Protein phosphatase 2Cm is a critical regulator of branched-chain amino acid catabolism in mice and cultured cells

Abstract

The branched-chain amino acids (BCAA) are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. In individuals with deficiencies in BCAA, these amino acids can be preserved through inhibition of the branched-chain-alpha-ketoacid dehydrogenase (BCKD) complex, the rate-limiting step in their metabolism. BCKD is inhibited by phosphorylation of its E1alpha subunit at Ser293, which is catalyzed by BCKD kinase. During BCAA excess, phosphorylated Ser293 (pSer293) becomes dephosphorylated through the concerted inhibition of BCKD kinase and the activity of an unknown intramitochondrial phosphatase. Using unbiased, proteomic approaches, we have found that a mitochondrial-targeted phosphatase, PP2Cm, specifically binds the BCKD complex and induces dephosphorylation of Ser293 in the presence of BCKD substrates. Loss of PP2Cm completely abolished substrate-induced E1alpha dephosphorylation both in vitro and in vivo. PP2Cm-deficient mice exhibited BCAA catabolic defects and a metabolic phenotype similar to the intermittent or intermediate types of human maple syrup urine disease (MSUD), a hereditary disorder caused by defects in BCKD activity. These results indicate that PP2Cm is the endogenous BCKD phosphatase required for nutrient-mediated regulation of BCKD activity and suggest that defects in PP2Cm may be responsible for a subset of human MSUD.

Figure 1. PP2Cm interacts with the BCKD complex and dephosphorylates E1α at Ser293.

(A) The PP2Cm immunocomplex was isolated using anti-FLAG antibody from a HEK293T cell line stably expressing PP2Cm-FLAG and separated on SDS-PAGE. Based on mass spectrometry, the molecular identity of specific protein species, as shown in a silver staining gel, was revealed as labeled. Lanes were run on the same gel but were nonadjacent. BCKDHA-E1α, branched-chain ketoacid dehydrogenase E1, α polypeptide; BCKDHB-E1β, branched-chain ketoacid dehydrogenase E1, β polypeptide; Dbt-E2, dihydrolipoamide branched-chain transacylase E2; Mr, molecular weight; CTRL, control sample from parental HEK293T cells without PP2Cm-FLAG expression following the same immunoprecipitation procedure. (B) Immunoblotting on the same SDS-PAGE gel, using specific antibodies for E2, E1α, and anti-(E1+E2) antibodies, which recognize E2, E1α, and E1β subunits to differing extents. (C) Immunoblotting on the same SDS-PAGE gel for pSer293 (pS293) E1α, total E1α, and PP2Cm is shown on separate rows as indicated (25). MEFs infected with adenoviral vector expressing PP2Cm (Adv-PP2Cm), at different MOI as indicated, were left untreated or treated with 5 mM BCKA mixture.

Figure 2. Generation of PP2Cm^–/– mice.

(A) Schematic illustration of gene-targeting strategy for the mouse PP2Cm gene. The NLS-LacZ (nuclear-localized lacZ) fragment was used to replace the coding sequence of PP2Cm in exon 2. (B) Real-time RT-PCR analysis of mRNA levels of BCKD kinase and PP2Cm in the heart, using primers against different PP2Cm exons as indicated. The relative mRNA levels were normalized against GAPDH and shown as relative levels to the signals from PP2Cm^+/+ exon 1. The results were generated from 3 independent experiments and are shown as mean ± SEM. (C) Immunoblot analysis of total heart lysate from PP2Cm^+/+ and PP2Cm^–/– mice using anti-PP2Cm–specific polyclonal antibody. H, HindIII; NSB, nonspecific band.

Figure 3. PP2Cm is the essential BCKD phosphatase in vitro.

(A) PP2Cm^+/+ and PP2Cm^–/– MEFs were first incubated in Krebs-Ringer-HEPES media at 37°C for 2 hours (basal conditions [Basal] and amino acid starvation [–AA]) and then treated with DMEM culture medium containing 5 mM each of the BCKA for an additional 30 minutes (treatment of BCKA [+KA]) as illustrated. Total cell lysates collected at different time points were subjected to immunoblotting analysis using anti–pSer293 E1α antibodies, BCKD kinase, and β-actin as indicated. PP2Cm protein was detected by immunoprecipitation and subsequent immunoblot using PP2Cm antibody as described (23). (B) PP2Cm^+/+ and PP2Cm^–/– MEFs were infected with MOI 50 of Adv-PP2Cm (WT) or Adv–PP2Cm-R236G (R236G) for 48 hours. Cells were challenged by media containing 5 mM each of the BCKA for 30 minutes, and total cell lysates were analyzed by immunoblotting with the antibodies as indicated. (C) PP2Cm^+/+ or PP2Cm^–/– MEFs were stimulated with media containing 5 mM each of the BCKA in culture medium for up to 8 hours. The pSer293 and total E1α were measured by immunoblotting as indicated. The last 2 lanes were loaded with additional PP2Cm^+/+ and PP2Cm^–/– samples from the 60-minute time point as internal controls.

