Phosphorylation of the histone deacetylase 7 modulates its stability and association with 14-3-3 proteins.

Class II histone deacetylases (HDACs) play a role in myogenesis and inhibit transcriptional activation by myocyte enhancer factors 2. A distinct feature of class II HDACs is their ability to shuttle between the nucleus and the cytoplasm in a cell type- and signal-dependent manner. We demonstrate here that treatment with the 26 S proteosome inhibitors, MG132 and ALLN, leads to detection of ubiquitinated HDAC7 and causes accumulation of cytoplasmic HDAC7. We also show that treatment with calyculin A, a protein phosphatase inhibitor, leads to a marked increase of HDAC7 but not HDAC5. The increase in HDAC7 is accompanied by enhanced interaction between 14-3-3 proteins and HDAC7. HDAC7 mutations that prevent the interaction with 14-3-3 proteins also block calyculin A-mediated stabilization. Expression of constitutively active calcium/calmodulin-dependent kinase I stabilizes HDAC7 and causes an increased association between HDAC7 and 14-3-3. Together, our results suggest that calcium/calmodulin-dependent kinase I-mediated phosphorylation of HDAC7 acts, in part, to promote association of HDAC7 with 14-3-3 and stabilizes HDAC7.

Acetylation and deacetylation of histone tails are critical mechanisms regulating gene expression. Acetylation of histone tails has been suggested to play a role in remodeling chromatin and to serve as signals for specific recognition by transcription factors and chromatin remodeling proteins (1)(2)(3)(4). Histone acetylation and deacetylation are catalyzed by two groups of enzymes, histone acetyltransferases and histone deacetylases (HDACs) 1 (5,6). Association and dissociation of sequence-specific DNA-binding transcription factors with histone acetyltransferases or HDACs in response to signaling represents an important mechanism regulating gene expression (7,8).
Mammalian class II HDACs include HDAC4, -5, -6, -7, -9, and -10 (5,9,10). HDAC4, -5, -7, and -9 constitute a subclass that contain a catalytic domain in their carboxyl-terminal region and an N-terminal noncatalytic region showing limited sequence homology with each other. Among these four HDACs, HDAC7 contains the most divergent sequence in its amino terminus (11). This region of HDAC7 possesses, however, three short regions containing sequences highly conserved in the other class II members. In addition, association of myocyte enhancer factors 2 with class II HDACs via the N-terminal region appears to be conserved among HDAC4, -5, -7, and -9 (12)(13)(14)(15)(16)(17)(18). Association of class II HDACs down-regulates myocyte enhancer factor 2-mediated transcription activity and thereby controls myoblast differentiation (16, 19 -22). The N-terminal region is also responsible for the recruitment of the transcriptional co-repressors, CtBP, BCL6, and HP1 (23)(24)(25). The carboxyl-terminal catalytic domain of HDAC4, -5, -7, and -9 contains over 80% amino acid sequence identity and is the site of SMRT (silencing mediator for retinoid and thyroid hormone receptors) and N-CoR (nuclear receptor co-repressor) association (11, 26 -28). One distinct feature of class II HDACs is their ability to shuttle between the nucleus and the cytoplasm. Nucleocytoplasmic shuttling of HDAC4, -5, and -7 is governed by an interplay between an N-terminal nuclear localization sequence and a C-terminal leucine-rich nuclear export sequence (12,29,30), 2 a putative CRM1 recognition sequence. Leptomycin B treatment blocks nuclear export of HDAC4, -5, and -7, suggesting a role for CRM1 in nuclear export of the class II HDACs. The N terminus of class II HDACs contains three highly conserved serine residues that are critical for nuclear export. Sequence analyses indicate that these three serines are putative targets of calcium/calmodulin-dependent kinases (CaMKs) and 14-3-3 proteins. 14-3-3 family proteins interact with phosphorylated serine or threonine-containing motifs present in proteins implicated in cell cycle progression, apoptosis, and regulation of gene expression (31)(32)(33). Exogenous expression of a constitutively active form of CaMK I and CaMK IV promotes nuclear export of HDAC7 and HDAC5, respectively (12,30). We proposed that CaMK I-mediated phosphorylation on these serine residues promotes association of class II HDACs with cytoplasmic 14-3-3 proteins (12). Mutational studies demonstrated that serine to alanine mutations abrogate both 14-3-3 binding and nuclear export (12,29). These studies establish a correlation between 14-3-3 binding and nuclear export of class II HDACs. However, it is not clear whether cytoplasmic class II HDACs associate with 14-3-3 proteins and, if they do, the functional significance of this association.
