Extracellular signal-regulated kinase (ERK) phosphorylates histone deacetylase 6 (HDAC6) at serine 1035 to stimulate cell migration

Abstract

Histone deacetylase 6 (HDAC6) is well known for its ability to promote cell migration through deacetylation of its cytoplasmic substrates such as α-tubulin. However, how HDAC6 itself is regulated to control cell motility remains elusive. Previous studies have shown that one third of extracellular signal-regulated kinase (ERK) is associated with the microtubule cytoskeleton in cells. Yet, no connection between HDAC6 and ERK has been discovered. Here, for the first time, we reveal that ERK binds to and phosphorylates HDAC6 to promote cell migration via deacetylation of α-tubulin. We have identified two novel ERK-mediated phosphorylation sites: threonine 1031 and serine 1035 in HDAC6. Both sites were phosphorylated by ERK1 in vitro, whereas Ser-1035 was phosphorylated in response to the activation of EGFR-Ras-Raf-MEK-ERK signaling pathway in vivo. HDAC6-null mouse embryonic fibroblasts rescued by the nonphosphorylation mimicking mutant displayed significantly reduced cell migration compared with those rescued by the wild type. Consistently, the nonphosphorylation mimicking mutant exerted lower tubulin deacetylase activity in vivo compared with the wild type. These data indicate that ERK/HDAC6-mediated cell motility is through deacetylation of α-tubulin. Overall, our results suggest that HDAC6-mediated cell migration could be governed by EGFR-Ras-Raf-MEK-ERK signaling.

Keywords: Cell Migration; ERK; Histone Deacetylase; MAP Kinases (MAPKs); Protein Phosphorylation.

FIGURE 1.

Thr-1031 and Ser-1035 are phosphorylated in HDAC6. A and B, the peptide was detected at 32.6 min in the total ion chromatogram (TIC) (A) with mass-to-charge ratio 1068.5189, triply charged, which represents an error of 6.8 ppm (B). C, the tandem mass spectrum (MS/MS) matched the following sequence, HQTPPTSPVQGTTPQISPSTLIGSLRTLE, indicating that Thr-1031 and Ser-1035 (highlighted in red) were phosphorylated; the detection of b₃, y₂₂, and y₂₃ is consistent with this localization. The assignment was made with Mascot with a score of 50 and Sequest XCorr 5.62. D, ion chromatograms for peptides containing both phosphorylated Ser-1035 and unmodified Ser-1035 were extracted using Xcalibur 2.0 (Thermo Scientific). The peak areas under the curves were used to calculate the percentage of phosphorylation. E, upper panel, the consensus ERK1/ERK2 recognition motif is shown. Lower panel, a stretch of HDAC6 amino acid sequence showing the conservation of mass spectrometry analyses identified two ERK1/ERK2 sites (Thr-1031 and Ser-1035) and a putative ERK1/ERK2 site (Ser-1045) among rat, mouse, dog, and human.

FIGURE 2.

HDAC6 is phosphorylated by ERK1 in vitro at Thr-1031 and Ser-1035. A, diagrams show full-length and C terminus of HDAC6 with putative phosphorylation sites. B, upper panel, in vitro kinase assays were performed with the indicated substrates and recombinant ERK1 as described under “Experimental Procedures.” The reactions were separated on 6% SDS-PAGE and analyzed by autoradiography. Lower panel, Coomassie Blue staining of the substrates shows the protein amount and purity for kinase assays.

FIGURE 3.

Characterization of anti-pSer-1035 (HDAC6) antibody. A, anti-FLAG-M2 bead-purified FLAG-HDAC6 protein from 293T cells was treated without (lane 1) or with (lane 2) calf-intestinal alkaline phosphatase (CIP) (30 units) (Takara) at 37 °C for 1.5 h. Western blot analysis (IB) was performed with anti-pSer-1035(HDAC6) antibodies (upper panel). The blot was stripped and reprobed with anti-FLAG antibodies (lower panel). B, ectopically expressed 5-μg empty vector, FLAG-HDAC6 wild type, or S1035A was immunoprecipitated (IP) from 293T cells with anti-FLAG-M2 beads. Western blot analysis was carried out using rabbit anti-pSer-1035(HDAC6) antibodies (upper panel). The blot was then stripped and reprobed with anti-FLAG antibodies (lower panel).

