HDAC6 Associates with the Repeat Region of Cortactin
To identify HDAC6 cytoplasmic substrates, we prepared cytoplasmic extracts from a HeLa S3-derived cell line stably expressing Flag-tagged HDAC6. Flag-HDAC6 and associated proteins were purified by anti-Flag immunoaffinity chromatography. As a control, mock purifications were performed using extracts prepared from HeLa cells expressing an empty Flag vector (). We found at least five polypeptides that specifically associated with Flag-tagged HDAC6. Tandem mass spectrometric analysis (MS/MS) of a 80–85 kDa polypeptide yielded the amino acid sequence LPSSPVYEDAASFK, which was identified as cortactin (Accession Q14247).
To determine if HDAC6 interacts with cortactin under normal physiologic conditions, co-immunoprecipitation of the endogenous proteins from a cytoplasmic extract was performed. As shown in , a significant fraction of cortactin could be co-precipitated with an anti-HDAC6 antibody but not with pre-immune serum. No cortactin was precipitated when the primary antibody was omitted. Using bacterially expressed and highly purified GST-cortactin and histidine-tagged HDAC6 expressed and purified from Sf9 insect cells, we found that HDAC6 interacts directly with cortactin in the absence of any other cellular proteins ().
To demonstrate specificity for the HDAC6-cortactin interaction, we used adenoviral infection to generate a HeLa cell line that overexpresses Flag-tagged HDAC5. HDAC5-containing protein complexes were then immunoprecipitated from HeLa extracts using a Flag-specific antibody. The presence of cortactin in these immunoprecipitates was analyzed by Western blotting. As shown in , the anti-Flag antibody specifically co-precipitated cortactin from cells expressing Flag-tagged HDAC6 but not from cells expressing Flag-tagged HDAC5. In a reciprocal experiment, an anti-cortactin antibody specifically co-precipitated Flag-tagged HDAC6, but not Flag-tagged HDAC5. Western blots verified the equivalent expression of the Flag-tagged HDAC6 and HDAC5 proteins in the infected cells. Further, overexpressed Flag-HDAC5 was active as determined by a comparison of acetylated histone H4 levels in Flag-HDAC5-overexpressing cells and in parental cells. Thus, Flag-HDAC5 appears to be a suitable negative control. In similar experiments, cortactin was not shown to interact with any other class II HDACs (data not shown). This finding suggests that the cortactin-HDAC6 interaction is highly specific.
To identify the HDAC6-binding region of cortactin, we performed serial deletions from the N- or C-terminus. The truncated sequences were then Myc-tagged and expressed in HeLa cells by transfection. These cells also expressed Flag-tagged HDAC6 as a result of adenoviral infection. Lysates from these cells were subjected to immunoprecipitation using a Flag-specific antibody followed by Western blotting using a Myc-specific antibody. As shown in , neither an N-terminal cortactin fragment (containing the acidic domain, aa 1–84) nor a C-terminal cortactin fragment (containing the α-helix, the proline-rich region, and the SH3 domain, aa 350–546) interacted with HDAC6. Results from GST pull-down assays further reveal that the repeat region alone binds HDAC6 (Figure S1
). Therefore, the cortactin repeat region, a region known to be responsible for interaction with F-actin (Wu and Parsons, 1993
), is necessary and sufficient for the interaction between cortactin and HDAC6.
To define the HDAC6 domain(s) required for interaction with cortactin, anti-Flag immunoprecipitates prepared from cells expressing Myc-cortactin and either Flag-tagged wildtype (1–1215) or deletion mutants were subjected to Western blot analysis using anti-Myc antibodies. As shown in , full-length HDAC6 and a C-terminal HDAC6 deletion mutant (1–840) bind cortactin. In contrast, an HDAC6 mutant (1–503) lacking both the C-terminal and the second deacetylase domain (DAC2) completely lost its ability to bind cortactin. Further deletion analysis indicates that DAC2 alone does not bind HDAC6 (data not shown), suggesting that both DAC1 and DAC2 are necessary, and neither domain alone is sufficient for cortactin binding.