Many transcription factors such as p53 and nuclear hormone receptors have been shown to be targets of ubiquitin-mediated proteolysis (34 -38). Ubiquitination is catalyzed by a set of enzymes, E1, E2, and E3, via three sequential steps (39,40). Ubiquitin is first activated by a single ubiquitin-activating enzyme, E1. Subsequently, one of several E2 enzymes (ubiquitin-conjugating proteins) transfers ubiquitin from E1 to a member of the ubiquitin-protein ligase family, E3, that is responsible for covalent attachment of ubiquitin to lysine residues in substrate proteins. Ubiquitin-marked proteins are then recognized by the 26 S proteosome and degraded. Regulation of protein degradation by the proteosome pathway depends primarily on the ubiquitination of targeted proteins. Recent studies have demonstrated the phosphorylation of substrate proteins implicated in ubiquitin (Ub)-mediated proteolysis (41)(42)(43).
In this study, we demonstrate that cytoplasmic HDAC7 is a target of ubiquitin-mediated proteolysis. Increased phosphorylation leads to a marked increase of endogenous and transfected HDAC7. This phosphorylation-dependent activity requires the N-terminal conserved serine residues of HDAC7. We show that phosphorylation promotes association of endogenous HDAC7 with 14-3-3 in the cytoplasm. Co-expression of a constitutively active CaMK I promotes HDAC7/14-3-3 interactions and results in an increase of HDAC7. Our data suggest a concerted regulation of HDAC7 activity by phosphorylation-mediated nuclear export and ubiquitin-regulated proteolysis.

MATERIALS AND METHODS
Plasmid Construction and Reagents-The plasmids pCMX, pCMX-mHDAC7 (WT)-HA, and pCMX-1F-14-3-3⑀ have been described previously (12). The plasmids pCMX-1F-mHDAC7 (wild type), pCMX-1F-mHDAC7 (S178A/S344A/S479A), and pCMX-1F-CaMK I were constructed by PCR of HDAC7 or SR␣-CaMK I expression vector (a kind gift from Dr. Anthony Means) and subcloned into pCMX-1F vector. HA-Ub ϩ 1 expression plasmids were kindly provided by Dr. Sajay Pimplikar. MG132 and calyculin A were purchased from Sigma. ALLN was from Calbiochem. Anti-HDAC4, -5, and -7 antibodies were purchased from Cell Signaling. Since class II HDACs are homologous to each other, we first determined the specificity of antibodies against HDAC4, HDAC5, and HDAC7. We found that anti-HDAC7 antibodies were specific for HDAC7 and did not cross-react with HDAC4 or HDAC5 (Fig. 1A). Similarly, HDAC4 and HDAC5 antibodies did not cross-react with their family members. For co-immunoprecipitation experiments, we used anti-HDAC7 antibodies generated by immunizing rabbits with GST fusion protein containing the first 254 amino acids of mouse HDAC7 followed by sequential purification by GST and GST-HDAC7-(2-254) affinity columns. The specificity of these antibodies was examined by Western blot analyses. We found that our purified antibodies were specific for HDAC7 and did not cross-react with family members HDAC4 or HDAC5 (Fig. 1B). We have also probed with preimmune serum and found no signals (data not shown). Anti-FLAG and anti-HA antibodies were from Sigma and Roche Applied Science, respectively. Anti-FLAG M2-agarose beads and anti-HA agarose beads were from Sigma and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. Anti-14-3-3, anti-ubiquitin, anti-glyceraldehyde-3-phos-phate dehydrogenase, anti-lamin B, and anti-␤-actin were purchased from Santa Cruz Biotechnology.
