Class IIa HDACs are constitutively phosphorylated and bound to 14-3-3 proteins.
Phosphorylation-dependent nuclear exclusion of class IIa HDACs is thought to be mediated by at least two signal-responsive families of kinases, the CaMKs and PKDs. However, accumulating evidence suggests that class IIa HDACs could also be phosphorylated in the absence of external stimulus and implies the existence of additional HDACs kinases distinct from CaMKs or PKD (3
As a step toward the identification of these new class IIa HDACs kinases, we first investigated whether endogenous HDAC7 could be phosphorylated under normal growing conditions. In vivo, HDAC7 is predominantly expressed in double-positive thymocytes (8
). Logically, we found that among numerous cell lines tested, T-cell hybridomas express high levels of the HDAC7 protein (data not shown). Do11.10 T-cell hybridoma cells were thus metabolically labeled with inorganic 32
P, and endogenous HDAC7 was recovered by immunoprecipitation. As shown in Fig. , HDAC7 appeared as a phosphorylated protein after SDS-PAGE analysis and autoradiography. Interestingly, phosphorylation of endogenous HDAC7 was almost completely abolished by treatment with staurosporine, a general serine/threonine kinase inhibitor (Fig. ).
FIG. 1. Class IIa HDACs are constitutively phosphorylated, bound to 14-3-3 proteins, and subjected to phosphorylation-dependent nucleo-cytoplasmic shuttling. (A) Do11.10 cells were labeled with [32P]orthophosphate and subsequently treated with staurosporine (+) (more ...)
According to the current model, phosphorylation of specific serine residues in class IIa HDACs creates docking sites for 14-3-3 proteins. To confirm the above observations, we thus examined the interaction between HDAC7 and 14-3-3 proteins in Do11.10 cells. Endogenous HDAC7 (Fig. , left) or ectopically expressed Flag-tagged HDAC7 (Fig. , right) was immunoprecipitated and analyzed for association with endogenous 14-3-3 proteins. Western blot analysis revealed a constitutive association between endogenous 14-3-3 proteins and endogenous or FLAG-HDAC7 (Fig. ). Interestingly, treatment with staurosporine resulted in a drastic reduction in the interaction between HDAC7 and 14-3-3 proteins. Similar observations were made with HDAC4 and HDAC5 in HEK293 cells (data available on request). These results thus suggest that class IIa HDACs exists as phosphoproteins in unstimulated cells and are constitutively associated with 14-3-3 proteins in a phosphorylation-dependent manner.
HDAC7 constitutively shuttles from the nucleus to the cytoplasm.
Phosphorylation of class IIa HDACs and association with 14-3-3 proteins controls their distribution between the nucleus and the cytoplasm. We thus examined the steady-state subcellular localization of GFP-HDAC4 and -HDAC7 in HeLa cells (Fig. ). Under normal growth conditions, the vast majority of cells showed predominantly cytoplasmic localization of both HDAC4 and HDAC7 (Fig. , No Treatment). In contrast, under hypophosphorylating conditions induced by staurosporine, both HDACs accumulated in the nucleus (Fig. ).