HDAC6 Regulates the Acetylation of Cortactin in vivo
The association between cortactin and HDAC6 suggests that cortactin may be a deacetylation target of HDAC6. To determine if cortactin is acetylated, immunoprecipitated Myc-tagged cortactin was subjected to Western blotting using an anti-acetyl-lysine specific antibody. As shown in , Myc-cortactin is acetylated in vivo. Similarly, endogenous cortactin was also found to be acetylated as assayed by high stringency immunoprecipitation of a whole cell extract either with an anti-acetyl-lysine antibody followed by Western blotting with an anti-cortactin antibody or anti-cortactin followed by anti-acetyl-lysine antibody (). Treatment of cells with the class I and II HDAC inhibitor, TSA, greatly increased the level of acetylated Myc-tagged () and endogenous () cortactin.
To confirm that cortactin is acetylated, we raised a rabbit polyclonal antibody that specifically recognizes acetylated cortactin. Western blot analysis using this antibody revealed that HDAC inhibition increased acetylation of both endogenous and overexpressed cortactin (). Interestingly, like TSA, treatment of cells with the class III deacetylase inhibitor nicotinamide also resulted in an increase of cortactin acetylation. However, unlike TSA, cortactin acetylation was unchanged in the presence of the potent HDAC inhibitor sodium butyrate, which does not affect HDAC6 (Hubbert et al., 2002
Next, using an alternative approach to demonstrate that cortactin is acetylated under physiologic conditions, we immunopurified cortactin from a NIH3T3 whole cell extract, separated the products on a 2-dimensional gel, and analyzed the acetylated cortactin by Western blot with an anti-acetyl-lysine antibody. As shown in , a significant fraction of endogenous cortactin was present in an acetylated form.
To examine the possible deacetylation of cortactin by HDAC6, we co-expressed Myc-tagged cortactin and either Flag-tagged HDAC6 or Flag-tagged HDAC5 in HeLa cells. Myc-immunoprecipitates were then analyzed by Western blot using an acetyl-lysine-specific antibody. As shown in , overexpression of HDAC6 resulted in a reduction in the level of acetylated cortactin. The total level of cortactin was unaffected. In contrast, the overexpression of HDAC5 or a catalytically defective HDAC6 did not alter levels of total or acetylated cortactin.
HDAC6 Deacetylates Cortactin in vivo
To verify that HDAC6 mediates the deacetylation of cortactin, we used an established A549 cell line (HD6KD) in which HDAC6 expression is specifically knocked-down by a retrovirus-mediated RNAi system (Kawaguchi et al., 2003
). Consistent with previous studies (Hubbert et al., 2002
), HD6KD cells contained more acetylated tubulin than control A549 cells (). More importantly, we found that HD6KD cells contained higher levels of acetylated cortactin than the control cells. A reciprocal experiment with a cell line stably overexpressing HDAC6 revealed that acetylated cortactin levels were markedly decreased in these cells as compared to the negative control parental cells.
Since HDAC6 has been reported to interact with SIRT2 (North et al., 2003
), we tested the effect of SIRT2 on cortactin acetylation levels. As shown in , similar to HDAC6 knockdown, SIRT2-knockdown resulted in an increase in cortactin acetylation suggesting that, in addition to HDAC6, SIRT2 might also regulate the state of cortactin acetylation.
Previous analysis of mouse tissues has shown that HDAC6 is highly expressed in testes (Seigneurin-Berny et al., 2001
). In addition, we have found that HDAC6 is highly expressed in human ovarian tumor tissues (data not shown). We examined the expression of HDAC6 in three different human ovarian cancer cell lines and found that HDAC6 expression in the OV2008 and SW626 cell lines was relatively high. On the other hand, the OVCAR3 cell line contained lower levels of HDAC6 (). Importantly, the acetylation level of cortactin was inversely proportional to the expression level of HDAC6. That is, endogenous cortactin was highly acetylated in the OVCAR3 cell line, but not in the OV2008 or SW626 cell lines. These data strongly suggest that HDAC6 is a chief regulator of cortactin acetylation.
Cortactin is Acetylated/Deacetylated in its Repeat Region
To examine the HAT-mediated acetylation of cortactin, Myc-tagged cortactin was co-expressed with either Flag-tagged PCAF or HA-tagged p300 in HeLa cells. Anti-Myc-immunoprecipitates prepared from these cells were then analyzed by Western blot using an acetyl-lysine-specific antibody. As shown in , PCAF expression increased the level of acetylated cortactin in a dose-dependent manner (left panel). However, overexpression of p300 or catalytic mutants of PCAF (Δ579–608 and Δ609–624) did not alter levels of acetylated cortactin.