Figure 4. PP2Cm is the essential BCKD phosphatase in vivo.

(A) Immunoblotting analysis of total tissue lysates from the liver (top panel) and heart (bottom panel) of PP2Cm^+/+ and PP2Cm^–/– mice, for both total and pSer293 E1α as indicated. (B) Representative immunoblots of liver lysate for pSer293 E1α, total E1α, and β-actin from PP2Cm^+/+ and PP2Cm^–/– mice 30 minutes after oral administration with either saline vehicle (Veh) or leucine (Leu) as described in Methods. (C–E) The relative levels of pSer293 E1α (C) and total E1α (D) and their ratio (E) from PP2Cm^+/+ and PP2Cm^–/– livers were quantified. The quantification of the pSer293 to total E1α ratio was normalized against vehicle-treated WT mice. All bar graphs represent mean ± SEM. *P < 0.01, saline-treated PP2Cm^–/– versus PP2Cm^+/+mice; ^#P < 0.01, leucine-treated versus saline-treated PP2Cm^+/+ mice.

Figure 5. Specific association of PP2Cm with the BCKD complex.

(A) Glutathione-S-transferase (GST) immunoprecipitation followed by anti-His or anti-GST immunoblotting, after incubation of GST-PP2Cm with His-tagged BCKD subunits as indicated. Five percent of the total input for the immunoprecipitation assay was also subjected to immunoblotting as shown. (B) COS7 cells were infected with adenoviruses expressing FLAG-tagged PP2Cm WT or mutants (H129A, R236G, D298A, and mitochondria-targeting defective [MD]) as indicated (MOI 50). Twenty-four hours later, total cell lysates were immunoprecipitated with M2 beads, followed by immunoblotting using anti-E1/E2 and anti-PP2Cm antibodies as indicated. The full-length PP2Cm (premature) and cleaved PP2Cm (mature) proteins were detected. (C) PP2Cm-FLAG was coexpressed with either HA-tagged WT E1α (E1α-HA) or mutants (Ser293A, Ser303A, and E1α-Ser293A/Ser303A) in HEK293T cells. Immunoblotting for E1α and PP2Cm in total cell lysates (input) or anti-FLAG immunoprecipitates are shown as indicated. (D–F) PP2Cm-FLAG–expressing 293T cells were treated with 5 mM BCKA for 1 hour (KA1H) or 3 days (KA3D). Total cell lysates were analyzed by immunoblot directly with the antibodies as labeled (D) or following immunoprecipitation with anti-E1α (E) or anti-FLAG (F) antibodies.

Figure 6. BCAA catabolic defects in PP2Cm-deficient mice.

(A) Plasma concentrations of BCAA in randomly fed PP2Cm^+/+ (n = 8) and PP2Cm^–/– (n = 9) mice. Results are presented as mean ± SEM. **P < 0.05, Student’s t test. (B and C) WT (n = 4) and PP2Cm^–/– (n = 4) mice were fasted for 6 hours and then i.p. administered a 150-mM leucine solution. Blood samples were collected and BCAA were measured at different time points as indicated. The changes in total (B) and baseline-corrected (C) plasma levels of BCAA upon leucine challenge are presented as mean ± SEM. *P < 0.05, Student’s t test. (D–G) Plasma levels of BCAA (D), KIC (E), α-keto-β-methylvalerate (KMV) (F), and α-ketoisovalerate (KIV) (G) in WT (black bars) and PP2Cm^–/– (white bars) animals 30 minutes after oral administration of 1.027 M leucine slurry (n = 5–6) or saline (n = 3) following overnight fast. Results are presented as mean ± SEM. *P < 0.05, ***P < 0.001, Student’s t test.

Figure 7. Oxidative stress in PP2Cm-deficient cells and tissues.

(A) Representative dichlorodihydrofluorescein diacetate (DCF-DA) fluorescence images of PP2Cm^+/+ and PP2Cm^–/– MEFs treated with or without 5 mM BCKA at 37°C for 30 minutes. Fluorescent signals were recorded digitally under the same setting, and the relative fluorescence intensity compared with the control is shown as labeled. Scale bars: 100 μm. (B) Electron spin resonance measurement of superoxide level in WT and PP2Cm^–/– MEFs under basal conditions, infected with (gray bars) or without (white bars) Adv-PP2Cm (MOI = 100). Results are presented as mean ± SEM. *P < 0.05, Student’s t test. (C) Electron spin resonance measurement of superoxide production in liver from randomly fed animals. Results are presented as mean ± SEM. *P < 0.05, Student’s t test. (D) Survival curve of offspring from the breeding of male PP2Cm^–/– mice and female PP2Cm^+/– mice fed a high-protein diet.