It is now well known that class IIa HDACs show differential subcellular localization depending on the cell line examined (11
). This suggests that the machinery controlling nuclear export of class IIa HDACs is differently effective in different cell types. To generalize the above observations, we examined the effect of staurosporine in Cos7 cells, where ectopically expressed class IIa HDACs have been shown to localize primarily in the nucleus (11
). In the majority of the transfected cells, GFP-HDAC7 was, for the most part, found in the nucleus, with only a small fraction of the protein present in the cytoplasm. However, in about 25% of the cells, HDAC7 was predominantly detected in the cytoplasm (Fig. , No treatment). Similarly to HeLa cells, treatment with staurosporine was associated with significant nuclear retention of HDAC7. Indeed, after 1 h of treatment, the protein was almost exclusively localized in the nucleus, and the proportion of cells with predominant cytoplasmic staining dropped to less than 5% (Fig. ). Of note, staurosporine also increased nuclear retention of GFP-HDAC7 in HEK293 cells, where it distributes equally between the nucleus and the cytoplasm in untreated cells (data available on request). Our observations so far clearly unraveled basal phosphorylation and 14-3-3 binding of class IIa HDACs and point toward a constitutive, phosphorylation-dependent nuclear efflux of these enzymes in various normally growing cell lines. To challenge this model, we examined the intracellular mobility of HDAC7 in Cos7 cells, which appear to be the least potent in exporting class IIa HDACs from the nucleus to the cytoplasm. GFP-HDAC7 was thus expressed in Cos7 cells, and its nuclear efflux was examined on live cells using the FLIP technology. For this purpose, the cytoplasm of transfected cells was selectively and repeatedly bleached, and loss of fluorescence was monitored in the nucleus. FLIP experiments revealed a substantial loss of nuclear fluorescence of GFP-HDAC7 in living Cos7 cells after bleaching of the cytoplasm (Fig. ). This loss of nuclear fluorescence was completely blocked by leptomycin B, an inhibitor of CRM1-dependent nuclear export (Fig. ). Quantification of the data confirmed these observations and showed a 50% decrease in nuclear fluorescence of GFP-HDAC7 wild type (wt) 25 min after cytoplasmic bleaching (Fig. , HDAC7wt). In accordance with the steady-state localization data (Fig. ), staurosporine treatment totally abolished constitutive nuclear export of HDAC7 (Fig. ). A similar effect was observed when the four 14-3-3 binding sites of HDAC7 (Ser155
, and Ser449
]) were mutated to alanines (Fig. , HDAC7ΔP). These results clearly demonstrate a constitutive efflux of HDAC7 from the nucleus to the cytoplasm under normal growing conditions. In addition, they are consistent with a model in which the dynamic nuclear export of class IIa HDACs is dependent on the constitutive phosphorylation of their conserved 14-3-3 binding sites. This thus implies the existence of additional class IIa HDACs kinases.
FIG.2. Class IIa HDACs are subjected to phosphorylation-dependent nucleocytoplasmic efflux. (A) Recombinant GFP-HDAC7 was transfected into Cos7 cells. Forty-eight hours posttransfection, cells were left untreated (No Treatment) or treated with staurosporine (more ...) An 85-kDa autophosphorylating kinase associates with the N terminus of class IIa HDACs.
In an attempt to identify the kinases responsible for the constitutive nuclear export of class IIa HDACs, we used the N terminus of HDAC7, which contains the four previously identified phosphorylatable serines (9
), in a GST pull-down assay. The material pulled down from unstimulated 293 cell extracts was analyzed by an IGK assay to determine the molecular weight(s) of associated cellular kinase(s). Bands of variable intensity were observed in the input lane, revealing the presence of several constitutively active kinases in the lysate. One band, with an apparent molecular mass of approximately 85 kDa was specifically detected in association with GST-HDAC7Nter (Fig. ). Identical results were obtained with extracts from Cos7, NIH 3T3, HeLa, Do11.10, and DPK cells (data not shown).
FIG. 3. The N terminus of class IIa HDACs associates with an 85-kDa autophosphorylating kinase. (A) The N terminus of HDAC7 was expressed as a GST fusion protein (GST-Nter) and incubated with total cellular extracts from unstimulated, normally growing HEK293 (more ...)
We then expressed the HDAC7 sequences surrounding each phosphorylatable serine residue as GST fusion proteins (respectively, GST-S155, GST-S181, GST-S321, and GST-S449) and examined their ability to recruit the 85-kDa kinase in a pull-down assay. Surprisingly, only the fusion protein corresponding to Ser155 specifically associated with the 85-kDa kinase (Fig. ). In addition, when Ser155 was mutated into alanine, association with the constitutively active kinase was greatly impaired, thus indicating that the interaction specifically involves Ser155 (Fig. ). To generalize our findings, we tested other class IIa HDACs for their ability to recruit the same 85-kDa kinase activity. Pull-down assays were carried out with GST fusion proteins corresponding to regions of HDAC4 and HDAC5 centered on Ser246 and Ser259, respectively, the residues corresponding to Ser155 of HDAC7. IGK assays revealed that an autophosphorylating kinase with a similar molecular mass of approximately 85 kDa was highly enriched in the material associated with GST-HDAC4, GST-HDAC5, and GST-HDAC7 but not with GST alone (Fig. ). These results show that members of class IIa HDACs can associate with a similar, if not identical, 85-kDa autophosphorylating kinase that is constitutively active in numerous mammalian cell lines.