Coimmunoprecipitation-For co-immunoprecipitations, HEK293 cells on 10 ϫ 10-cm plates were transfected with 10 g of the appropriate plasmids using either CaPO 4 or liposomes. Cells were harvested after 48 h, washed with 1ϫ PBS, and resuspended in resuspension buffer (1 ml of 50 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors (Roche Applied Science)). Cells were sonicated and cleared by centrifugation for 15 min at 14,000 rpm. The supernatants were kept as whole cell extract. After preclearing by incubation with protein A/G-agarose (Santa Cruz Biotechnology), immunoprecipitations were carried out using M2-agarose beads (Sigma) for 2 h at 4°C. After washing four times with resuspension buffer, samples were boiled in SDS loading buffer, separated on SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the appropriate antibodies. For in vivo ubiquitination of HDAC7, after treatment by 26 S proteosome inhibitors, HEK293 cells were lysed in radioimmune precipitation buffer. Co-immunoprecipitation was also performed in radioimmune precipitation buffer.
Fractionation of Nuclear and Cytoplasmic Extracts-Nuclear and cytoplasmic fractions were prepared according to Ref. 44. Briefly, cells were lysed in hypotonic lysis buffer (20 mM HEPES, pH 7.8, 0.2 mM EDTA, 20% glycerol, 10 mM NaCl, 1.5 mM MgCl 2 , 0.1% Triton X-100, 25 mM NaF, 25 mM ␤-glycerophosphate, 1 mM dithiothreitol with added protease inhibitors) for 30 min on ice. Nuclei were pelleted by centrifugation at 800 ϫ g for 3 min. The resulting supernatant was centrifuged at 14,000 rpm for 15 min at 4°C to yield cytoplasmic extracts. The pelleted nuclei were washed once with lysis buffer and resuspended in lysis buffer, briefly sonicated, and centrifuged at 17,000 ϫ g for 15 min at 4°C. The supernatants were kept as nuclear extracts. Immunoprecipitation was carried out using 14-3-3 antibodies followed by SDS-PAGE and Western blot analyses probed with 14-3-3 and purified HDAC7 antibodies.
Inhibitor Studies-For endogenous proteins, subconfluent HEK293 cells were treated with 10 nM calyculin A for 1 h followed by cell extract preparation or fractionation. Transfected cells were treated with 20 nM calyculin A for 1 h before harvest, and the expression level of transfected proteins was examined. HEK293 cells or HEK293 cells transfected with expression vectors expressing HA-HDAC7 were treated with 20 M MG132 or 25 nM ALLN 48 h posttransfection for the indicated time before harvesting cells.

Inhibitors of 26 S Proteosome Stabilize Cytoplasmic
HDAC7-To determine whether class II HDACs are subject to proteosome-mediated degradation, HEK293 cells were treated with ALLN and MG132, inhibitors of the 26 S proteosome. Cells were harvested after 2-4 h of treatment, and extracts were subjected to Western blot analyses with antibodies against class II HDACs (Fig. 2). We found that all three HDACs were stabilized by ALLN and MG132, although the degree of stabilization differed. The stabilization of HDAC4 and -5 was modest but reproducible. In contrast, stabilization of HDAC7 was more dramatic. Subcellular fractions were prepared for Western blot analyses probed with anti-HDAC7 antibodies. We found that MG132 caused an accumulation of HDAC7 in the  1-3), anti-HDAC4 (lanes 4 -6), anti-HDAC5 (lanes 7-9), and anti-HDAC7 (lanes 10 -12) antibodies. B, specificity of HDAC7 antibodies. A rabbit polyclonal anti-HDAC7 antiserum was raised against GST-HDAC7-(2-254) and affinitypurified. Purified HDAC7 antibodies were used to probe HEK293 whole cell extracts (lane 1). In vitro transcribed and translated (TNT) HA-HDAC4, -5, and -7 were resolved on SDS-PAGE, followed by Western blot analyses probed with anti-HDAC7 (lanes 2-4) antibodies.
