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

Abstract

The nature of protein phosphatases that are active against the phosphorylated proteins of glycogen metabolism was investigated in rabbit skeletal muscle and liver. Six 32P-labelled substrates corresponding to the major phosphorylation sites on glycogen phosphorylase, phosphorylase kinase, glycogen synthase and inhibitor-I were used in these studies. The results showed that the four protein phosphatases defined in the preceding paper, namely protein phosphatases-I, 2A, 2B and 2C [Ingebritsen, T. S. and Cohen, P. (1983) Eur. J. Biochem. 232, 255-2611 were the only significant enzymes acting on these substrates. The four enzymes can 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-2Ao (0.12 M NaCI), 2A1 (0.2 M NaCI) and 2Az (0.28 M NaCI) that had apparent molecular weights of 210000, 210000 and 150000 respectively. Protein phosphatase-2Ao was a completely inactive enzyme whose activity was only expressed after dissociation to a 34000-M,,,,,, catalytic subunit by freezing and thawing in 0.2 M 2-mercaptoethanol. This treatment also dissociated protein phosphatases 2A1 and 2Az to more active 34000-Mr~,,,~ catalytic subunits. The catalytic subunits derived from protein phosphatases-2Ao, 2A1 and 2Az possessed identical substrate specificities, preferentially dephosphorylated the r-subunit of phosphorylase kinase, were unaffected by inhibitor-I and inhibitor-2 and were inhibited by similar concentrations of ATP. The properties of protein phosphatases-2Al and 2Az 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-cellulose in the same fraction as protein phosphatase-2Ao. These activities were resolved by gel filtration, the Mr(app) of protein phosphatase-2B being 98 000. Protein phosphatase-2B was completely inhibited by 100 pM trifluoperazine, which did not affect the activity of protein phosphatase-2Ao or any other protein phosphatase. Freezing and thawing in 0.2 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 phosphatase-2Al. These activities were completely resolved by gel filtration, since the Mr(,,,) of protein phosphatase-2C was 46000.Two forms of protein phosphatase-1 can be identified by chromatography on DEAE-cellulose, namely protein phosphatase-1 itself and the Mg . ATP-dependent protein phosphatase. Both these species were eluted at 0.16 M NaCl just ahead of protein phosphatases-2C and 2Al. 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 phosphatases described in the literature as enzymes active on the phosphorylated proteins of glycogen metabolism can now b...

Multisite phosphorylation of the glycogen‐binding subunit of protein phosphatase‐1G by cyclic AMP‐dependent protein kinase and glycogen synthase kinase‐3

“…Protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A) account for all the phosphatase activity in mammalian skeletal muscle extracts towards the sites 3 region of glycogen synthase [2,3]. Two lines of evidence suggest that PP-1 is the enzyme that dephosphorylates these serines in vivo.…”

Protein phosphatase 2A is associated in an inactive state with microtubules through 2A1-specific interaction with tubulin

“…The first peak found at 0n31 M NaCl, was judged to be PP2A1 from the NaCl concentration for the elution and the sensitivity to okadaic acid [18,38,39]. PP1 or PP2A0 reported to elute at 0n25 M NaCl or less [38,40] was hardly detected possibly because of loss of the unstable enzymes and\or omission of the activation procedures used for these enzymes [40]. When the postmicrotubule supernatant was chromatographed under the conditions identical with those used in Figure 1A, the total activity in the peak of PP2A1 was found to be about 40 % of that of PP2A1 isolated from the brain extract.…”

On the 6‐phosphofructo‐1‐kinase phosphatase activity of protein phosphatase 2C and its dimeric nature

“…According to [9] the first peak eluting with the void volume represents phosphatase 2Ar and the second the Mg2+-dependent phosphatase 2C. No peak of PFK-phosphatase activity emerged with Mrcapp.)…”

“…After loading this supernatant on a DEAE-cellulose column and washing with 80 mM NaCl, the protein phosphatases were eluted with 200 mM NaCI. This eluate should contain PFKphosphatase activity as well as phosphatase 2C as this enzyme emerges from DEAE-cellulose at 120 mM [Z] or 200 mM NaCl [9], while PFKphosphatase elutes at 120-150 mM NaCl fl]. The proteins were concentrated by ammo~~rn sulfate precipitation (0.39 g/ml) and ~hromatograp~ed on a Sephadex G-100 column.…”

“…phosphatases obtained from 7-9 experiments are 71400 [9], 86400 [9] and 87000 [8] with respect to catalase and 75 150 181, 91400 [8] and 91500 [7] with respect to alcohol dehydrogenase as standard enzymes. Accordingly, the klr values of the protein 44500 (not shown) indicating that the 90-kDa enzyme consists of two identical subunits.…”