During thymic education, negative selection of T cells is associated with the activation of a specific apoptotic program. One of the crucial mediators of negative selection in thymocytes and T cell hybridomas is the orphan nuclear receptor Nur77, whose expression is rapidly induced during TCR-mediated apoptosis. Expression of Nur77 is tightly controlled by several proteins, amongst which the transcription factor MEF2D plays a central role by alternatively recruiting repressors or activators. We recently identified HDAC7, a class IIa HDAC, as a major MEF2D-associated transcriptional repressor of Nur77 in unstimulated thymocytes (11
). Derepression of Nur77 and induction of apoptosis occurs through TCR-mediated phosphorylation of HDAC7 and subsequent nuclear exclusion. To investigate the involvement of protein kinases in the TCR-induced nuclear exclusion of HDAC7, we used several serine/threonine protein kinase inhibitors. However, kinase inhibitor studies should always be regarded with a degree of caution. Indeed, Gö6976 was also reported to be an efficient inhibitor of MAPKAP-K1b, MSK1, PKCα, and CHK1, although at a slightly higher concentration (1 μM; reference 22
). Thus, we resorted to a more specific approach based on the use of a dominant negative mutant of PKD1. Our results provide strong evidence that PKD1 contributes to Nur77 expression by phosphorylating HDAC7 in response to TCR signaling during negative selection of thymocytes. However, additional experiments will be required to confirm this model.
Previous work identified KidIns220 (kinase D–interacting substrate of 220 kD), a lipid raft–associated protein selectively expressed in brain and neuroendocrine cells, as an interacting partner and a substrate for PKD (23
). However, the true biological effect of KidIns220 phosphorylation remains to be elucidated. PKD was also shown to phosphorylate S351 of the RAS effector RIN1 (24
). Interestingly, the authors suggested that phosphorylation of serine 351 by PKD might create a 14-3-3 binding site in RIN1. Remarkably, the sequence around S351 of RIN1 (SL346
) perfectly fits with the LXR/KXXS consensus motif that is also present in the four PKD sites identified in this study in HDAC7. We and others had previously associated phosphorylation of S155, S321, and S449 of HDAC7 with 14-3-3 binding and inactivation through nuclear export (11
). Thus, our findings provide additional evidence that PKD may function as a regulator of 14-3-3 binding to several cellular proteins. Identification of additional endogenous substrates of PKD should confirm this hypothesis.
It has been proposed that PKD function may also be dependent on the intracellular localization of the kinase (25
). In B cells, antigen receptor stimulation rapidly activates PKD, which transiently translocates to the plasma membrane and then redistributes to the cytosol (26
). Based on the B cell model, it is thought that PKD would localize first at the plasma membrane and then in the cytosol during TCR activation of T cells. Here, we show that stimulation of the TCR induces PKD-dependent phosphorylation of a nuclear protein, HDAC7, which then shuttles from the nucleus to the cytoplasm. Thus, our results imply that active PKD would be found, at least in some conditions, in the nucleus of TCR-stimulated thymocytes. This would be reminiscent of the nucleo-cytoplasmic shuttling observed for PKD in response to G protein–coupled receptor agonists or oxidative stress (27
). These mechanisms await further studies to be fully defined in T cells.