The 85-kDa autophosphorylating kinase activity includes hPar-1 kinases, C-TAK1 and EMK.
The apparent molecular weight observed in IGK assays precludes the autophosphorylating kinase(s) from being a member of the CaMK or PKD families. To identify this new class IIa HDACs-associated kinase, we screened the human kinome for constitutively active serine/threonine protein kinases with apparent molecular sizes between 80 and 100 kDa (23
) and showing autophosphorylation in an IGK assay. This search identified members of the PKC, RSK, MARK/Par-1, and MSK families. We then tested if any of these kinases could interact with Ser155
of HDAC7. Pull-down assays were carried out with GST-S155 or GST-A155 and GST as a control and analyzed by sequential Western blotting with antibodies directed against PKC family members, RSK1/2, the MARK/Par-1 kinase C-TAK1, and MSK1/2. Among these, GST-S155 specifically associated with endogenous C-TAK1 (Fig. ). More importantly, mutation of Ser155
into alanine greatly reduced the amount of bound C-TAK1. Because these results strongly suggested that C-TAK1 could be the 85-kDa autophosphorylating kinase described above, we then tested the ability of C-TAK1 to associate with other class IIa members. As expected, GST fusion proteins corresponding to Ser246
of HDAC4 and HDAC5, respectively, were also able to specifically recruit endogenous C-TAK1 in pull-down assays (Fig. ).
FIG. 4. MARK/Par-1 kinases, C-TAK1 and EMK, associate with the N terminus of HDAC7. (A) GST fusion proteins corresponding to Ser155 of HDAC7 (GST-S155), to the serine-to-alanine mutant (GST-A155), or to GST alone were incubated with HEK293 cell extracts in a (more ...)
Other MARK/Par-1 family members include the two closely related hPar-1c and hPar-1b/EMK that phosphorylate the microtubule-associated proteins (MAPs) MAP2, MAP4, and Tau (10
). We next investigated if EMK could participate in the 85-kDa kinase activity described above. Pull-down reactions prepared with GST-S155 and GST-A155 were then tested for the presence of EMK. Western blot analysis revealed that while endogenous EMK associated with GST-S155, the interaction was greatly impaired by mutation of Ser155
into alanine (Fig. ).
C-TAK1 phosphorylates serine 155 of HDAC7 in vitro.
We next asked whether C-TAK1 could directly phosphorylate the N terminus of HDAC7, which contains the four phosphorylatable serines involved in nucleocytoplasmic shuttling of HDAC7 (i.e., Ser155
, and Ser449
). For this purpose, the N- or C-terminal domains of HDAC7 were expressed as GST fusion proteins and incubated with purified recombinant C-TAK1 in an IVK assay. Since C-TAK1 has been shown to phosphorylate Cdc25C on serine 216 (32
), we used GST-Cdc25C wt and GST-Cdc25C(S216A) as controls. By comparison with GST-Cdc25C wt, the N terminus of HDAC7 was very efficiently phosphorylated by recombinant C-TAK1 (Fig. ). In contrast, C-TAK1 was unable to phosphorylate the C terminus of HDAC7 or the Cdc25C(S216A) mutant (Fig. ).
FIG. 5. C-TAK1 and EMK specifically phosphorylates Ser155 of HDAC7 in vitro. (A) The C- and N-terminal domains of HDAC7 and wild-type or S216A mutant Cdc25C were produced as GST fusion proteins [GST-HDAC7Cter, GST-HDAC7Nter, GST-Cdc25Cwt, and GST-Cdc25C(S216A), (more ...)