cytoplasmic but not the nuclear fraction (Fig. 3A). Additionally, we found that whole cell extracts and the cytoplasmic fraction of HEK293 cells transfected with a plasmid encoding HA-HDAC7 contained increased amounts of HA-HDAC7 in the presence of MG132 (Fig. 3, B and C). These results indicate that class II HDACs can be stabilized by 26 S proteasome inhibitors and that proteolysis of both endogenous and transfected HDAC7 is mediated, in part, by the 26 S proteasome.
Ectopic Expression of Ubiquitin Decreases the Expression of HDAC7-To test whether Ub has a role in proteasomal degradation of HDAC7, we tested the ubiquitination of this protein.
Whole cell extracts were prepared from cells treated with ALLN or MG132 followed by immunoprecipitation with anti-HDAC7 antibodies and Western blot analysis using anti-Ub antibodies. As shown in Fig. 4A, immunoprecipitated HDAC7 proteins were ubiquitinated in both ALLN-treated (lane 4) and MG132-treated cells (lanes 5 and 6). In contrast, immunoprecipitation in the absence of anti-HDAC7 antibodies did not yield ubiquitination signals (lanes 1 and 2). Furthermore, cotransfection of a Ub frameshift mutant, Ub ϩ1 (which blocks 26 S proteasome activity) (45) leads to a dramatic increase in HDAC7 (Fig. 4B) and the detection of a ubiquitinated protein whose size was similar to that of FLAG-HDAC7 (Fig. 4C, left  panel, lane 4). Immunoprecipitation of Ub ϩ1-containing ly-sates with M2 (anti-FLAG antibodies)-agarose beads followed by blotting with anti-Ub antibodies demonstrated that FLAG-HDAC7 was ubiquitinated (right panel, lane 4).
To further demonstrate that HDAC7 is capable of being ubiquitinated in vivo, HA-Ub and FLAG-HDAC7 expression plasmids were co-transfected into HEK293 cells, and IP was carried out using M2-agarose beads. Fig. 5 shows the expression of ubiquitinated cellular proteins (upper panel) and FLAG-HDAC7 (bottom panel). MG132 treatment led to an accumulation of ubiquitinated cellular proteins and an increased level of FLAG-HDAC7. M2 beads co-precipitate FLAG-HDAC7 species that can be detected by anti-HA antibodies (lane 4). These co-precipitated species represent FLAG-HDAC7 species covalently linked to Ub, indicating that HDAC7 is a target of the ubiquitination machinery. The addition of MG132, which inhibits proteosomal degradation of HDAC7, leads to an accumulation of ubiquitinated HDAC7 (lanes 5 and 6). Together, our results strongly suggest that HDAC7 is subjected to Ub-mediated proteolysis.
