Activation of brain protein phosphatase-1(I) following cardiac arrest and resuscitation involving an interaction with 14-3-3 gamma

Abstract

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-1(I), 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-1(I) 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 14-3-3gamma, Rab GDP dissociation protein beta, PFTAIRE kinase, and C-TAK1 kinase. PP-1(I) purified from ischemic brain contained significantly less 14-3-3gamma than PP-1(I) purified from control brain, and purified 14-3-3gamma directly inhibited the catalytic subunit of PP-1 and reconstituted PP-1(I). These findings suggest that activation of brain PP-1(I) following global cerebral ischemia in vivo involves dissociation of 14-3-3gamma, a novel inhibitory modulator of PP-1(I). This identifies modulation of PP-1(I) by 14-3-3 in global cerebral ischemia as a potential signaling mechanism-based approach to neuroprotection.

Fig. 1

Identification of protein phosphatase (PP)-1_I activity in pig brain. (a) Phosphorylase phosphatase activity was determined in soluble pig brain extracts in the absence or presence of Mg²⁺/ATP and phospho-T34-DARPP-32, as indicated. PP-1 activity is defined as phospho-T34-DARPP-32-sensitive phosphorylase phosphatase activity; the residual activity is primarily because of PP-2A. (b) The activity of PP-1_I is defined as Mg²⁺/ATP-dependent PP-1 activity, or the difference between PP-1 activity measured in the absence (basal PP-1) or presence (basal PP-1 + PP-1_I) of Mg²⁺/ATP. Mean ± SD (n = 3).

Fig. 2

Activation of brain protein phosphatase (PP)-1_I by global cerebral ischemia. Total activity of PP-1_I was determined in soluble brain extracts from control pigs and pigs subjected to 10 min of cardiac arrest followed by resuscitation and reperfusion for 2 h. PP-1_I activity was quantified as described in Fig. 1. Mean ± SD (n = 3). *p < 0.05 versus control (Student t-test).

Fig. 3

Quantification of protein phosphatase (PP)-1_I following partial purification of brain extracts from control and ischemic brain. The activity of PP-1_I is defined as the activity in the presence of Mg²⁺/ATP minus activity in the absence of Mg²⁺/ATP. (a) Soluble brain extracts from a control pig and from a pig subjected to 10 min of cardiac arrest followed by resuscitation and reperfusion for 2 h were prepared as described in Experimental procedures and loaded onto a DEAE Sepharose column. PP-1_I activity from control (■) and ischemic (●) brain was determined. Data are shown for a single representative experiment (n = 3). (b) Active fractions from the DEAE Sepharose column were pooled as indicated by the bar in (a) and loaded onto a poly-L-lysine agarose column. PP-1_I activity from control (■) and ischemic (●) brain was determined. (c) Total PP-1_I activity from the poly-L-lysine agarose chromatography step from control or ischemic brain was quantified by determining the area under the peak of activity. Mean ± SD (n = 3). *p < 0.05 versus control (Student t-test).

Fig. 4

Identification of protein components of purified protein phosphatase (PP)-1_I by mass spectrometry. (a) Purified PP-1_I from control (C) and ischemic/reperfused (IR) pig brain was subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The bars on the left indicate the migration of marker proteins. The bars on the right indicate the migration of the major proteins present in purified PP-1_I. (b) Protein bands were excised from the gel and digested with trypsin, and tryptic peptides were analyzed by matrix-assisted laser desorption time of flight mass spectrometry (MALDI ToF MS). The molecular masses and amino acid sequences of the tryptic peptides identified for each major protein are shown. (a and b) migrated as a doublet; analysis of both bands yielded peptides derived from C-TAK1.

Fig. 5

Analysis of purified protein phosphatase (PP)-1_I by gel filtration. Purified PP-1_I from control (left) and ischemic (right) brain were analyzed on a Superdex 200 column (1.0 × 60 cm) at a flow rate of 0.75 mL/min with collection of 1.25 mL fractions. The arrows indicate the void volume (Vo) and the elution positions of the marker proteins.

Fig. 6

Identification of a protein phosphatase (PP)-1_I : inhibitor-2 (I-2) : 14-3-3γ complex in control brain. (a) Purified control brain PP-1_I was immunoblotted with anti-PP-1α_cat (lane 1), anti-14-3-3γ (lane 2), or anti-I-2 (lane 3). (b) Purified control brain PP-1_I was immunoprecipitated with anti-PP-1α_cat followed by immunoblotting with anti-14-3-3γ (left) or anti-I-2 (right). (c) Purified control brain PP-1_I was immunoprecipitated (IP) with anti-14-3-3γ followed by immunoblotting (IB) with anti-PP-1α_cat (left) or anti-I-2 (right).

Fig. 7

Inhibition of protein phosphatase (PP)-1 catalytic subunit α (PP-1α_cat) and reconstituted PP-1α_cat : inhibitor-2 (I-2) by 14-3-3γ. Purified PP-1α_cat (■) and reconstituted PP-1α_cat : I-2 (●) were pre-incubated with various amounts of 14-3-3γ for 10 min and then assayed using [³²P]phospho-Bad as substrate.

Fig. 8

Quantification of 14-3-3γ in purified protein phosphatase (PP)-1_I from control and ischemic brain. (a) PP-1_I was purified to near homogeneity from control (C) and ischemic/reperfused (IR) pig brain as described in Experimental procedures, analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and stained with Sypro Ruby which identified six major bands (a–f). (b) Densitometric scans of gels obtained from PP-1_I purified from control (top) or ischemic (bottom) pig brain. The migration of 14-3-3γ is indicated by an asterisk. (c) Quantification of proteins present in PP-1_I purified from control (C) and ischemic brain (IR) pig brain. Relative protein amounts were quantified as peak area obtained by densitometric scans of stained gels. Mean ± SD (n = 3). *p < 0.05 versus control (Student t-test). (d) Immunoblot analysis of 14-3-3γ in purified PP-1_I from control (C) and ischemic (IR) brain. (e) Densitometric scans of immunoblots of purified PP-1_I from control (top) or ischemic (bottom) pig brain. (f) Quantification of immunoreactive 14-3-3γ in purified PP-1_I from control (C) or ischemic (IR) pig brain as peak area. Mean ± SD (n = 3). *p < 0.05 versus control (Student t-test).