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. 2011 Jun;31(11):2349-63.
doi: 10.1128/MCB.01205-10. Epub 2011 Mar 28.

Acetylation of a conserved lysine residue in the ATP binding pocket of p38 augments its kinase activity during hypertrophy of cardiomyocytes

Vinodkumar B Pillai  1 , Nagalingam R Sundaresan, Sadhana A Samant, Don Wolfgeher, Chinmay M Trivedi, Mahesh P Gupta
Affiliations

Acetylation of a conserved lysine residue in the ATP binding pocket of p38 augments its kinase activity during hypertrophy of cardiomyocytes

Vinodkumar B Pillai et al. Mol Cell Biol. 2011 Jun.

Abstract

Like phosphorylation, acetylation of lysine residues within a protein is considered a biologically relevant modification that controls the activity of target proteins. During stress of cells, massive protein acetylation takes place. Here, we show that p38 mitogen-activated protein kinase (MAPK), which controls many biological functions during stress, is reversibly acetylated by PCAF/p300 and HDAC3. We identified two acetylated lysine residues, K152 and K53, located in the substrate binding domain and in the ATP-binding pocket of p38, respectively. Acetylation of lysine 53 enhanced the activity of p38 by increasing its affinity for ATP binding. The enhanced acetylation and activation of p38 were found to be in parallel with reduced intracellular ATP levels in cardiomyocytes under stress, as well as in vivo models of cardiac hypertrophy. Thus, our data show, for the first time, that p38 activity is critically regulated by, in addition to phosphorylation, reversible acetylation of a lysine residue, which is conserved in other kinases, implying the possibility of a similar mechanism regulating their activity.