Hydrophobic residues at −5, +1, and + 5, as well as an arginine at the −3 position relative to the phosphorylated serine seem to be crucial for optimal phosphorylation by C-TAK1 (30
). Except for the presence of a hydrophobic residue at the +5 and +1 positions, the four phosphorylation sites previously identified in the N terminus of HDAC7 match this consensus phosphorylation motif (Fig. ). These observations suggest that C-TAK1 could directly phosphorylate HDAC7 and identify Ser155
, and Ser449
as putative target sites for C-TAK1. To test this hypothesis, GST fusion proteins corresponding to each serine residue (respectively, GST-S155, GST-S181, GST-S321, and GST-S449) were evaluated as potential substrate for C-TAK1 in an IVK assay. Surprisingly, only Ser155
was efficiently phosphorylated by recombinant C-TAK1 (Fig. ).
EMK and C-TAK1 display site preference among the four serine residues of HDAC7.
To confirm and extend these observations, we performed an exhaustive analysis of C-TAK1 target sites in the N terminus of HDAC7. This region of HDAC7 was incubated with [γ-32
P]ATP and recombinant C-TAK1 in vitro. A control reaction was performed in parallel with PKD, which phosphorylates Ser155
, and Ser449
in vitro (9
). Labeled proteins were digested with trypsin, and the resulting peptides were separated by HPLC. Radioactive fractions were then analyzed by mass spectrometry to identify the phosphorylated residue(s). Labeling with PKD led to four major radioactive peaks (Fig. ), which corresponded to the formerly identified Ser155
, and Ser449
. In contrast, after phosphorylation with C-TAK1, the HPLC profile exhibited a single major phosphorylation peak (Fig. ). Mass spectrometry analysis showed that this peak corresponded to phosphorylated Ser155
. In contrast to PKD, which targets all four serine residues implicated in the nuclear-cytoplasmic shuttling of HDAC7, these results demonstrate that C-TAK1 specifically phosphorylates Ser155
Our results showed that EMK can associate with the N-terminal Ser155
of HDAC7 (Fig. ). In addition, sequences around this serine residue, which are conserved in all class IIa HDACs (Ser246
, and Ser220
of HDAC4, HDAC5, HDAC7, and HDAC9, respectively), match with the KXGS motif phosphorylated by EMK in Tau (10
). We therefore investigated whether EMK, similarly to C-TAK1, could phosphorylate Ser155
of HDAC7. As expected, the entire N-terminal domain of HDAC7, which contains all four 14-3-3 binding sites, was very efficiently phosphorylated by purified C-TAK1 or EMK in IVK assays (Fig. , GST-Nter). More importantly, when the sole Ser155
was mutated to alanine, phosphorylation by both kinases was totally abolished (Fig. , GST-NterS155A). Taken together, these results demonstrate the specificity of C-TAK1 and EMK for Ser155
over the three other serine residues previously involved in the nuclear cytoplasmic shuttling of HDAC7.
HDAC7 is phosphorylated on Ser155 in vivo.
We next examined the phosphorylation status of Ser155 on endogenous HDAC7 in vivo. For this purpose, we first developed an antibody that specifically recognizes the phosphorylated form of HDAC7 Ser155 (data of antibody characterization available on request). Total extracts from Do.11.10 T-cell hybridomas, which express high levels of endogenous HDAC7, were examined by Western blotting with the phospho-specific antibody for Ser155. As shown in Fig. , strong basal phosphorylation of HDAC7 was consistently observed at Ser155 in normally growing cells (Fig. , α-pS155). More importantly, Ser155 phosphorylation was totally lost when cells were treated with staurosporine.
FIG. 6. HDAC7 is phosphorylated on Ser155 in vivo. (A) Total cell lysates were prepared from Do11.10 cells which were first treated with staurosporine (+) for 1 h or left untreated (−). Phosphorylation of endogenous HDAC7 was detected by Western (more ...)
Because sequences around Ser155 of HDAC7 are highly conserved in other members of the class IIa, the phospho-specific antibody against Ser155 also recognizes the corresponding phosphorylated serines in HDAC4 and HDAC5 (Ser246 and Ser259, respectively). To generalize our observations to other members of the class IIa, FLAG-tagged versions of HDAC4, HDAC5, and HDAC7 were transiently expressed in HEK293 cells and examined by Western blotting using the phospho-specific antibody (Fig. , α-pS155). Confirming our observations on endogenous HDAC7, all three class IIa HDACs showed basal phosphorylation of their respective serine residue, which was significantly reduced upon treatment with staurosporine.