Phosphorylation Regulates the Steady State Level of HDAC7 and Promotes HDAC7/14-3-3 Association-Ubiquitination is not the only effector of protein degradation. Phosphorylation has been implicated in the regulation of protein stability in several systems (41). To investigate whether phosphorylation FIG. 3. Cytoplasmic HDAC7 is stabilized by 26 S proteosome. A, cytoplasmic HDAC7 is stabilized by MG132. Nuclear (N) and cytoplasmic (C) fractions were prepared, fractionated on SDS-PAGE, and analyzed by Western blotting probed with anti-HDAC7. Anti-actin and anti-lamin B were used as cytoplasmic and nuclear markers, respectively. B, transfected HA-HDAC7 is stabilized by MG132. Whole cell extracts were prepared, and Western blots were probed with anti-HA (upper panel) and anti-glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (lower panel) antibodies, respectively. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. C, cytoplasmic HA-HDAC7 in transfected cells is stabilized by MG132. Western blots were performed as in A except that fractions were prepared from cells transfected with HA-HDAC7 (B). As a control, HDAC7 (S-A), which was shown to constitutively localize in the nucleus, was included for fractionation (lanes 9 and 10).  ALLN (lanes 2, 6, and 10) or 20 M MG132 (lanes 3, 4, 7, 8, 11, and 12) for the times indicated. Anti-HDAC antibodies were used to probe the protein expression levels of class II HDACs. Quantitation of protein levels was performed using Kodak Digital Science TM Image station 440CF. The actin expression level was used as an internal loading control to normalize expression of HDACs. The ratios (HDAC/actin) of untreated samples were set as 1 (lanes 1, 5, and 9). The numbers (top) indicate the -fold changes in HDAC expression after inhibitor treatment. The data are the average of two Western blots. might control the steady state level of HDAC7, we conducted inhibitor studies using calyculin A, an inhibitor of serine and threonine phosphoprotein phosphatases. As shown in Fig. 6A, calyculin A treatment of HEK293 cells led to a marked increase of HDAC7 but not HDAC5 or ␤-actin. Cell fractionation demonstrated that cytoplasmic HDAC7 was the target of phosphorylation-mediated stabilization (Fig. 6B, lanes 1 and 3). Since 14-3-3 proteins associate with phosphoserine/phosphothreo-nine-containing motifs in HDAC7, we tested whether hyperphosphorylation induced by calyculin A can promote HDAC7/ 14-3-3 interaction. Immunoprecipitation experiments were conducted and showed that the increase of HDAC7 correlated with an enhancement of HDAC7/14-3-3 association (Fig. 6C,  compare lanes 1 and 3). Similar to endogenous HDAC7, transfected FLAG-tagged HDAC7 was also stabilized by calyculin A (Fig. 6D, lane 2). However, the S178A/S344A/S479A HDAC7

FIG. 4. Protein ubiquitination is involved in HDAC7 proteolysis.
A, endogenous HDAC7 is ubiquitinated. HEK293 cells were treated with or without MG132, as in Fig. 2. Whole cell extracts were subject to immunoprecipitation with anti-HDAC7 antibodies (lanes 3-6) followed by Western blot analyses probed with anti-Ub antibodies. As controls, immunoprecipitation was also performed without HDAC7 antibodies (lanes 1 and 2). B, Ub ϩ1 mutant stabilizes HDAC7 expression. Increasing amounts of an Ub ϩ1 expression construct was co-transfected with FLAG-HDAC7. Whole cell extracts were prepared and followed by Western blot analysis. ␤-Actin was used as internal loading control. C, co-expression of Ub ϩ1 leads to the accumulation of ubiquitinated HDAC7. FLAG-HDAC7 expression plasmid was transfected into HEK293 cells with (lanes 2 and 4) or without (lanes 1 and 3) Ub ϩ1 expression plasmid. Whole cell extracts (left panel) and M2-agarose bead precipitates (right panel) were prepared for Western blot analyses probed with FLAG (lanes 1 and 2) and Ub (lanes 3 and 4) antibodies, respectively. WCE, whole cell extracts.
