Regulation of protein phosphatase 1I by Cdc25C-associated kinase 1 (C-TAK1) and PFTAIRE protein kinase

Abstract

Protein phosphatase 1I (PP-1I) is a major endogenous form of protein phosphatase 1 (PP-1) that consists of the core catalytic subunit PP-1c and the regulatory subunit inhibitor 2 (I-2). Phosphorylation of the Thr-72 residue of I-2 is required for activation of PP-1I. We studied the effects of two protein kinases identified previously in purified brain PP-1I by mass spectrometry, Cdc25C-associated kinase 1 (C-TAK1) and PFTAIRE (PFTK1) kinase, for their ability to regulate PP-1I. Purified C-TAK1 phosphorylated I-2 in reconstituted PP-1I (PP-1c. I-2) on Ser-71, which resulted in partial inhibition of its ATP-dependent phosphatase activity and inhibited subsequent phosphorylation of Thr-72 by the exogenous activating kinase GSK-3. In contrast, purified PFTK1 phosphorylated I-2 at Ser-86, a site known to potentiate Thr-72 phosphorylation and activation of PP-1I phosphatase activity by GSK-3. These findings indicate that brain PP-1I associates with and is regulated by the associated protein kinases C-TAK1 and PFTK1. Multisite phosphorylation of the I-2 regulatory subunit of PP-1I leads to activation or inactivation of PP-1I through bidirectional modulation of Thr-72 phosphorylation, the critical activating residue of I-2.

Keywords: Cell Signaling; Enzyme; Enzyme Mechanism; Inhibitor 2; Ischemia; Protein Phosphorylation.

FIGURE 1.

ATP-dependent activation of native PP-1_I. A, native PP-1_I purified 1120-fold from pig brain (14) was incubated without (■) or with (●) ATP/Mg²⁺ for 5 min prior to initiation of the phosphatase assay by the addition of [³²P]phosphorylase a. Incubation with ATP/Mg²⁺ increased phosphatase activity ∼5-fold. B, chromatography of purified PP-1_I on Superdex 200 showing elution at M_r ∼160,000. PP-1_I was assayed using [³²P]phosphorylase a as a substrate following preincubation with ATP/Mg²⁺ and GSK-3β. C, SDS/PAGE analysis of brain PP-1_I showing copurification of six major proteins (n = 3).

FIGURE 2.

Reconstitution of core PP-1_I from purified recombinant PP-1c and I-2. A, chromatography of recombinant PP-1α_cat on Superdex 200 showing elution at M_r 40. PP-1 activity was assayed using [³²P]phosphorylase a as a substrate. B, SDS/PAGE analysis of PP-1c eluted from Superdex 200. C, chromatography of reconstituted PP-1_I on Superdex 200 showing elution at M_r 75. PP-1_I was assayed using [³²P]phosphorylase a as a substrate following preincubation with ATP/Mg²⁺ and GSK-3β. D, SDS/PAGE analysis of reconstituted PP-1_I eluted from Superdex 200 showing coelution of PP-1c and I-2 at M_r ∼70,000 (n = 3).

FIGURE 3.

Reconstituted PP-1_I is not activated by C-TAK1 or PFTK1. Recombinant I-2 was incubated with Mg²⁺/[γ-³²P]ATP (control (Ctl) and control IgG immunoprecipitate (Ctl IP)) or with Mg²⁺/[γ-³²P]ATP plus C-TAK1 (C-TAK1) or PFTK1 (PFTK1 IP), all including 100 nm tautomycin, for 30 min at 30 °C. Reactants were separated by SDS/PAGE, and the gels were stained, destained, dried, and subjected to autoradiography. Representative autoradiogram (A, top panel) and quantitative densitometric analysis (A, bottom panel) of I-2 phosphorylation (pI-2) (relative to background phosphorylation by control or IgG immunoprecipitate) are shown. Data are mean ± S.E. (n = 3). ***, p < 0.001 versus control (Student's t test). B, reconstituted PP-1_I (0.2 μg) assayed in the presence of Mg²⁺/ATP alone (Ctl) or Mg²⁺/ATP plus GSK-3β (0.01 μg), C-TAK1 (0.02 μg), or GSK-3β and C-TAK1. C, reconstituted PP-1_I (0.2 μg) assayed for phosphatase activity in the presence of Mg²⁺/ATP (control), Mg²⁺/ATP plus IgG IP (control), Mg²⁺/ATP plus GSK-3 β (0.01 μg), or PFTK1 immunoprecipitate. C-TAK1 or PFTK1 did not activate reconstituted PP-1_I. However, prior phosphorylation of I-2 by C-TAK1 reduced subsequent activation of PP-1_I by GSK-3 β (Β). Data are mean ± S.E. (n = 3). ***, p < 0.001 versus control (one-way analysis of variance).

