We set out to generate a powerful genetic knockout system for studying PKD function in lymphocytes. As previously described, both PKD1 and PKD3 family members are expressed in DT40 B cells, where they are strongly and rapidly activated in response to BCR engagement (22
). We could not detect expression of the PKD2 protein in DT40 cell extracts, however (data not shown). Therefore, we made DT40 B cells lacking either PKD1 or PKD3 by targeting critical exons encoding key motifs essential for enzymatic activity (Fig. ) (see Materials and Methods). As both PKD1 and PKD3 can phosphorylate similar peptide substrates in vitro, raising the possibility of functional redundancy in vivo, we also generated PKD1/PKD3 double deficient DT40 B cells (Fig. ) (see Materials and Methods).
Integration of the targeting constructs into correct gene loci was confirmed by the absence of detectable PKD1 and/or PKD3 proteins in the resulting knockout cell lines, as shown by Western blot analysis of whole-cell extracts with isoform-specific antibodies (Fig. ). The PKD1 polyclonal antibody used was directed against a C-terminal epitope of PKD1/2 that is not present in PKD3. As the amino acid sequence of this epitope is fully conserved between PKD1 and PKD2, this confirmed the absence of PKD2 expression in the DT40 B cell line. As shown in Fig. , loss of either PKD1 or PKD3 partially reduces the amount of detectable, phosphorylated PKD in phorbol ester-treated cell extracts, indicating that PKD1 and PKD3 both make significant contributions to the total PKD pool present in DT40 B cells, whereas no activated PKD was detectable in the PKD1/PKD3 double deficient DT40 B cells (Fig. ). These data reveal that the expression and activation of PKD1 and PKD3 are independently regulated in vivo, as the expression and phorbol ester-induced phosphorylation of PKD1 was intact in the PKD3−/− cells and vice versa.
Initial characterization revealed that the PKD1−/− and PKD3−/− DT40 B cells were generally normal: cell surface BCR levels were similar to those of wild-type DT40 B cells, and population-doubling times were similar to those of wild-type cells (within the range of 11 to 13 h) (data not shown). In addition, long-term culture of PKD1−/−3−/− cells showed that the absence of any PKD expression had no adverse effect on cellular growth rates (data not shown), indicating that loss of the total cellular PKD pool does not effect basal DT40 cell proliferation/survival. Further characterization of the PKD1−/−3−/− cells will be described elsewhere.
To date, one proposed downstream target for PKDs in lymphocytes is the class II HDACs (7
). The BCR regulates key transcriptional changes in B lymphocytes that control effector function, and a large body of work has focused on the direct regulation of transcription factor activity by the BCR. However, little is known about BCR-mediated signaling events that regulate chromatin acetylation/deacetylation. Inhibitors of HDACs have indicated a role for HDACs in controlling B-cell differentiation and survival (2
). HDACs are also implicated in the repression of specific lytic viral genes and upregulation of latent membrane protein 1 during latent Epstein-Barr virus infection of B cells (3
). However, little is known about how HDACs are regulated in B lymphocytes; in particular, it is unclear whether the BCR is functionally coupled to class II HDACs in vivo.
We therefore initially investigated whether activation of the BCR complex or DAG signals (which would activate a PKC-PKD signaling pathway) could regulate class II HDACs in B cells. To assess class II HDAC regulation, we transiently expressed a GFP-tagged HDAC5 protein in avian DT40 B cells or in murine A20 B cells and used a phosphospecific HDAC5 antibody that specifically recognizes HDAC5 molecules phosphorylated at the regulatory serine 259 site (46
). Low-level basal phosphorylation of HDAC5 at this key site was consistently observed in resting DT40 B cells, which was rapidly and strongly enhanced after BCR cross-linking or following stimulation with the DAG-mimetic PDBu, and remained elevated over a prolonged time period (Fig. ). Both the basal and BCR-/PDBu-induced phosphorylations of HDAC5 were lost when a HDAC5 mutant (in which the regulatory serine residues were replaced by nonphosphorylatable alanine residues) was expressed in DT40 cells (data not shown). Antigen receptor activation, or DAG signals alone, also induced robust and sustained phosphorylation of HDAC5 in a murine A20 B-cell line (Fig. ).