FIGURE 4.

HDAC6(Ser-1035) phosphorylation is targeted by ERK pathway in vivo. A, HDAC6 phosphorylation is decreased by MEK inhibitor treatment. MEFs were treated with MEK inhibitor U0126 or PD98059 with the indicated concentration for 1 h. Endogenous HDAC6 was immunoprecipitated (IP) by anti-HDAC6 antibodies, and the phosphorylation status of HDAC6 was measured by immunoblotting (IB) using anti-phosphoserine/threonine antibodies. The blot was then stripped and reprobed with anti-HDAC6 antibodies. Anti-pERK and anti-ERK Western blotting analyses were also performed as indicated. The bands of pHDAC6 were quantified by densitometry and are shown in the bar graph. Student's t test was performed with * indicating p < 0.05. Error bars, S.D. B, Ras-induced HDAC6 phosphorylation is attenuated by dominant negative MEK and ERK. 293T cells were transfected with the indicated plasmids. HA and FLAG double-tagged HDAC6 was immunoprecipitated by anti-HA-agarose beads, and phosphorylation of HDAC6 was examined by immunoblotting with anti-phosphoserine/Thr antibodies. The membrane was then stripped and reblotted with anti-HA antibodies to detect the immunoprecipitation efficiency of HDAC6, HA-MEK(K97A), and HA-Ras(G12V). The expression of GST-DN-ERK1 was examined by anti-ERK Western blotting. Anti-pERK, anti-ERK, and anti-β-tubulin Western blotting analyses were also performed. C, Ser-1035 site is a major phosphorylation site of HDAC6 targeted by the EGFR-Ras-Raf-MEK-ERK signaling pathway. 293T cells were transiently transfected with the indicated plasmids. Following transfection, cell lysate was immunoprecipitated using anti-FLAG M2 beads, and the phosphorylation status of HDAC6 (pHDAC6) was determined with anti-pSer-1035 (HDAC6) Western blotting analysis. The blot was then stripped and reprobed with anti-FLAG antibody to examine the immunoprecipitation efficiency. The levels of EGFR, HA-Ras(G12V), GST-Braf(V600E), and HA-MEK(S218/222D) were examined by Western blotting analyses using the indicated antibodies. The anti-β-tubulin Western blotting analyses were also carried out.

FIGURE 5.

EGF promotes HDAC6(Ser-1035) phosphorylation via ERK1. A, EGF promotes HDAC6 Ser-1035 phosphorylation. 293T cells were transfected with indicated plasmids then serum-starved overnight followed by EGF treatment prior to harvest. Wild type or mutant HDAC6 was immunoprecipitated (IP) by anti-HA antibodies followed by anti-pSer/Thr Western blotting (IB) analyses. The blot was then stripped and reprobed by anti-HA antibodies. The total cell lysates were also subjected to anti-pERK, anti-ERK and anti-β-tubulin Western blotting analyses. B, HeLa cells were serum-starved for 24 h followed by pretreatment with vehicle or 10 μm U0126 for 1 h prior to EGF treatment. The Western blotting analyses were performed with the indicated antibodies. C, 293T cells were transfected with the indicated plasmids. The cells were serum-starved for 24 h followed by 10-min EGF treatment prior to harvest. The Western blotting analyses were performed with the indicated antibodies.

FIGURE 6.

ERK1/2 interacts with HDAC6. A, HeLa S3 cells were fractionated into nuclear and cytoplasmic fractions as described by Dignam et al. (30). Immunoprecipitation (IP) was performed using control (anti-IgG) or anti-HDAC6 antibodies followed by anti-ERK and anti-HDAC6 Western blotting analyses. B, diagrams show HDAC6 full-length and HDAC6 deletion constructs. C, GST beads or GST bead-bound proteins generated from B were incubated with HeLa S3 nuclear or cytoplasmic fraction, and GST pulldown assays were performed followed by anti-ERK1/2 Western blotting (IB) analyses. Protein expression is indicated by Coomassie Blue staining.

FIGURE 7.