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Figures

Fig. 1.
Fig. 1.
Cardiomyocyte stress induces p38 acetylation. (A) Expression levels of phospho (Pho)-p38, Pho-JNK, Pho-ERK, and Pho-AKT after mechanical stretching (MS) of cardiomyocytes. (B, C) Cardiomyocytes were subjected to 10% MS for 4 h or treated with 1 μM angiotensin-II for 24 h. Cell lysate was subjected to immunoprecipitation (IP) with an anti-p38 antibody. The resulting immune complex was analyzed by IB with antibodies against acetyl lysines (Ac-K) and p38. Arrow points to acetylated p38 band. The sizes of the molecular weight markers are indicated on the right. (D) HeLa cells were treated overnight with 500 ng of TSA. Cell lysate was subjected to IP with p38 antibody, and the complex was analyzed by IB using anti-Ac-K and anti-p38 antibodies. (E, F) Quantification of ATP levels in MS and Ang-II treated cardiomyocytes. (G) ATP levels in HeLa cells treated with TSA. C, control. Results shown are mean ± standard error (SE). n = 4.
Fig. 2.
Fig. 2.
p38 is acetylated by PCAF/p300. (A) Histidine-tagged p38 on beads was subjected to in vitro acetylation using GST-PCAF or GST-p300. Beads were separated, washed, and subjected to SDS-PAGE and IB with indicated antibodies. The sizes of the molecular weight markers are indicated on the left. (B to D) HeLa cells were overexpressed with indicated plasmids. Cell lysate was subjected to IP with indicated antibodies, and the resulting beads were analyzed by IB using antibodies shown on the right. (E) HeLa cells were subjected to PCAF knockdown by use of scrambled or PCAF-specific siRNA. Cell lysates were subjected to IP with anti-p38 or anti-HA antibodies. The resulting beads were analyzed by IB with the use of indicated antibodies. (F) IB analysis of whole-cell lysate (WCL) showing PCAF knockdown in scrambled or PCAF-specific siRNA-treated cells. (G) HeLa cell lysate was subjected to IP with anti-p38 or anti-HA antibodies, and the resulting beads were analyzed by IB with the use of indicated antibodies.
Fig. 3.
Fig. 3.
HDAC3 binds to and deacetylates p38. (A) HeLa cells were cotransfected with indicated plasmids. Cell lysate was subjected to IP with HA antibody, and the resulting complex was analyzed by IB with anti-Flag antibody. Blot was stripped and reprobed with anti-HA antibody. The sizes of the molecular weight markers are indicated on the left. (B) Cells were cotransfected with indicated plasmids, and the cell lysate was subjected to IP with the indicated antibody. The resulting beads were analyzed by IB with the antibody given on the right. (C) HeLa cell lysate was subjected to IP with anti-p38 or anti-HA antibodies, and the resulting beads were analyzed by IB with the use of anti-HDAC3 and anti-p38 antibodies. (D) Cells were cotransfected with indicated plasmids in different combinations. Cell lysate was subjected to IP with HA antibody, and the resulting complex was analyzed by IB with anti-Ac-K antibody. Arrowhead points to acetylated p38 band. Shown at bottom is IB of the WCL depicting input of HDAC1, -3, and -5 in lanes 2, 3, and 4 from the left, respectively. (E) Histidine-tagged p38 on beads was subjected to in vitro acetylation using GST-PCAF. Beads were washed and incubated with different HDACs in a deacetylation reaction buffer. Deacetylation of p38 was examined by IB with use of indicated antibodies.
Fig. 4.
Fig. 4.
Acetylation enhances kinase activity of p38 by promoting its affinity for ATP. (A) Phospho-p38 (Pho-p38) was acetylated in vitro with PCAF. Acetylated Pho-p38 and nonacetylated Pho-p38 were incubated with ATF2 in a kinase buffer containing 200 μM ATP. At indicated time points, reaction was stopped and samples were analyzed by IB with use of anti-Pho-ATF2 antibody. Blot was stripped and probed with anti-Pho-p38 antibody. Same blot was stained with Coomassie blue to verify equal loading of ATF2. (B) ATP-binding ability of acetylated, nonacetylated, or inhibitor-bound (SB; SB203580) Pho-p38 was determined by incubation with different amounts of [32P]ATP as indicated. Values are mean ± SE of 3 experiments. (C) In a kinase buffer, ATF2 (1 μg) was incubated with acetylated or nonacetylated Pho-p38 at various concentrations of ATP, as indicated. Samples were analyzed by IB with use of anti-Pho-ATF2 antibody. Blot was stripped and probed with anti-Pho-p38 antibody and later stained with Coomassie blue to verify equal loading of ATF2. (D) Experiment similar to that described in the legend to panel C was done with more points analyzed between 50 and 200 μM ATP. (E) IB showing acetylation of Pho-p38, utilized in assays described in the legend to panels A, C, and D. (F) HeLa cells were overexpressed with HA-p38 with or without PCAF. HA-p38 was subjected to IP and incubated with GST-ATF2 (1 μg) in kinase buffer with different amounts of ATP as indicated. Samples were analyzed by IB with use of anti-Pho-ATF2 antibody. Blot was stripped and probed with anti-GST, anti-Pho-p38 antibodies, and, later, anti-p38 antibody. (G) Quantification of activity of acetylated and nonacetylated p38. Mean ± SE; n = 3. (H) HeLa cells were cotransfected with indicated vectors. Cell lysate was subjected to IP p38, and the resulting beads were assayed for activity to phosphorylate the substrate ATF2. ATF2 phosphorylation was detected by IB with indicated antibodies.
Fig. 5.
Fig. 5.
Acetylation does not change the ability of p38 to bind to ATF2. (A) Annotation of a representative tandem mass spectrum of trypsin-digested p38 depicting acetylation of K152. (B) HeLa cells were overexpressed with HA-p38 (WT) or HA-p38K152R (K152R). Cell lysate was subjected to IP p38. The resulting p38 on beads was assayed for its ability to phosphorylate the substrate ATF2. Phosphorylation of ATF2 was detected by IB. The same blot was stripped and reprobed with indicated antibodies. (C) ATF2 (GST-ATF2) in increasing concentrations (0.01 to 2 μg) was incubated with acetylated or nonacetylated Pho-p38 in a kinase buffer. The samples were analyzed by IB with use of anti-Pho-ATF2 antibody. Blot was stripped and reprobed with anti-GST and later with anti-Pho-p38 antibodies to verify equal loading of proteins. (D) Histidine-tagged p38 on beads was acetylated in vitro with PCAF. Beads were washed and incubated with decreasing concentrations of GST-ATF2 (2, 1, and 0.5 μg) or GST (2 μg) in a kinase buffer for 30 min. Beads were washed, and samples were analyzed by IB with indicated antibodies.
