Antigen receptor regulated signalling pathways in PKD-null DT40 B cells
To further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells. Initial experiments revealed that surface expression of the BCR was reduced in PKD1/3-/- (and in PKD1/3-/-:Flag-PKD3+ve) cells compared to wild-type DT40 B cells (Fig. 3A and data not shown). Nevertheless, BCR-crosslinking of PKD1/3-/- cells was sufficient to induce the activation of a number of signalling cascades, similar to that observed in wild-type cells (Fig. 3B). Hence, BCR-induced activation of the Akt, mTOR/p70 S6 kinase (as shown by S6 ribosomal protein phosphorylation) and MAPK signalling pathways was clearly detectable in PKD1/3-null B cells (Fig. 3B). Furthermore, enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1/3-null B cells (data not shown). We did observe that the strength of BCR (but not phorbol ester)-induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1/3-/- B cells compared to wild-type B cells (Fig. 3B). One interpretation of this data is that PKD enzymes may modulate Erk activation. Indeed, PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines [33-35]. However, BCR-induced Erk phosphorylation was also reduced in PKD1/3-/--Flag-PKD3+ B cells (data not shown) suggesting that reduced BCR levels on the surface of PKD1/3-/- (and PKD1/3-/--Flag-PKD3+) B cells may itself impact on the strength of activation of this specific intracellular signalling pathway. 
To search for other potential PKD targets that may show defective regulation in PKD1/3-/- DT40 B cells, we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes (LxRxxpS/T) [36]. As shown in Fig. 3C, phorbol ester- and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1/3-/- cells and is therefore independent of PKD enzymes. However, pretreatment of both wild-type and PKD1/3-/- DT40 B cells with GF109203X, a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS/T motifs. Thus loss of PKD1/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS/T motifs. This result is perhaps not surprising as LxRxxS/T motifs also act as good substrates for other serine/threonine kinases such as MAPKAPK2. However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera. 
BCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival, proliferation and function. In this context, it has been proposed that PKD family members control of gene transcription through activation of the NFkappaB transcription factor. Thus, PKD-mediated activation of NFkappaB occurs downstream of a variety of different signals, including mROS/oxidative stress, lysophosphatidic acid and the Bcr-Abl oncogene [17,21,23,30,37]. Furthermore, expression of an activated PKD1 mutant enhances HPK1-mediated NFkappaB activation [38]. In B cells, NFkappaB is known to be regulated via DAG and PKCbeta [39,40] but whether PKDs are key intermediaries for NFkappaB regulation has not been explored. 
The data (Fig. 4A) show that NFkappaB transcriptional activity was strongly induced in both wild-type and PKD1/3-/- DT40 B cells in response to either phorbol ester or BCR stimulation. In contrast, BCR and phorbol ester-induced NFkappaB transcriptional activity was abolished in PKCbeta-/- DT40 B cells (Fig. 4A), although strong activation of PKD kinases (as assessed by autophosphorylation of PKD1 at S916) was observed in the PKCbeta-/- cells (Fig. 4B). Thus, PKD kinases are neither essential nor sufficient to mediate BCR-induced NFkappaB activation in DT40 B cells and hence do not participate in DAG/PKC mediated control of NFkappaB. 