Cortactin is Acetylated Primarily in its Repeat Region
Although PCAF has been reported to be located both in the nucleus and cytoplasm of cells (Wong et al., 2004
), most studies on PCAF so far have focused on its nuclear functions. Because cortactin is a cytoplasmic protein, to rule out the possibility that PCAF acetylation of cortactin is a result of over-expression and consequently mis-localization, we re-examined the subcellular localization of endogenous PCAF both by cellular protein fractionation and by immunostaining. As shown in Figure S2
, endogenous PCAF is indeed present in both the nuclear and cytoplasm of 293T and NIH3T3 cells, confirming the possibility that cortactin could be a true physiological substrate of PCAF.
To determine if PCAF directly acetylates cortactin, an in vitro acetylation assay was performed using GST-cortactin and the catalytic domains of either PCAF or p300. Consistent with the in vivo results, these experiments showed that PCAF, but not p300, can acetylate cortactin in vitro ().
To map the region(s) of cortactin acetylated by PCAF in vitro, the following three GST-tagged cortactin fragments were prepared: the N-terminal acidic region (1–84), the repeat region (84–330), and the C-terminal region (331–546) (see diagram in ). Together, these fragments cover the entire cortactin protein sequence. As determined by in vitro acetylation assays, PCAF was able to acetylate the repeat region of cortactin, but not the N-terminal acidic or the C-terminal regions (). These data suggest that the repeat region of cortactin is the primary site of acetylation.
To determine if the cortactin repeat region alone is sufficient to serve as a HDAC6 substrate, we infected HeLa cells that express Flag-(84–330) with adenoviruses that express either Flag-HDAC6 or GFP as control, prepared cell lysates, and assayed acetylation levels by immunoprecipitation with anti-Flag and Western blotting with anti-acetyl-lysine antibodies. As shown in , acetylation level of cortactin repeat region diminishes significantly in the presence of overexpressed HDAC6.
Identification of Acetylated Lysines in Cortactin
To identify the sites of acetylation on cortactin, in vitro acetylation assays were performed using GST-cortactin, the PCAF catalytic domain, and acetyl CoA. To verify cortactin acetylation, an aliquot of each reaction mixture was analyzed by Western blotting using an anti-acetyl-lysine antibody (data not shown). The remainder of the reaction mixture was resolved by SDS-PAGE, and the polypeptide band corresponding to cortactin was excised and analyzed by LC tandem mass spectrometry (LC-MS/MS). Of the 50 lysines in cortactin, 11 were found to be acetylated. Of these 11 acetyl-lysines, eight (K87, K161, K189, K198, K235, K272, K309, and K319) were present in the cortactin repeat region (). We also mapped the in vivo sites of acetylation on cortactin by focusing on the repeat region. For these analyses, we transfected 293T cells with a plasmid encoding the Flag-tagged repeat region of cortactin. To maximize acetylation, cells were treated with 400 ng/ml TSA for 12 h. Following this treatment, cellular extracts were prepared from these cells, and the extracts were subjected to immunoprecipitation using a Flag-specific antibody. The resulting immunoprecipitates were then resolved by SDS-PAGE, and the band corresponding to the cortactin repeat region was excised from the gel and analyzed by LC-MS/MS. Finally, using a similar strategy, we immunopurified endogenous cortactin protein using anti-cortactin antibody and subjected the purified product to LC-MS/MS analysis.
Identification of Acetylation Sites of Cortactin in vitro and in vivo
To assess their contributions to the overall acetylation status of cortactin, all eight of the lysines that were identified as the PCAF in vitro
acetylation sites as well as K124 (a residue detected both in the purified Flag-tagged repeat region and endogenous cortactin) were mutated to glutamine. This mutant, referred to as 9KQ, was examined using an in vitro
acetylation assay. As shown in , while PCAF effectively acetylated wildtype GST-cortactin, acetylation of the GST-9KQ mutant was nearly undetectable. Coomassie blue gel staining verified that similar amounts of wildtype cortactin and 9KQ were present in both assays. Furthermore, the lysine to glutamine change does not affect the binding of cortactin to HDAC6 (Figure S3
). Similarly, in transiently transfected HeLa cells, the 9KQ mutant was also much less efficiently acetylated than wild-type cortactin (). Thus, the repeat region is most likely the primary (if not the only) cortactin region acetylated in vitro
and in vivo
The secondary structure of the individual repeats of cortactin repeat region predicts an α-helical region within the carboxyl-half of each subunit (Wu and Parsons, 1993
). Previous computer modeling of this helical region suggested that highly conserved lysine residues may be positioned on the same face of the α-helices, a conformation that would contribute to a positively-charged helical surface. In our predicted model, all of the above-described acetylated lysines in the repeat domain (with the exception of K189 and K319) are in a loop rather than in the helical region (). Additionally, the acetylated lysines are present at two ends of the helices, a conformation that could result in the formation of two “charged patches”. In its deacetylated state, this charged patch (or the positively-charged loop surface) of cortactin is likely to contribute to F-actin binding.
