Two lines of evidence suggest an involvement of mHDAC6 in the Ub-dependent signaling processes. First, two proteins copurified with mHDAC6 showed striking sequence homology to yeast proteins involved in Ub-dependent protein degradation. The first one, p97/VCP/Cdc48p, an AAA-ATPase, was shown to participate in different functions, depending on its partner proteins (29
). Its role in Ub-dependent protein degradation was first suggested in yeast, where it was found in a complex with UFD3 (12
) and then with E4/UFD2 (22
). Moreover, p97/VCP/Cdc48p was recently shown to form a complex with the mammalian homologue of yeast UFD1 (27
). Finally, p97/VCP/Cdc48 was found to specifically associate with the ubiquitinated forms of IκBα and to probably control its proteasome-dependent degradation (10
). The second protein, PLAP, is a mammalian homologue of yeast UFD3. PLAP is involved in the activation of several phospholipases (30
). In yeast, UFD3 seems to play a role in the control of the concentration of free Ub in cells (19
). Indeed, UFD3 mutants have reduced levels of intracellular free Ub. The degradation of the UFD substrates in these mutants could be restored by overexpressing Ub. It is not clear how UFD3 may control the concentration of cellular Ub in yeast. Since UFD3/PLAP is capable of activating phospholipases, specifically phospholipase A2 (PLA2) (30
), one may expect the involvement of these enzymes in the control of the free cellular Ub level. Interestingly, there is one hint in the literature regarding the participation of PLA2 in the control of Ub concentration. Indeed, it has been shown that a membrane-bound form of Ub was associated with budded virions of baculovirus and possessed a phospholipid anchor, which could be removed by PLA2 treatment (14
). Therefore, PLA2 could release Ub from the membrane. Although the massive presence of cellular Ub in a membrane-bound form has not been evidenced, it is possible that PLAP/UFD3 participates in the release of this putative pool through the activation of PLA2. In the yeast UFD3 mutant, the absence of this PLAP-like activity might be responsible for the observed decrease in the amount of intracellular free Ub.
The second line of evidence in favor of the involvement of mHDAC6 in protein ubiquitination relies on the analysis of the structure of mHDAC6 itself. Indeed, a Ub carboxyl-terminal hydrolase-like zinc finger (ZnF-UBP) domain was found in the C-terminal region of mHDAC6. This particular domain with unknown function is shared with a number of Ub-specific proteases (1
). Here we showed that this finger specifically mediated the interaction of mHDAC6 with Ub. Besides mHDAC6, we have also identified eight other Ub-binding proteins in the mouse testis cytosolic extract. Several are Ub C-terminal hydrolases, which very probably directly interact with Ub. One of them, the Ub carboxy-terminal hydrolase 5, also contained a ZnF-UBP domain (not shown). Four noncharacterized proteins were also on the list, and two of them showed a specific structural motif suggesting a relationship with Ub signaling pathways. Indeed, both AK011826
and AWPI possess a Ub-like domain (not shown), which is also found in several proteins containing a small region of limited sequence identity to Ub (9
Most interestingly, we also found TIP120 protein among the Ub-associated proteins. TIP120, a TBP-interacting protein, activates the basal level of transcription from all three classes (I, II, and III) of promoters (25
). TIP120 is also part of a complex containing several proteasomal ATPases (24
). TIP120 is therefore thought to participate in both transcriptional regulation and proteasome-dependent protein degradation. Here we report a novel property of TIP120: its ability to interact with Ub.
In the literature, besides enzymes directly involved in protein ubiquitination, other proteins with Ub-binding activity have been described. Indeed, several proteins involved in diverse functions share a potential Ub-binding domain termed UBA. Two plant de novo methyltransferases (7
), as well as Rad23, involved in DNA repair (32
), and the mammalian protein, p62 (35
), are among these proteins. The UBA domain very probably links the activity of these proteins to that of the Ub-dependent signaling pathway. Indeed, the UBA domain was originally defined in various components of the Ub-dependent protein degradation pathway, such as Ub C-terminal hydrolases (UCHs), Ub-conjugating enzymes (E2), and Ub protein ligases (E3) (16
). In the case of Rad23, it has recently been shown that UBA mediated the specific binding of Ub (5
) and that this interaction inhibited multi-Ub chain formation (28
Here we found that the interaction of mHDAC6 with Ub led to the dissociation of the mHDAC6 complex and notably to the release of p97/VCP/Cdc48. Interestingly, it has been reported that Ub binding by yeast E4/UFD2 resulted in the release of Cdc48p (22
). Therefore, mHDAC6 shares three properties with the yeast E4/UFD2: (i) Ub binding, (ii) interaction with p97/VCP/Cdc48, and (iii) release of this partner after the interaction with Ub. However, it is not clear how mHDAC6 participates in the control of protein ubiquitination. Our data show that free Ub, and even a Ub sequence inserted in the middle of a protein (GST-Ub-H4) (Fig. ), can recruit mHDAC6. The latter suggests that once a protein is monoubiquitinated, it becomes a potential target for mHDAC6. After the recruitment of mHDAC6 by a monoubiquitinated protein, at least two scenarios can be considered. First, mHDAC6 would deacetylate critical lysines on the substrate protein (and/or partner proteins) and to allow their ubiquitination. Indeed, the deacetylation of specific lysines may allow their subsequent ubiquitination. Second, mHDAC6 would control the activity of the ubiquitination machinery by deacetylating them. Our in vitro assays suggested that the Ub-mHDAC6 complex would have to be dissociated for the ubiquitination of substrate proteins to take place. Since the Ub-mHDAC6 interaction seems to dissociate the mHDAC6-p97/VCP/Cdc48p complex, one may assume that p97/VCP/Cdc48p could play a reverse role and would help mHDAC6 to release Ub. In agreement with this hypothesis, it has been suggested that the basic activity of p97/VCP/Cdc48p would be protein unfolding or disassembly of protein complexes (29
Our experiments also suggest another property of mHDAC6. Indeed, the isolated C-terminal domain of mHDAC6 seems to have a better Ub-binding capacity than the full-length protein (Fig. A and B). This may suggest that the Ub-binding activity of mHDAC6 could be dependent on its conformation, which could itself be controlled by posttranslational modifications or its interaction with other partner proteins.
Unfortunately, there is no hint in the literature about a possible mechanism linking protein acetylation to the Ub signaling pathway. However, a relationship between protein acetylation and stability has recently been demonstrated in the case of E2F, which shows an increased half-life when acetylated (26
). Our work therefore strongly suggests a link between two key posttranslational protein modifications—acetylation and ubiquitination—both modifying lysine residues.