In fibroblast and epithelial cells, PKD has been implicated in many important cellular processes, including function and organization of the Golgi apparatus (30
), normal and tumor cell proliferation (33
), cancer cell invasion (35
), and apoptosis (36
). Several interesting observations initially suggested a key role for PKD in lymphoid cells. B cell receptor or TCR engagement rapidly induces activation of PKD in B cells, peripheral blood T lymphoblasts, and Jurkat cells (38
). However, very little information is available for the true biological function of PKD in T cells. In this study, we provide direct experimental evidence supporting a role for PKD in T cells, where it regulates Nur77 expression and the rate of apoptosis in response to TCR engagement. Recently, an elegant study used the CD2 promoter and locus control region to express a membrane- or cytosol-targeted active PKD in transgenic mice T cells (39
). Expression of active PKD in the cytosol had a dramatic effect on thymocyte cellularity, with transgenic mice having reduced cell numbers and lacking DP and single positive (SP) compared with control littermates. This was partially explained by the capacity of cytosolic PKD to suppress rearrangement and expression of TCRβ subunits, a mechanism reminiscent of allelic exclusion. However, even in the presence of a functional TCR complex, cytosolic PKD prevented development of DP and SP thymocytes (39
). This suggests that expression of constitutively active PKD in the cytoplasm could perturb thymocyte maturation downstream of TCR rearrangement. In light of our new findings, this perturbation could be partially due to an early derepression of the Nur77 promoter and premature engagement of negative selection programs in early DP thymocytes. Indeed, the cytosolic-targeted PKD used in the transgenic mice study was generated by deletion of the DAG-binding domain, which targets the protein to the membrane. One could imagine that this mutant would retain its capacity to locate to the nucleus and induces expression of Nur77 in the absence of TCR stimulation. Interestingly, decreased thymic cellularity, absence of DP and SP thymocytes, and reduced TCR αβ expression were amongst the phenotypes observed in thymi from Nur77-transgenic mice (8
Multiple studies have demonstrated the role of Ca2+
signaling in TCR-induced transcriptional activation of Nur77 (7
). However, published data indicate that Ca2+
-independent pathways also contribute significantly to Nur77 expression (41
). At moderate levels, Nur77 mRNA and protein are rapidly induced in thymocytes stimulated with phorbol ester (7
). This observation can now be interpreted in light of our current results. Indeed, activation of PKD by phorbol ester would induce phosphorylation-dependent nuclear export of HDAC7. Subsequently, this would lead to the establishment of a chromatin environment favorable to Nur77 transcription and transactivation by other Ca2+
-independent coactivators such as ERK5 (42
). Maximal induction of Nur77 would require Ca2+
-dependent inactivation of additional repressors, such as Cabin-1 (10
), and recruitment of Ca2+
) coactivators, such as NFAT. This coordinated control through release of repressors and recruitment of coactivators would constitute a tightly regulated mechanism whereby T cells can activate Nur77 transcription to a high level in a relatively short time.
The ability of recombinant CaMK to induce class IIa HDAC nuclear export in transfected cells suggested that CaMK might be involved in the transduction of extracellular stimuli through HDAC inactivation (15
). CaMKI has been shown to induce nuclear export of HDAC7 when transfected into Hela cells (17
). However, here we provide in vitro and in vivo experimental evidence that signal-responsive kinases other than CaMKs are involved in this process. We identified PKD1 as the kinase responsible for the direct phosphorylation and nuclear exclusion of HDAC7 after TCR stimulation of thymocytes. Interestingly, in these cells, activation of the CaMKs with ionomycin did not alter the subcellular localization of HDAC7, suggesting that HDAC7 is not responsive to the CaMK pathway in T cell hybridomas. This further suggests that class IIa HDACs might be regulated by different signaling pathways depending on the cellular context and/or the extracellular stimuli.
Previous data from us and others supported a role for S155, S321, and S449 in the nuclear exclusion mechanism of HDAC7 (11
). Data presented in this study identified S181 as an additional phosphorylation site in the amino terminus of HDAC7. Although the role of S181 as a true 14-3-3 binding site remains to be directly tested, sequences flanking S181 are very similar to those around S321 and S449 and match the optimal binding motif for 14-3-3. Interestingly, although S155, S321, and S449 are conserved amongst other members of the class IIa, S181 is uniquely present in HDAC7. This could support a model in which each member of the class IIa HDAC might be regulated differentially through phosphorylation.
Intriguingly, all four phosphorylation sites within HDAC7 are not equivalent. We and others have already shown that S155 was the most critical for HDAC7 nucleo-cytoplasmic regulation (11
). Sequence comparison showed that S155 is the most divergent of the four phosphorylation sites (refer to ). In addition, here we show that S155 in vitro phosphorylation by PKD1 is uniquely insensitive to an L150A substitution. The identification of multiple phosphorylation sites in HDAC7 and the fact that they present specific properties raise the interesting possibility that combinatorial phosphorylation of these sites would differentially impact HDAC7 function and regulation.
Class IIa HDACs regulate myogenesis and cardiac hypertrophy (18
). As PKD had previously been implicated in cardiac activity (44
and unpublished data) and hypertrophy (45
), it is intriguing to speculate that it could identically control these biological processes by promoting nuclear export of class IIa HDACs.