increased phosphorylase phosphatase about threefold, but decreased glycogen synthase phosphatase activity. Similar results were obtained with the glycogen plus microsomal fraction from rabbit skeletal muscle or the glycogen-bound form of protein phosphatase-1 purified to homogeneity from the same tissue. Therefore the divergent effects of trypsin on glycogen synthase phosphatase and phosphorylase phosphatase activities are an intrinsic property of protein phosphatase-1. It is concluded that the major protein phosphatase in both the glycogen and microsomal fractions of rat liver is a form of protein phosphatase-1, and that this enzyme accounts for virtually all the glycogen synthase phosphatase and phosphorylase phosphatase activity associated with these subcellular fractions.