Acetylation of Cortactin Impedes its Interaction with F-Actin
To determine the effect of cortactin acetylation status on cortactin interactions with F-actin, F-actin co-sedimentation assays were performed. For these experiments, GST-fused cortactin repeat regions were expressed in and purified from E. coli cells. This purified protein was then incubated with polymerized rabbit muscle F-actin. As shown in , following F-actin sedimentation, the non-acetylated GST-tagged cortactin repeat region was bound to (i.e., co-sedimented) F-actin in vitro; whereas, immunopurified, PCAF-acetylated GST-tagged cortactin repeat region was not.
Acetylation of Cortactin Reduces its Interaction with F-actin
To examine this effect in more detail, we prepared two sets of GST-tagged cortactin mutants. In the first set, each lysine of the cortactin repeat region was individually mutated to glutamine. Although glutamine cannot be acetylated, its neutral charge mimics the acetylation of lysine. Each of these point mutants were able to bind to F-actin as efficiently as wild-type cortactin (). Likewise, a charge-preserving cortactin mutant in which all nine of the repeat region lysines were mutated to arginine (9KR) was able to efficiently bind to F-actin. In sharp contrast, a charge-neutralizing cortactin mutant in which all nine of the repeat region lysine residues were mutated to glutamine (9KQ) was not able to bind F-actin. In the second set of mutants, the lysines of the cortactin repeat region were progressively mutated to glutamine beginning at either the amino or carboxyl end (). Mutation of less than three lysines at either terminal end did not significantly alter the ability of cortactin to bind to F-actin. However, mutation of more than four of these lysines dramatically reduced the F-actin binding activity of cortactin; the more residues mutated, the less binding detected. The effect was not limited to mutations at the N-terminal or the C-terminal end of cortactin, because mutation within the internal repeats (6KQ) resulted in similar decrease in F-actin binding when compared to N6KQ or C6KQ (). Thus, the acetylation of multiple lysine residues of the cortactin repeat region attenuates its actin-binding ability in vitro. This effect was also observed in vivo using cells overexpressed with the cortactin repeat region. For example, HeLa cells treated with TSA had more acetylated Flag-tagged cortactin and more cortactin in the supernatant (i.e., not bound to F-actin) than did untreated cells (). Consistent with this finding, inhibition of HDAC6 either by treatment of cells with HDAC inhibitors or HDAC6 siRNA decreased endogenous cortactin-F-actin association ().
Acetylation Causes Aberrant Cortactin Localization and Cell Motility
In response to growth factor stimulation or small GTPase, Rac1 activation, cortactin translocates from the cytosol to the cell periphery, where it interacts with and enhances the formation of F-actin (Weed et al., 1998
; Weed et al., 2000
). Because acetylation/deacetylation is a key determinate of cortactin binding to F-actin, we examined whether Rac1 has an effect on the level of cortactin acetylation. NIH3T3 cells were transfected with plasmids expressing Myc-cortactin and various amounts of constitutively active Rac1 (HA-Rac1G12V). Anti-Myc immunoprecipitates prepared from these cells were subjected to Western blot analysis using an anti-acetyl-lysine antibody. As shown in , consistent with our observation that deacetylated cortactin binds better to F-actin, Rac1 activation clearly resulted in cortactin deacetylation.
Acetylation of Cortactin Prevents its Localization to Membrane Ruffles and Inhibits Cell Motility
To further analyze whether the acetylation of cortactin affects its subcellular location, we co-transfected NIH3T3 cells with constitutively active Rac1 (HA-Rac1G12V) and either Flag-tagged wildtype, 9KQ mutant, or 9KR mutant cortactin. Cells were immunostained with anti-Flag and anti-HA antibodies followed by Alexa-594 and Alexa-488 conjugated secondary antibodies. Consistent with previous studies, wildtype cortactin was present in the membrane ruffles in cells expressing active Rac1. Similarly, the 9KR cortactin mutant, which is capable of F-actin binding, translocated to the cell periphery in the presence of active Rac1. In contrast, the 9KQ cortactin mutant, which is not capable of F-actin binding, remained cytoplasmic. Quantification of the percentage of cells expressing Rac1 and the wildtype, 9KQ, or 9KR cortactin that show a leading edge is presented in Table S1
. Together, these results suggest that the acetylation of cortactin inhibits, while deacetylation may be required for, its Rac-mediated translocation to the cell periphery.