Substitution of Ser-1035 to alanine or aspartate does not affect HDAC6 enzymatic activity toward core histones. A, 293T cells were transiently transfected with the plasmids as indicated. HDAC6 wild type or mutant complexes were immunoprecipitated by anti-FLAG M2 agarose beads and then subjected to HDAC assay as described under “Experimental Procedures.” The purified HDAC6 wild type or mutant complexes used in the HDAC assays are also shown by Coomassie Blue staining. NS, not significant. B, Sf9 cells were transduced by the indicated viruses. HDAC6 wild type or mutant proteins were purified by anti-FLAG M2 agarose and then subjected to HDAC assays as in A. The purified HDAC6 wild type or mutant proteins used in the HDAC assays are also shown by Coomassie Blue staining. Data shown are representative of at least three repeated experiments. NS stands for not significant. C, 293T cells were transiently transfected with HA-HD6-F alone or HA-HD6-F and HA-Ras(G12V). Anti-HA immunoprecipitated (IP) samples were subjected to HDAC assay as described in A. The phosphorylation status of HDAC6 was examined by anti-HA immunoprecipitation followed by anti-pSer/Thr Western blotting (IB) analyses. The expression of HA-HD6-F and HA-Ras(G12V) was also examined by anti-HA Western blotting analyses. D, GST-HD6 proteins were purified from Sf9 cells and subjected to a cold ERK1 assay (lane 2) or not (lane 1). The reactions were then subjected to HDAC assays as shown in the bar graph. The phosphorylation of GST-HDAC6 was examined by anti-pSer/Thr Western blotting analysis. The amount of HDAC6 was examined by anti-HDAC6 Western blotting analysis.

FIGURE 8.

S1035A mutant of HDAC6 displays decreased TDAC activity in vivo. A, HDAC6(S1035A) mutant exhibits decreased TDAC activity in vivo compared with the wild type. CHO and H1299 cells were transfected with the indicated plasmids. After 36 h, cells were harvested and lysed. Western blotting (IB) analyses were performed using the indicated antibodies. B, 293T cells were transiently transfected with the indicated shRNA to knock down ERK1 or/and ERK2. shRNA vectors against ERK1 (TRCN0000006150; Sigma) and ERK2 (TRCN0000010040; Sigma) were used to knock down ERK1 and ERK2, respectively. pLKO.1-puro empty vector was used as a control. These vectors were transfected individually or in combination as indicated into 293T cells for 24 h followed by the same transfections for another 24 h prior to harvesting. Western blot analyses were then performed with the indicated antibodies. C, GST-HDAC6 protein was purified from Sf9 cells. The proteins was either subjected to ERK1 kinase assay (lane 2) or not followed by TDAC assays as described under “Experimental Procedures.” Ac-tubulin was detected by anti-Ac-tubulin Western blot analysis. The HDAC6 and pHDAC6 proteins were examined by Coomassie Blue staining. The anti-pSer/Thr Western blot analysis was carried out to examine the phosphorylation of HDAC6.

FIGURE 9.

Active Raf promotes cell migration via HDAC6. A, c-RAF-BXB plasmid was transfected into HDAC6 WT or KO MEFs. Cell migration assays were performed as described under “Experimental Procedures.” The snapshots of the migratory cells are shown as indicated. B, the stained migratory cells were de-stained in 2% SDS and then quantified at A_{560 nm}. A bar graph was made after the quantification. C, anti-pERK, anti-ERK, anti-mHDAC6, and anti-β-tubulin Western blot (IB) analyses were performed using the cell lines described in A and B. NS refers to not significant. ** indicates p < 0.01.

FIGURE 10.

S1035A mutant of HDAC6 exerts decreased migration potential in MEFs. A, cell migration assays were carried out using HDAC6 KO MEFs cells stably expressing empty vector, HA-HD6-F, or HA-HD6(S1035A)-F as described under “Experimental Procedures.” Snapshots of the migratory cells are shown as indicated. B, the stained migratory cells were de-stained in 2% SDS then quantified at A_{560 nm}. The bar graph represents the quantification. NS refers to not significant. ** indicates p < 0.01. C, Western blot (IB) analyses were performed using the indicated antibodies and the cell lines described in A. D, the working model shows how the EGF-EGFR-Ras-Raf-MEK-ERK signaling cascade regulates cell migration via HDAC6.