FIG. 5. Transfected HDAC7 is a target for ubiquitination. HA-Ub and FLAG-HDAC7 expression plasmids were co-transfected into HEK293 cells. Cells were treated with MG132 48 h posttransfection for the indicated times. Whole cell extracts were prepared and subjected to immunoprecipitation (IP) using M2-agarose beads. Immunoprecipitates were resolved by 7.5% SDS-PAGE, followed by Western blot analyses. Western blots of whole cell extracts (lanes 1-3) were probed with anti-HA (top panel; to detect ubiquitinated cellular proteins) or anti-FLAG (bottom panel; to detect FLAG-HDAC7) antibodies. Immunoprecipitates were probed with anti-HA (top panel; to detect FLAG-HDAC7-(Ub) n ) or anti-FLAG (bottom panel; to detect FLAG-HDAC7) antibodies (lanes 4 -6). Note that we also detected smeared bands whose molecular weights were less than full-length FLAG-HDAC7. These bands are probably ubiquitinated, degraded HDAC7. The major species in lane 6 are most likely ubiquitinated FLAG-HDAC7-(Ub) n . The asterisk (right, top panel) represents an uncharacterized form of FLAG-HDAC7. The dot is likely to be FLAG-HDAC7. M2 beads, anti-FLAG antibody-conjugated agarose beads; WEC, whole cell extracts; IB, immunoblot. mutant that is defective in 14-3-3 association and is constitutively nuclear (12) was not stabilized by calyculin A (Fig. 6D,  lanes 3 and 4). These data suggest that phosphorylation increased the steady state level of HDAC7 and promoted the association between HDAC7 and 14-3-3 proteins in the cytosol. Furthermore, our data indicate that the residues Ser 178 , Ser 344 , and Ser 479 are critical for phosphorylation-dependent stabilization of HDAC7.
Calyculin A and MG132 Inhibit Distinct Pathways Leading to Stabilization of HDAC7-To examine whether calyculin A and MG132 exert their inhibitory effects on the same pathway, cells were treated with MG132 (Fig. 7A, lane 2), calyculin A (lane 3), or their combination (lane 4). We found that treatment of both MG132 and calyculin A had an additive effect on the expression of FLAG-HDAC7 (lane 4). Immunoprecipitation was used to examine whether FLAG-HDAC7 is ubiquitinated in the presence of MG132, calyculin A, or their combination (Fig. 7B,  upper panel). Whereas MG132 treatment resulted in significant accumulation of ubiquitinated HDAC7 (Fig. 7B, lower  panel, lane 2), calyculin A treatment only had a slight effect (lane 3). These results suggest that MG132 and calyculin A mediate stabilization of HDAC7 through distinct pathways.
Co-expression of CaMK I Increases Steady State Level of HDAC7 and Promotes HDAC7/14-3-3 Association-Since elevated phosphorylation up-regulates the steady state level of HDAC7 and because CaMK I is responsible for phosphorylation of the conserved serine residues of HDAC7 (12), we tested whether CaMK I was capable of regulating HDAC7 stability. As shown in Fig. 8A, ectopic expression of CaMK I or 14-3-3⑀ increased HDAC7 accumulation (top panel, lanes 2 and 3). Furthermore, co-expression of CaMK I and 14-3-3⑀ results in an additive of accumulated HDAC7 (lane 4). Immunoprecipitation with anti-HA antibodies demonstrated that HDAC7 associates with higher levels of 14-3-3 proteins in the presence of CaMK I (bottom panel, compare lanes 6 and 8), suggesting that CaMK I promoted HDAC7/14-3-3 association (lanes 6 and 8). GST pull-down assays were carried out to examine the association between 14-3-3 and HDAC7 (Fig. 8B). GST-14-3-3⑀ inter-acted more strongly with HDAC7 co-transfected with CaMK I. These data suggest that phosphorylation-dependent stabilization of HDAC7 is partly mediated by activation of CaMK I signaling, which in turn promotes HDAC7 association with 14-3-3.