It has been reported that growth factor-induced membrane ruffling is due to activation of Rac1 (Kozma et al., 1995
; Nobes and Hall, 1995
). We found that the treatment of cells with EGF resulted in a decrease in cortactin acetylation (). To determine if HDAC6 is involved in the Rac1-mediated translocation of cortactin, we stimulated serum-starved NIH3T3 cells with EGF and monitored the subcellular localization of cortactin and HDAC6 by immunofluorescence microscopy. We found that HDAC6 was translocated to the cell periphery together with cortactin upon EGF stimulation (). This result suggests that HDAC6 interacts with and deacetylates cortactin at the cell periphery. Further analysis demonstrated that, upon EGF stimulation, 9KR mutant translocates to the cell periphery suggesting that the charge-preserving mutant could dislodge the HDAC6-cortactin association. Also, as can be seen in Figure S4
, consistent with the notion that cortactin must be deacetylated in order to translocate to the membrane ruffle or leading edges, the localization of the 9KQ mutant is unchanged in the presence of EGF.
A previous study has shown that overexpression of HDAC6 increases the chemotactic motility of NIH3T3 cells (Hubbert et al., 2002
). To confirm this result, transwell assays were performed with 293T cells expressing either HDAC6 siRNA or control siRNA. The results clearly indicate that chemotactic cell motility decreases in cells depleted of HDAC6 (). To examine the effect of cortactin acetylation status on chemotactic cell migration, parental NIH3T3 cells and NIH3T3 cells stably expressing either wildtype or mutated cortactins were seeded in the upper chamber of migration plates. As shown in , the percentage of motile cells was decreased among cultures expressing cortactin mutants that do not bind F-actin (N8KQ and 9KQ) than in cells expressing either wildtype, N1KQ mutant, or 9KR mutant with intact F-actin binding capability. Western blot analyses indicate that cortactin mutants are expressed in comparable levels as wildtype cortactin (Figure S5
). These results strongly argue that cortactin deacetylation is critical for the regulation of cell migration.
In complementary experiments, we determined if cortactin mutants could rescue the phenotype caused by cortactin knockdown. Cortactin expression was assessed by a Western blot (Figure S5
). As expected, a HT1080 human fibrosarcoma cell line in which cortactin protein expression was reduced by more than 95% (cortactin KD; Bryce et al., 2005
) exhibited slower migration compared to parental cells (control) (). Interestingly, the cell motility defect in cortactin KD cells was effectively rescued by the introduction of wildtype or 9KR mutant cortactin, but not by the 9KQ mutant, underscoring the significance of deacetylation in cortactin function.
To further demonstrate that acetylation/deacetylation of cortactin affects cell motility, we performed migration assays on three different ovarian cancer cell lines that were determined to possess different levels of acetylated cortactin (). As shown in , OV2008 and SW626 cells that express high levels of HDAC6 and contain hypoacetylated cortactin show faster migration when compared to OVCAR3 that expresses low level of HDAC6 and contain hyperacetylated cortactin. Next, we used siRNA to knock-down HDAC6 expression in SKOV3 cells, an ovarian cancer cell line with high levels of HDAC6 protein (). We then compared the migratory properties of the HDAC6 knock-down cells with the parental cells treated with control siRNA. As expected, cells with partially depleted HDAC6 (HDAC6KD) displayed a slower migration phenotype.
Finally, using an alternative approach to assess the effects of acetylation/deacetylation of cortactin on cell motility, we established an MDA-MB-231 cell line that stably expresses the 9KQ mutant. Using a live-cell imaging technique, we then measured the actual distance and velocity these cells traveled under random motility compared to the parental cell line. As shown in , movement velocity was significantly decreased in cells overexpressing the 9KQ mutant. These results are consistent with the transwell assay results and unequivocally confirm that cells that express the charge-neutralizing acetylation mutant of cortactin (9KQ) travel less distance and move slower than the parental cell line.