Fig. 6.
Fig. 6.
Acetylation of p38 at lysine 53 augments its ATP-binding ability. (A) GST proteins (GST, GST-p38, GST-p38K53R, GST-p38K53R,54R, and GST-p38K54R) were subjected to phosphorylation in vitro by MKK6 and then tested for their ability to phosphorylate ATF2. The reaction mix was analyzed by IB with use of anti-Pho-p38 and anti-Pho-ATF2 antibodies. For loading control, blot was stained with Coomassie blue. (B) ATP-binding ability of acetylated, nonacetylated, or inhibitor-bound p38 was measured directly by incubating GST proteins with 15 μCi [32P]ATP. Values are mean ± SE of 3 experiments. (C) HeLa cells were overexpressed with wild-type p38 or p38K152R mutant. p38 was IP, and the resulting beads were assayed for ATP-binding ability by incubating them with 15 μCi [32P]ATP. Beads treated with SB served as a control. Values are mean ± SE of 3 experiments. (D) His-p38 and His-p38K53R were subjected to acetylation with PCAF. His beads were separated and analyzed by IB. Top portion of the blot was probed with anti-PCAF, and bottom portion with anti-Ac-K and later for anti-p38 antibodies. (E) HeLa cells were overexpressed with indicated plasmids in different combinations. Cell lysate was subjected to IP p38 with HA antibody, and the resulting complex was analyzed by IB with anti-Ac-K and anti-HA antibodies. WCL was also analyzed by IB to determine PCAF expression. (F) Nonphosphorylated and phosphorylated p38 was subjected to acetylation by PCAF in vitro. The resulting acetylated p38 was then tested for its ability to phosphorylate the substrate ATF2. The samples were analyzed by IB with use of indicated antibodies.
Fig. 7.
Fig. 7.
Acetylation of JNK1 enhances its kinase activity. (A) Table showing conserved lysines (K underlined) in the ATP-binding pocket of p38, JNK1, and JNK2. (B) HeLa cells were transfected with plasmids synthesizing Flag-JNK1 and/or PCAF in different combinations, as indicated. Cell lysate was subjected to IP with Flag antibody, and the resulting complex was analyzed by IB with use of anti-Ac-K antibody. Arrow points to acetylated JNK1 band. The blot was stripped and probed with anti-Flag antibody. (C) Pho-JNK1 was subjected to in vitro acetylation with PCAF. In a kinase buffer, acetylated and nonacetylated Pho-JNK1 was incubated with GST-ATF2 with various amounts of ATP as indicated. The reaction was stopped by addition of Laemmli buffer, and samples were analyzed by IB with indicated antibodies.
Fig. 8.
Fig. 8.
p38 acetylation promotes stress-induced apoptosis. (A) HeLa cells were treated overnight with 500 nM TSA. Next day, cells were treated with anisomycin (Ani) (500 ng/ml) for 4 h in the presence or absence of TSA. p38 and JNK inhibitors, 5 μM SB203580 (SB), and 10 μM SP600125 (SP), respectively, were added either separately or together 1 h prior to treatment of cells with Ani. Extent of apoptosis was measured by estimating the percentage of annexin V-positive cells by FACS analysis. (B) Bar graph shows results (mean ± SE) of 3 independent experiments. (C) Cells subjected to p38 knockout (KO) were treated with TSA and Ani, as described in the legend to panel A. (D) Quantification of death of cells described in legend to panel C (mean ± SE; n = 3). (E) IB showing knockdown of p38 in HeLa cells. (F) IB showing acetylation of p38 in Ani-treated HeLa cells. Shown in bottom panel is loss of acetylation signal when Ac-K antibody was first incubated with acetyl-BSA, thus validating specificity of the anti-Ac-K antibody used. (G) Quantification of ATP in HeLa cells treated with Ani for the indicated time points. *, statistically significant (P < 0.05).
Fig. 9.
Fig. 9.
HDAC3 deficiency promotes p38-mediated apoptosis under stress. (A) HDAC3 was knocked down from 293T cells by the use of a plasmid expressing shRNA against HDAC3. Cells were treated with Ani (500 ng/ml) for 4 h. p38 and JNK inhibitors, SB (5 μM) and SP (10 μM) were added either separately or together 1 h prior to treatment of cells with Ani. Extent of apoptosis was measured by estimating the percentage of annexin V-positive cells by FACS analysis. (B) Bar graph shows results (mean ± SE) of 3 independent experiments. (C) IB showing knockdown of HDAC3 in 293T cells. (D) Cell lysate of 293T cells subjected to HDAC3 knockdown was subjected to IP p38. The resulting beads were analyzed by IB with the use of indicated antibodies. (E) HeLa cells were overexpressed with p38 or mutant p38 (mt-p38; p38K53R) alone or together with PCAF or HDAC3. Cells were subsequently treated with Ani (500 ng/ml) for 2 h, and cell death was measured. (F) Bar graph shows results (mean ± SE) of 3 independent experiments. (G) Expression levels of p38, PCAF, and HDAC3 in HeLa cells used for experiments described in the legend to panel E. *, statistically significant (P < 0.05).
Fig. 10.
Fig. 10.
p38 is acetylated and activated during cardiac hypertrophy. (A) HW/BW ratio of control (C; vehicle treated) and isoproterenol (ISO)-treated mice. (B) Quantification of ATP levels in the heart lysates of control and ISO-treated mice. Mean ± SE, n = 5. (C) Endogenous p38 was subjected to IP from the heart lysate, and it was analyzed for acetylation and phosphorylation by IB with use of anti-Ac-K, anti-Pho-p38, and anti-p38 antibodies. (D) IB showing ATF2 phosphorylation in hearts of control and ISO-treated mice. Results of two mice in each group are shown. (E) p38 was subjected to IP from heart lysate of control and ISO-treated mice and was assayed for its activity to phosphorylate the substrate ATF2 at 50 and 100 μM ATP. Phosphorylation of ATF2 was detected by IB with use of anti-Pho-ATF2 antibody. (F) Quantification of p38 activity. Mean ± SE; n = 3. (G) p38 was subjected to IP from heart lysate of control (C) and cardiac-specific HDAC3-expressing transgenic (T) mice and was assayed for its activity to phosphorylate ATF2 at 50 and 100 μM ATP. Phosphorylation of ATF2 was detected by IB with use of indicated antibodies. (H) IB showing reduced acetylation of p38 in the heart lysate of HDAC3 transgenic mice, compared to controls. *, statistically significant (P < 0.05).
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