FIG. 2. Regulation of HDAC5 phosphorylation by the B-cell antigen receptor complex. (A and B) Avian DT40 B cells or (C) murine A20 B cells were transfected with a GFP-HDAC5 expression construct as described in Materials and Methods. Twenty-four hours after transfection, (more ...)
Class II HDAC-repressed gene targets in B cells are unknown. Therefore, to investigate the role of PKD enzymes in the regulation of class II HDACs, we used a reporter construct linked to the Nur77 promoter (52
), which is regulated by class II HDACs following antigen receptor triggering in T cells (7
). As demonstrated in Fig. , the activity of the Nur77 reporter was low in untreated wild-type DT40 B cells and was strongly induced in response to either phorbol ester treatment or BCR cross-linking (Fig. ). Strikingly, activation of the Nur77 reporter in response to either phorbol esters or BCR triggering was clearly defective in PKD1−/−
B cells (Fig. ). These data suggest that PKD enzymes are required for the correct regulation of class II HDACs in DT40 B cells. Currently, class II HDAC-repressed gene targets in B cells that are regulated by PKDs are unknown, but this is under investigation.
FIG. 3. Analysis of class II HDAC function and phosphorylation in PKD1−/−3−/− double knockout DT40 B-cell lines. (A) PKD1−/−PKD3−/− double knockout cells, transfected with the Nur77-CAT reporter (more ...)
Many signaling molecules other than class II HDACs have been shown to regulate the Nur77 promoter, including Creb and NF-κB (6
). Therefore, to determine if PKD enzymes are indeed required to directly regulate class II HDACs, we investigated whether PKD enzymes were required for the regulation of HDAC5 phosphorylation in DT40 B cells. As discussed previously, inducible phosphorylation of the regulatory serine residues in class II HDACs is required to relieve their repressive effects on gene transcription. As observed above, low basal phosphorylation of HDAC5 was detectable in untreated, wild-type DT40 B cells, which was rapidly enhanced in a sustained manner following BCR stimulation (Fig. ). Strikingly, both the basal and BCR-induced HDAC5 phosphorylations were almost completely abolished in PKD1−/−
B cells, even after prolonged stimulation (Fig. ). Phorbol ester-induced HDAC5 phosphorylation was also almost completely abolished in the PKD1−/−
B cells (see Fig. and data not shown). We went on to investigate the requirement for PKD enzymes in the regulation of another class II HDAC, HDAC7. Similar to our observations for HDAC5, basal phosphorylation of HDAC7 was consistently observed in wild-type DT40 B cells, which was enhanced in response to phorbol ester treatment or BCR triggering (Fig. ). Again, both basal and stimulus-induced phosphorylations of HDAC7 were significantly reduced in PKD1−/−
B cells (Fig. ). However, the BCR could regulate other downstream signals in PKD1−/−
B cells, as assessed by the activation of the Akt/PKB serine kinase (Fig. ).
FIG. 5. Expression of either PKD1 or PKD3 is sufficient to rescue HDAC5 and HDAC7 phosphorylation in PKD1−/−3−/− double knockout DT40 B cells. (A) Basal phosphorylation of HDAC5 is restored in PKD1−/−3−/− (more ...)
Given the dysregulation of HDAC5 and HDAC7 in PKD-null DT40 B cells, we went on to address the issue of functional redundancy between PKD1 and PKD3 in this context. To date, the majority of work indicating functional roles for PKD enzymes in vivo have focused on the PKD1 isoform. Although all three PKD isoforms exhibit similar sequence specificity requirements in vitro, they may have distinct functions in vivo, particularly as they can be found localized in distinct subcellular compartments within the same cell (reference 40
and data not shown [M. Spitaler and D. A. Cantrell]). We initially analyzed HDAC5 phosphorylation in single-knockout DT40 B cells lacking either PKD1 or PKD3. We observed that BCR- and phorbol ester-induced phosphorylation of HDAC5 in the PKD3−/−
B cells was comparable to that observed in wild-type DT40 cells (Fig. ). HDAC5 phosphorylation in the PKD1−/−
cells appeared elevated basally and was not further enhanced in response to BCR triggering, although the stronger stimulus PDBu did increase HDAC5 phosphorylation in these cells (Fig. ). These data indicate that loss of a single PKD isoform does not affect HDAC5 phosphorylation: the essential role for PKDs as HDAC kinases in DT40 B cells is revealed only by the loss of both PKD1 and PKD3.