The above findings suggest that phosphorylation of Ser155 in HDAC7 (or Ser246 and Ser259 in HDAC4 and HDAC5, respectively) might be important for constitutive nuclear export. To test this hypothesis, we fractionated extracts from HEK293 cells transiently transfected with FLAG-tagged HDAC4 or HDAC7 into nuclear and cytoplasmic fractions. Confirming our immunofluorescence data (data available on request), comparable amounts of HDAC4- or HDAC7-FLAG were found in both fractions (Fig. ). However, Western blot analysis with the phospho-specific antibody revealed that HDAC7-phosphorylated at Ser155 and HDAC4-phosphorylated at Ser246 were highly enriched in the cytoplasm (Fig. ). To confirm and extend these observations, we performed a similar experiment in HeLa and Cos7 cells expressing FLAG-tagged HDAC7. In accordance with the immunofluorescence data (Fig. and ), HDAC7 localized primarily in the cytoplasm of HeLa cells, where it is phosphorylated on Ser155 (Fig. ). In contrast, HDAC7 was found almost exclusively in the nucleus of Cos7 cells, and no phosphorylation of Ser155 could be detected (Fig. ). Taken together, these results establish a strong correlation between phosphorylation of Ser155 and cytoplasmic localization of HDAC7.
EMK and C-TAK1 alter nuclear export of class IIa HDACs and regulate their repressive activity.
Our results so far strongly suggest that in the absence of extracellular stimuli, EMK and C-TAK1 control localization of class IIa HDACs by phosphorylating their most N-terminal 14-3-3 binding sites (i.e., Ser246, Ser259, and Ser155 in HDAC4, HDAC5, and HDAC7, respectively). In Cos7 cells, class IIa HDACs are mainly found in the nucleus, probably because the mechanisms controlling their nuclear export are poorly efficient in these cells. To test our model, we examined the steady-state localization of HDAC7 in the presence of overexpressed EMK or C-TAK1 in Cos7 cells. As observed before, HDAC7 was mainly found in the nucleus of Cos7 cells when expressed alone (Fig. ). In contrast, coexpression of EMK and C-TAK1 induced dramatic cytoplasmic accumulation of HDAC7. However, we did not observe convincing colocalization of HDAC7 with either MARK members in the cytosol (Fig. , merged). To generalize these observations, we tested the effect of EMK and C-TAK1 on the subcellular localization of HDAC4. By comparison with HDAC7, HDAC4 was even more sensitive to cytoplasmic retention by MARK kinases, with approximately 80% of cells showing predominant cytoplasmic staining (Fig. ). We next tested whether cytoplasmic accumulation of class IIa HDACs induced by MARK/Par-1 kinases resulted directly from an increase in their nuclear export. FLIP experiments revealed a remarkable increase in the nucleocytoplasmic efflux of HDAC7 by both EMK and C-TAK1, resulting in less than 10% of the initial fluorescence left in the nucleus after 25 min (Fig. ).
FIG. 7. EMK and C-TAK1 control nuclear export of class IIa HDACs and regulate their repressive activity. (A) Cos7 cells were transfected with expression vectors for GFP-HDAC7 and FLAG-tagged EMK or C-TAK1. The intracellular localization of HDAC7 was detected (more ...)
In T cells, we have shown that HDAC7 associates with MEF2D to repress the Nur77 promoter and that this inhibitory action is relieved by T-cell receptor signaling which induces HDAC7 phosphorylation and cytoplasmic relocalization. (8
). As EMK and C-TAK1 promote nuclear export of class IIa HDACs, both kinases would be expected to overcome the inhibitory activity of HDAC7 over the Nur77 promoter, even in the absence of T-cell receptor signaling. To address this question, we used the isolated Nur77 promoter in reporter assays. As expected, the transcriptional activity of the Nur77 promoter was strongly inhibited by HDAC7 (Fig. ). Overexpression of EMK or C-TAK1 totally reversed the repressive effect of wild-type HDAC7. Of note, EMK increased the transcriptional activity of Nur77 up to twofold above levels observed in the absence of HDAC7.