FIGURE 4.

MS/MS spectra of the tryptic phosphopeptide from I-2 phosphorylated by C-TAK1. I-2 was phosphorylated with C-TAK1, subjected to SDS/PAGE, excised, and digested with trypsin. Liberated peptides were then analyzed by LC electrospray ionization MS/MS. MS/MS spectra of a phosphopeptide (m/z = 1313.5³⁺) were obtained and analyzed. b and y ions are marked in the spectra. The amino acid sequence of the phosphopeptide is shown at the top. Subfragment ion y₂₇²⁺ (p(STPY) HSMMGDDEDACSDTEATEAMAPDILAR, m/z = 1790.1) was identified in the phosphopeptide preparation, indicating that the site phosphorylated by C-TAK1 is within STPY. The phosphorylated residue was confirmed as Ser-71 by phospho-amino acid analysis. C*, acrylamide-modified cysteine.

FIGURE 5.

Phosphorylation of I-2 by C-TAK1 inhibits subsequent phosphorylation by GSK-3β. I-2 (2 μg) was incubated with Mg²⁺/[γ-³²P]ATP (control (Ctl), top left panel), Mg²⁺/[γ-³²P]ATP plus GSK-3β (0.02 μg) (top center panel), or Mg²⁺/ATP plus C-TAK1 (0.02 μg) for 30 min and then with Mg²⁺/[γ-³²P]ATP plus GSK-3β (0.02 μg) (top right panel). Reactants were separated by SDS/PAGE, and the gels were stained with Coomassie Blue, destained, dried, and subjected to autoradiography (top panels) and quantitative analysis (bottom panel). Prior phosphorylation of I-2 by C-TAK1 reduced subsequent phosphorylation by GSK-3β. Data are mean ± S.E. (n = 3). ***, p < 0.001 versus control (one-way analysis of variance).

FIGURE 6.

PFTK1 phosphorylates I-2 at Ser-86. A, stoichiometry of the phosphorylation of I-2 and Rb (positive control) by PFTK1. PFTK1 phosphorylated I-2 to one-fourth the stoichiometry of Rb phosphorylation. Data are mean ± S.E. (n = 3). B, recombinant I-2 WT and five Ser/Thr → Ala mutants were incubated with Mg²⁺/[γ-³²P]ATP and PFTK1 for 1 h at 30 °C. The I-2 reactants were separated by SDS/PAGE, and the gels were stained with Sypro protein stain and subjected to autoradiography. Phosphorylation was calculated as a percentage of WT band density from autoradiograms and normalized to Sypro protein staining. Densitometric analysis of I-2 WT and mutant phosphorylation by PFTK1 are shown. Data are mean ± S.E. (n = 2). C, representative autoradiograms (top panel) and quantitative densitometric analysis (bottom panel) from in vitro phosphorylation assays of I-2 WT and S86A mutant I-2 phosphorylated by PFTK1, CK1, or CK2. IP, immunoprecipitate. D, phosphorylation of I-2 WT by PFTK1, CK1, or CK2 in the presence of the CK1 inhibitor D4476 (D) or the CK2 inhibitor TBB (T). N, no inhibitor. Data are mean ± S.E. (n = 4). ***, p < 0.001 versus WT or without inhibitor (Student's t test).

FIGURE 7.

Schematic models of PP-1_I activation relative to I-2 phosphorylation. I-2 inhibition (inactive) of PP-1c is relieved when Thr-72 of I-2 is phosphorylated (active). Prior phosphorylation of Ser-71 reduces phosphorylation of Thr-72 (low) and, therefore, reduces PP-1_I activity. In contrast, prior phosphorylation of Ser-86 results in increased phosphorylation of Thr-72 (high) and, therefore, increases PP-1_I activity. Under ischemic conditions, with increased PFTK1 activity and reduced CTAK1 activity, both of these phosphorylation events would lead to enhanced phosphorylation of I-2 Thr-72 (ischemia) and PP-1_I activity (very high). Stoichiometry of phosphorylation is proportional to symbol size.