FIG. 4. HDAC5 phosphorylation remains intact in single PKD1−/− or PKD3−/− knockout DT40 B cells. Wild-type (WT) and PKD1−/− or PKD3−/− knockout DT40 cells were transfected with a GFP-HDAC5 expression (more ...)
To further investigate the functional redundancy of PKD1 and PKD3 in the control of HDAC5 phosphorylation, we expressed a PKD3 transgene (under the control of a doxycycline-inducible promoter) in our PKD1−/−3−/− B cells. We observed that basal phosphorylation of HDAC5 was restored in PKD1−/−3−/− cells that inducibly expressed the Flag-PKD3 transgene but not in PKD1−/−3−/− cells that were cultured in the absence of doxycycline and thus lacked Flag-PKD3 expression (Fig. , −Dox, +Dox). Importantly, phorbol ester- and BCR-induced HDAC5 phosphorylation was also restored to wild-type levels in PKD1−/−3−/− DT40 cells that inducibly expressed the Flag-PKD3 transgene (Fig. , +Dox) but again, not in PKD1−/−3−/− cells cultured in the absence of doxycycline (Fig. , −Dox). We also observed that doxycycline-induced expression of the Flag-PKD3 transgene was sufficient to increase basal and BCR/phorbol ester-induced phosphorylation of HDAC7 in the PKD1−/−3−/− cells (Fig. , −Dox, +Dox).
Transient reconstitution of the PKD1−/−3−/− double knockout cells with wild-type GFP-tagged PKD1 also rescued both basal and phorbol ester/BCR-induced HDAC5 phosphorylation compared to control, GFP-transfected cells (Fig. ). Strikingly, expression of kinase-dead PKD1 in the double knockout cells did not rescue HDAC5 phosphorylation (either in untreated or in stimulated cells), whereas expression of constitutively activated PKD1 resulted in a high basal phosphorylation of HDAC5 that could not be further enhanced by external stimuli (Fig. ). Identical results were observed when the PKD1/3 double knockout cells were transfected with activated and kinase-dead versions of PKD3 (data not shown).
Phosphorylation of class II HDACs controls their nuclear export and thus dictates whether HDACs can exert their repressive effects on target gene expression. Having shown that PKDs were required for class II HDAC phosphorylation, we therefore looked to see if there was a role for PKDs in controlling HDAC cellular localization. We quantified the subcellular distribution of GFP-tagged HDAC5 or HDAC7 in unstimulated and stimulated wild-type and PKD1−/−3−/− DT40 B cells by confocal microscopy. Predominantly, unstimulated wild-type DT40 B cells showed nuclear localization of GFP-HDAC5 and GFP-HDAC7 (Fig. ). Following BCR engagement or phorbol ester treatment, both HDAC5 and HDAC7 were observed to consistently redistribute from the nucleus to the cytosol. However, there was no significant nuclear exclusion of HDAC5 or HDAC7 in BCR- or phorbol ester-stimulated PKD1−/−3−/− DT40 B cells (Fig. ). The data in Fig. thus show that in PKD1/3-null B cells, HDAC5 and HDAC7 do not redistribute from the nucleus to the cytosol in response to BCR or phorbol ester signaling.
FIG. 6. Class II HDAC nuclear export in PKD1−/−3−/− double knockout DT40 B-cell lines. Wild-type (WT) or PKD1−/−3−/− double knockout cells (cultured with or without doxycycline to induce Flag-PKD3 (more ...)
We went on to determine whether reintroduction of PKD3 in the PKD1/3 knockout cells could restore normal HDAC5/7 localization patterns. As shown in Fig. , triggering of the BCR or phorbol ester stimulation in PKD1−/−3−/− B cells that expressed a PKD3 transgene could now induce nuclear exclusion of HDAC5 and HDAC7. Thus, the rescue of HDAC5 and HDAC7 phosphorylation after reexpression of PKD3 in the double knockout cells was accompanied by normal nuclear exclusion of these class II HDACs in response to phorbol ester or BCR stimulation (Fig. versus Fig. ).