To further assess the functional consequences of phosphorylation of class IIa HDACs by MARK/Par-1 kinases, we examined the ability of EMK and C-TAK1 to activate c-jun expression, another class IIa HDAC-repressed gene (45
). For this purpose, EMK and C-TAK1 were independently expressed in Cos7 cells, and levels of endogenous c-jun were examined by Western blotting. As expected, ectopic expression of EMK or C-TAK1 was associated with a marked increase in c-jun levels (Fig. ).
Taken together, these data demonstrate that the MARK/Par-1 kinases EMK and C-TAK1 are physiologically relevant kinases for class IIa HDACs and strongly support a novel role for these kinases in gene regulation.
EMK and C-TAK1 regulate phosphorylation and cytoplasmic localization of class IIa HDACs in the absence of extracellular stimuli.
We have previously shown that PKD efficiently phosphorylates Ser155
of HDAC7, even when the leucine residue at position −5 (Leu150
) is replaced with an alanine (9
). Interestingly, all C-TAK1 substrates identified so far have a leucine at position −5 of the targeted serine (Fig. ). While testing the importance of this leucine residue in EMK and C-TAK1 target recognition, we found out that the substitution of HDAC7 Leu150
to alanine totally abolished phosphorylation of Ser155
by both kinases (data available on request).
Based on these results, we generated an HDAC7 mutant specifically deficient for phosphorylation by EMK/C-TAK1 where Leu150 was mutated to alanine [HDAC7(L150A)]. We examined the in vivo phosphorylation of Ser155 in the context of this mutant using the phospho-specific antibody. As observed above (Fig. ), HDAC7wt showed strong basal phosphorylation of Ser155 and robust association with 14-3-3 proteins (Fig. ). Interestingly, the L150A mutation totally inhibited phosphorylation of Ser155. In addition, the same mutation also reduced association with 14-3-3 proteins.
FIG. 8. EMK and c-TAK1 phosphorylate Ser155 of HDAC7 in vivo. (A) FLAG-tagged versions of wild-type HDAC7 (HDAC7wt) or the HDAC7(L150A) mutant were transiently expressed in HEK293 cells, immunoprecipitated, and examined by Western blot analysis with antibodies (more ...)
Since the HDAC7(L150A) mutant is not phosphorylated on Ser155, it should consequently be impaired in its ability to exit the nucleus. To verify this hypothesis, we examined the subcellular localization of the L150A mutant in HeLa cells, where HDAC7 is very efficiently exported from the nucleus. Indeed, as shown above (Fig. ), wild-type HDAC7 was cytoplasmic in the majority of normally growing HeLa cells (Fig. ). By contrast, only 30% of HeLa cells expressing the L150A HDAC7 mutant showed predominant cytoplasmic staining (Fig. ). Interestingly, FLIP experiments revealed a clear difference between the abilities of wild-type HDAC7 and the L150A mutant to exit the nucleus. For wild-type HDAC7, half of the nuclear fluorescence was lost in about 10 min after bleaching the cytoplasm, and 35% of the initial nuclear fluorescence was left after 25 min (Fig. ). In contrast, the loss in nuclear fluorescence was much slower for HDAC7(L150A), which reached a plateau of ~65% of its initial value after 25 min (Fig. ). These experiments show that the L150A HDAC7 mutant is greatly impaired in its ability to exit the nucleus, which results in an altered steady-state subcellular localization. These results thus strongly suggest that EMK and/or C-TAK1 specifically target Ser155 of HDAC7 to control its nuclear export.
To establish this hypothesis more firmly, we used RNAi to inhibit endogenous C-TAK1 and EMK activities in HeLa cells. A combination of siRNAs directed against both C-TAK1 and EMK reduced the endogenous levels of both kinases (Fig. , left). Coincident with this reduction, we observed a substantial decrease in the phosphorylation of Ser155 in HDAC7 (Fig. , right). As expected, knockdown of EMK and C-TAK1 also altered subcellular distribution of HDAC7. Indeed, when HeLa cells were cotransfected with GFP-HDAC7 and siRNAs against EMK and C-TAK, the proportion of cells showing a predominant cytoplasmic staining decreased significantly (Fig. ).