DISCUSSION
Both class I and class II HDACs have been shown to be targets of protein modifications such as sumoylation and ubiquitination. Sumoylation has been implicated in modulating HDAC1-mediated transcriptional repression (46,47) and plays a role in nuclear import of HDAC4 (48). Furthermore, HDAC6 is implicated in the control of protein ubiquitination (49). In this study, we have shown that the steady state levels of HDAC4, -5, and -7 are sensitive to MG132 and ALLN, inhibitors of the 26 S proteosome. We further demonstrated that HDAC7 was subject to Ub-mediated proteolysis and that this FIG. 6. Phosphorylation regulates HDAC7 stability. A, HEK293 cells were treated with calyculin A for 1 h. Total cell extracts were isolated, and SDS-PAGE was conducted for Western blot analyses probed with HDAC7 or HDAC5 antibodies. As a control, anti-␤-actin antibodies were used to normalize the loading. B, calyculin A causes accumulation of endogenous cytoplasmic HDAC7; same as A, except that cytoplasmic (C) and nuclear fractions (N) were used. C, calyculin A enhances association between HDAC7 and 14-3-3 proteins. Cytoplasmic fractions (C, lanes 1 and 3) were used for immunoprecipitation with 14-3-3 antibodies (lanes 1 and 3) and probed with HDAC7 (top) or 14-3-3 antibodies (bottom). Beads alone were used as controls (lanes 2 and 4). D, conserved serine residues are critical for calyculin A-mediated stabilization of HDAC7; same as A except that extracts were prepared from cells transfected with wild-type or serine mutant (HDAC7 S178A/S344A/S479A). proteolysis was modulated by phosphorylation of HDAC7. Our results are the first to demonstrate that phosphorylation regulates HDAC protein stability.
We noted that the degrees of MG132-mediated stabilization of class II HDACs were quite different. In contrast to HDAC7, the levels of HDAC4 and HDAC5 were only moderately elevated by MG132. We speculate that this observation may be related to the subcellular distribution of class II HDACs. Indeed, we previously reported that the subcellular localizations of HDAC5 and HDAC7 are differentially regulated (12). In all of the cell lines examined, including HEP-G2, MCF7, and HEK293 cells (data not shown), a larger fraction of HDAC5 was present in the nucleus than HDAC7. In the current study, we further demonstrated that calyculin A treatment resulted in accumulation of HDAC7 but not HDAC5. It is possible that these observations are interconnected. Nevertheless, our data suggest that despite their sequence similarity, the mechanisms regulating the activities of HDAC5 and HDAC7 are distinct.
Previously, we and others have shown that nuclear export of class II HDACs requires conserved serine residues in the N terminus of class II HDACs (12,29,50). Mutations of these serine residues abolished 14-3-3 and HDAC7 association and also caused the accumulation of nuclear HDAC7. These results suggest a strong correlation between 14-3-3 binding and nuclear export or inhibition of nuclear import of HDAC7. Our present studies demonstrate that phosphorylation causes accumulation of HDAC7 and enhanced association of HDAC7 with 14-3-3 proteins. Furthermore, the putative 14-3-3 binding sites within HDAC7 are critical for phosphorylation-mediated stabilization of HDAC7. These data suggest that 14-3-3 proteins may be involved in stabilization of HDAC7. This hypothesis is further supported by the observation that HDAC7 forms FIG. 9. Models for phosphorylationdependent stabilization of HDAC7. Two mechanisms may account for phosphorylation-dependent stabilization of HDAC7. A, hyperphosphorylated HDAC7 is a preferred substrate for Ub-mediated proteolysis. However, 14-3-3 proteins bind phosphorylated HDAC7 on Ser 178 , Ser 344 , and Ser 479 and inhibit E3 ligase recognition and/or ubiquitination of HDAC7. B, alternatively, hypophosphorylated HDAC7 is a preferred substrate for Ub-mediated proteolysis. Phosphorylation of HDAC7 by CaMK I or blocking phosphatase activity by calyculin A treatment enhanced 14-3-3 binding. In both models, binding of 14-3-3 with HDAC7 can also block nuclear import of HDAC7. The immune pellets were subject to Western blot analyses (IB) probed with anti-HA (HDAC7) and anti-FLAG (14-3-3⑀) antibodies, respectively. B, in vitro GST pull-down assays were used to examine the interaction between 14-3-3⑀ and HDAC7. Note that an equal concentration of HDAC7 (lanes 1 and 2) was loaded for pull-down assays.
