The Proteink Phosphatases Involved in Cellur Regulation. 2. Glycogen Metabolism

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Abstract
The nature of protein phosphatases that are active against the phosphorylated proteins of glycogen metaboolism was investigated in rabbit skeletal muscle and liver. Six32P-labelled substrates corresponding to the major phosphorylation sites on glycogen phosphorylase, phosphorylase kinase, glycogen synthase and inhibitor-1 were used in these studies. The results showed that the four protein phosphatases defined in the preceding paper, namely protein phosphatases-1, 2A, 2B and 2C [Ingebritsen, T. S. and Chohen, P. (1983) Eur. J. Biochem. 132, 255–261]were the only singificant enzymes acting on these subvstrates. The four enzymes cna be conveniently separated and identified by a combination of ion-exchange chromatography and gel filtration and by the use of specific inhibitors. Three species of protein phosphatase-2A were resolved on DEAE-cellulose, termed protein phosphatases-2A0 (0.12 M NaCl), 2A1 (0.2 M Nacl) and 2A2 (0.28 M NaCl) that havd apparent molecular weights 210000, 210000 and 150000 respectively. Protein phosphatase-2A0 was a completely inactive enzyme whose activity was onhly expressed after dissociation to a 34000-Mr(app) catalytic subunit by freezing and thawing in 0.2 M 2-mercaptoethanol. This treatment also dissocaiated protein phosphatases 2A1 and 2A2 to more active 34000-Mr(app) catalytic subunits. The catalytic sununits derived from protein phosphatases-2A0, 2A2 and 2A2 possessed identical substrate specificities, preferentially dephosphorylated the α-subunit of phosphorylase kinase, were unaffected by inhibitor-a and inhibitor-2 ans were inhibited by similar concentrations of ATP. The properites of protein phosphatases-2A1 and 2A2 were very similar to those of the catalytic subunits, except that they were less sensitive to inhibition by ATP. Protein phosphatase-2B was eluted from DEAE-cellulosde in the same fraction as protein phosphatase-2A0. These activities were resolved by gel filtration, the Mr(app) of protein phosphatase-2B being 98000. Protein phosphatase-2B was completely inhibited by 100 μM trifluoperazine, which did njot affect the activityu of protein phosphatase 2A0 or any other protein phosphatse. Freezingf and thawing in 02 M 2-mercaptoethanol resulted in partial inactivation of protein phosphatase-2B. Protein phosphatase-2C was eluted from DEAE-cellulose at the leading edge of the peak of protein phosphatse-2A1. These activitiews were completely resolved by gel filtration, since the Mr(app) of protein phosphatase-2C was 46000. Two forms of protein phosphatse-1 can be identified by chromatography on DEAE-cellulsoe, namely protein phosphatse-1 itself and the Mg. ATP-dependent protein phosphatse. Both thse species were eluted at 0.16 M NaCl just ahead of protein phosphatases-2C and 2A1. These enzymes did not interfere with measurements of type-2 protein phosphatases, since it was possible to block their activity with inhibitor-2. Most, if not all, of the protein phosphatasses described in the literature as enzymes active on the phosphorylated proteins of glycogen metabolism can now be explained as protein phosphatases 1, 2A, 2B or 2C. The subunit structures of these four enzuyme are reviewed.