Altogether, these data strongly suggest that MARK/Par-1 kinases EMK and C-TAK1 phosphorylate class IIa HDACs on their most upstream 14-3-3 binding site and regulate their constitutive nuclear export in vivo.
The 14-3-3 binding sites are hierarchically phosphorylated in HDAC7.
The biological significance behind the specific constitutive phosphorylation of HDAC7 Ser155 (and corresponding HDAC4 Ser246, HDAC5 Ser259, and HDAC9 Ser220) by MARK/Par-1 kinases remained elusive. To address the role of Ser155 phosphorylation in the signal-independent nuclear efflux of HDAC7, we examined the dynamic nuclear export of an HDAC7 protein where Ser155 was mutated into alanine [HDAC7(S155A)]. Results from FLIP analysis were compared with data obtained with wild-type HDAC7 and the HDAC7ΔP mutant. Surprisingly, mutation of Ser155 alone had an effect comparable to mutating simultaneously the four serine residues and almost completely abolished constitutive nuclear export, as demonstrated by a constant postbleach relative nuclear fluorescence in HDAC7(S155A)-expressing cells (Fig. ). This observation demonstrates that Ser155 plays a dominant role in HDAC7 constitutive phosphorylation, association with 14-3-3, and nuclear export.
FIG. 9. The 14-3-3 binding sites in HDAC7 are hierarchically phosphorylated. (A) Cos7 cells were transfected with constructs expressing GFP fusion proteins corresponding to wild-type HDAC7 (HDAC7wt), a mutant of HDAC7 in which the residues Ser155 was mutated (more ...)
To further address the importance of Ser155, we examined the phosphorylation pattern of the HDAC7(S155A) mutant protein in vivo. For this purpose, wild-type and S155A mutant HDAC7 proteins were metabolically labeled with [32P] orthophosphate, affinity purified, and trypsin digested for HPLC analysis. As shown in Fig. , wild-type HDAC7 was found to be constitutively phosphorylated on five major sites, among which the four serine residues previously implicated in subcellular trafficking of HDAC7, i.e., Ser155, Ser181, Ser321, and Ser449 were identified. Very unexpectedly, the S155A mutant exhibited a drastically different phosphorylation pattern, as the alanine mutation at Ser155 also resulted in a complete loss of phosphorylation at Ser181 (Fig. ).
We next tested whether phosphorylation of Ser181 could be also dependent on the two other 14-3-3 sites, Ser321 or Ser449. HDAC7 mutants, where each of the phosphorylatable serine residues was independently mutated to alanine [HDAC7(S155A), HDAC(S181A), HDAC7(S321A), and HDAC7(S449A)] were thus examined by Western blotting with antibodies specific for the phosphorylated forms of Ser155 and Ser181 (Fig. ) (data of antibody characterization available on request). As expected, phosphorylation of Ser155 was detected for all constructs except for HDAC7(S155A). Basal phosphorylation of Ser181 was undetectable after staurosporine treatment (data not shown) or in the HDAC7(S181A) mutant, confirming the specificity of the phospho-Ser181 antibody (Fig. ). In accordance with the HPLC data, phosphorylation of Ser181 was totally abolished when Ser155 was mutated into alanine (Fig. ). Phosphorylation of Ser181 was uniquely dependent on Ser155 as it was unaltered by the S321A or S449A mutations. These observations thus confirm that the presence and/or the phosphorylation of Ser155 are required for subsequent phosphorylation of Ser181 in vivo.
To discriminate between both hypotheses, we examined the phosphorylation levels of Ser181 in the HDAC7(L150A) mutant, where Ser155 is present but poorly phosphorylated (Fig. ) both by Western blotting and HLPC analysis (Fig. , respectively). Interestingly, the L150A mutant also showed a concomitant reduction of Ser181 phosphorylation (Fig. ). These results demonstrate that basal phosphorylation of Ser155 is required for phosphorylation of Ser181 and raised the exciting possibility that class IIa HDACs may be regulated through a process of hierarchical phosphorylation.