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The intracellular signaling mechanisms that couple transient cerebral ischemia to cell death and neuroprotective mechanisms provide potential therapeutic targets for cardiac arrest. Protein phosphatase (PP)-1 is a major serine/threonine phosphatase that interacts with and dephosphorylates critical regulators of energy metabolism, ionic balance, and apoptosis. We report here that PP-1I, a major regulated form of PP-1, is activated in brain by approximately twofold in vivo following cardiac arrest and resuscitation in a clinically relevant pig model of transient global cerebral ischemia and reperfusion. PP-1I purified to near homogeneity from either control or ischemic pig brain consisted of the PP-1 catalytic subunit, the inhibitor-2 regulatory subunit, as well as the novel constituents 14-3-3γ, Rab GDP dissociation protein β, PFTAIRE kinase, and C-TAK1 kinase. PP-1I purified from ischemic brain contained significantly less 14-3-3γ than PP-1I purified from control brain, and purified 14-3-3γ directly inhibited the catalytic subunit of PP-1 and reconstituted PP-1I. These findings suggest that activation of brain PP-1I following global cerebral ischemia in vivo involves dissociation of 14-3-3γ, a novel inhibitory modulator of PP-1I. This identifies modulation of PP-1I by 14-3-3 in global cerebral ischemia as a potential signaling mechanism-based approach to neuroprotection. [ABSTRACT FROM AUTHOR]

The effects of polyamines on the oligomeric forms of protein phosphatase-1. (1G), protein phosphatase-2A (2A0, 2A1 and 2A2) and their free catalytic subunits (1C and 2Ac) has been studied using homogeneous enzymes isolated from rabbit skeletal muscle. Spermine increased the activity of protein phosphatase-2A towards eight of nine substrates tested. Half-maximal activation was observed at 0.2 mM with optimal effects at 1–2 mM. Above 2 mM, spermine became inhibitory. The most impressive activation of protein phosphatase-2A was obtained with glycogen synthase, especially when phosphorylated at sites-3 (8–15-fold with protein phosphatase-2A1) and phenylalanine hydroxylase (6–7-fold with protein phosphatase-2A1) as substrates. Activation of protein phosphatases 2A0 2A1 and 2A2 was greater than that observed with 2Ac. Spermine was a more potent activator than spermidine, while putrescine had only a small effect. Qualitatively similar results were obtained with five other substrates, although maximal activation was much less (1.3–3-fold with protein phosphatase+2A1). The rate of dephosphorylation of glycogen phosphorylase was decreased by spermine, inhibition being more pro- nounced with protein phosphatase-2Ac than with 2A0, 2A1 and 2A2. Spermine (I50 = 0.1 mM with protein phosphatase-2Ac) was a more potent inhibitor than spermidine (I50 = 0.9 mM) or putrescine (I50 = 8 mM). Partially purified preparations of protein phosphatases-2A0, 2A1 and 2A2 from from rat liver were affected by spermine in a similar mariner to the homogeneous enzymes from rabbit skeletal muscle. Spermine did not activate protein phosphatase-1 to the stone extent as protein phosphatase-2A. Greatest stimulation (2.5-fold) was again observed with glycogen synthase labelled in sites-3, with half-maximal activation at 0.2 mM and optimal effects at 1–2 mM spermine. Spermine was a much more effective stimulator than spermidine, while putrescine was ineffective. Very similar results were obtained with protein phosphatases 1G and 1c. With four other substrates maximal activation by spermine was < 1.5-fold, while the dephosphorylation of glycogen synthase (labelled in site-2), phosphorylase kinase, pyruvate kinase and glycogen phosphorylase were inhibited. Spermine (I50 = 0.04 mM) was a more potent inhibitor of the dephosphorylation of glycogen phosphorylase than spermidine (I50 = 0.9 mM) or putrescine (I50 = 9 mM). There was some resemblance between the effects of Mn2+ and spermine on the activity of protein phosphatase- 2A, although with the substrates inhibitor-1 and phenylalanine hydroxylase, activation by Mn2+ was greater than that observed with spermine, while with glycogen synthase the converse was true. Mn2+ could not mimic the activating effect of spermine on dephosphorylation of glycogen synthase (labelled in sites-3) by protein phosphatase 1. By contrast, Spermine hardly affected the dephosphorylation of inhibitor-1 by phosphatase-1, a reaction that was almost completely dependent on Mn2+. Physiological concentrations of Mg2+ (1.0 mM) neither mimicked, nor influenced, the response to spermine with either protein phosphatase-1 or 2A. The effects of spermine appear to result from interaction of the polyamine with both the protein phosphatases and their substrates. The possibility that activation of protein phosphatase-1 and/or 2A by spermine underlies the relatively specific dephosphorylation of sites-3 of glycogen synthase by insulin is discussed. [ABSTRACT FROM AUTHOR]