Phosphorylation is associated with the stability of several proteins. The peptidyl-prolyl cis-trans-isomerase, Pin1, binds a phosphorylated serine/threonine-containing motif with a distinct specificity (followed by a proline residue, (S/T)P) (52). Interestingly, Pin1 is able to regulate stability of its interacting partners (53). Recently, it was shown that Pin1 potentiates p53 transcription activity by stabilizing p53, although the detailed mechanism involved is not clear (54 -56). Conversely, ␤-catenin has been shown to be a target of Ub-mediated proteolysis in a phosphorylation-dependent manner (57,58). Phosphorylation of ␤-catenin enhanced its interaction with ␤TrCP, a phosphorylation-dependent E3 ligase, which promotes its degradation. These observations prompted us to investigate the role of phosphorylation in the stability of HDAC7.
Our current study demonstrates that when activated, CaMK I may stabilize cytoplasmic HDAC7. Consistent with this observation, calyculin A treatment led to a marked increased accumulation of cytoplasmic HDAC7, and this activity depended on the conserved serine residues, Ser 178 , Ser 344 , and Ser 479 , which are CaMK I target sites. The fact that the CaMK I consensus recognition site overlaps with the 14-3-3 binding site suggests a possible coordinate action of these molecules. Our results suggested that CaMK I in cooperation with 14-3-3 proteins stabilizes HDAC7. It remains possible that kinases other than CaMK I may have a similar effect on the steady state levels of HDAC7.
Two models may account for the mechanism underlying proteolysis of HDAC7. In the first, hyperphosphorylated HDAC7 is a preferred substrate for Ub-mediated proteolysis (Fig. 9A). It has been recently suggested that ␤TrCPs, ubiquitin E3 ligases, specifically target phosphorylated substrates for degradation (59,60). If this were the case for HDAC7, the steady state level of cytoplasmic HDAC7 (presumably phosphorylated at conserved serines) would depend on the abundance of cytoplasmic 14-3-3 proteins. Once bound by 14-3-3 proteins, phosphorylated HDAC7 would be protected from proteolysis through a mechanism yet to be elucidated.
In the second model, unphosphorylated HDAC7 may be subject to Ub-mediated proteolysis (Fig. 9B). Two lines of evidence favor the latter model. First, our fractionation experiments demonstrate that Ub-mediated degradation of HDAC7 occurs in the cytoplasm where the conserved serine residues of HDAC7 are probably phosphorylated. Second, calyculin A and ectopic expression of a constitutively active form of CaMK I do not promote degradation; instead, they markedly increased the steady state levels of HDAC7. These observations favor the model in which ubiquitin-mediated proteolysis degrades unphosphorylated HDAC7 but does not exclude the first model. It will be of interest to identify the putative E3 ligase responsible for HDAC7 ubiquitination and subsequent proteolysis.
Our observation that calyculin A causes stabilization of cytoplasmic HDAC7 is surprising, since phosphorylation of Ser 178 /Ser 344 /Ser 479 is thought to promote CaMK I-dependent cytoplasmic retention of HDAC7, and cytoplasmic HDAC7 is subject to proteosome-mediated degradation. Based on our current and previous studies, we hypothesize that phosphorylation of the conserved serine residues (Ser 178 /Ser 344 /Ser 479 ) may serve dual functions. It may promote export of nuclear HDAC7, and it may stabilize cytoplasmic HDAC7. This mechanism ensures that newly exported HDAC7 will not be degraded once transported to the cytoplasm. Alternatively, it provides a mechanism to protect HDAC7 from proteolysis and store newly synthesized HDAC7 in the cytoplasm until there is a signal directing HDAC7 to the nucleus. The steady state level of cytoplasmic HDAC7 may vary in different cell types, depending on the abundance of signaling molecules such as CaMK I and 14-3-3 and therefore may explain differential subcellular distribution of HDAC7 in different cell types.