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, 2A2 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 gets, suggest that the subunit structures of the enzymes are AB′C2 (2A0), ABC2 (2A 1) 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. [ABSTRACT FROM AUTHOR]

Homogenous preparations of the catalytic subunit of protein phosphatase-1 and inhibitor-2 can be combined to produce an inactive enzyme that consists of a 1:1 complex between these two proteins. This species is indistinguishable from the Mg-ATP-dependent protein phosphatase in that preincubation with glycogen synthase kinase-3 and Mg-ATP is required to generate activity. Activation results from the phosphorylation of inhibitor-2. The molar concentrations of protein phosphatase-1 and inhibitor-2 in rabbit skeletal muscle (0.25-0.5 µM) are similar. Incubation of the reconstituted Mg-ATP-dependent protein phosphatase with chymotrypsin is accompanied by limited proteolysis of inhibitor-2 and the loss of its phosphorylation site(s). This species can be activated by glycogen synthase kinase-3 and Mg-ATP provided that inhibitor-2 is added. This exogenous inhibitor-2 appears to displace the fragments of inhibitor-2 from the enzyme that were generated by chymotryptic digestion. These experiments may explain the report [Yang, S. D., Vandenheede, J. R. and Merlevede, W. (1981) J. Biol. Chem. 256, 10231-10234] that inhibitor-2 can function as an 'activator' as well as an inhibitor of the Mg-ATP-dependent protein phosphatase. Incubation of the catalytic subunit of protein phosphatase-t with sodium fluoride or sodium pyrophosphate converted the enzyme to an inactive form that could be partially reactivated by munganese ions, but not by glycogen synthase kinase-3 and Mg-ATP. Conversely, the reconstituted Mg-ATP-dependent protein phosphatase could only be activated by glycogen synthase kinase-3 and Mg-ATP, and not by manganese ions. It is concluded that the conversion of protein phosphatase-1 to a manganese-lint dependent form is a quite separate phenomenon from the formation of the Mg-ATP-dependent protein phosphatase. Inhibitor-2 can inactivate protein phosphatase-1 by a second mechanism that is not reversed by preincubation with glycogen synthase ... [ABSTRACT FROM AUTHOR]

The signaling pathways that couple cerebral ischemia to cell death and protective mechanisms have not been elucidated. Protein phosphatase-l (PP-1) interacts with and dephosphorylates multiple protein regulators of cellular energy metabolism, ionic balance and apoptotic cell death. We report that a Mg2+/ATP-dependent form of PP-1 associated with inhibitor-2 (PP-1I) is activated in brain by ∼2-fold following cardiac arrest and resuscitation in an intact animal model of severe transient global cerebral ischemia. PP-1I from control non-ischemic and ischemic pig brain was purified to near homogeneity. The properties, subunit compositions and regulation of PP-1I purified from control nonischemic pig brain was distinct from PP-1I purified from ischemic pig brain. Six major proteins (C-TAK1, Rab GDP dissociation protein b, protein phosphatase-la catalytic subunit [PP-1acat], 14-3-3g and inhibitor-2 [1-2] were identified by SDS/PAGE followed by MALDI ToF mass spectrometry from the non-ischemic brain. PP-lacat, 1-2 and 14-3-3g existed as a macromolecular complex as confirmed by coimmunoprecipitation. Both PP-1acat and reconstituted PP-1acat: phosphol-2 were inhibited directly by 14-3-3g. PP-1I purified from ischemic brain contained less 14-3-3g than that purified from nonischemic brain indicating that activation of PP-1I following cardiac arrest and resuscitation involves dissociation of inhibitory 14-3-3g. This is the first demonstration of PP-1I activation in a clinically relevant model of global cerebral ischemia in vivo. [ABSTRACT FROM AUTHOR]