In view of the heterogeneity of PKN isoform expression, to dissect the relative potential of isoforms to control migratory behaviour we screened 22 cell lines for their patterns of PKN isoform expression to establish a suitable model. All cell lines express at least one isoform and the relative levels of individual PKNs showed considerable variation in particular for PKN3. In the tumour cell line 5637 with relatively enriched expression of PKN2 compared to other cells tested, we were able to detect a strong phenotype just by depleting PKN2, with PKN1 depletion having a weaker effect and PKN3 none at all. The results show that unlike the situation reported in prostate cancer cells 
, PKN1 and PKN2 can contribute both to cell migration in wound healing assays as well as 3D invasion of tumour cells in transwells. Thus the PKN isoform contribution to cell motility in part reflects their expression in particular cell systems.
Using the 5637 cell model which is enriched for PKN2 expression, rescue of endogenous PKN2 depletion with the ectopic expression of an siRNA resistant PKN2 confirmed the specificity of the observed effects of knock down. Importantly, this phenotype could not be rescued by simple overexpression of PKN1 or 3. Since we had concluded that in principle any one of these PKN isoforms can contribute to migratory behaviour (albeit in other cell types), this inability to rescue with a heterologous isoform suggested that specific regulatory inputs might distinguish their action. To test this, we constructed a series of chimeric PKN molecules and derived stable cell lines for which we could assess the migratory behaviour ±endogenous PKN2 knock-down. These studies indicated that the regulatory domain of PKN2 was critical for rescue of migration in 5637 cells. Thus, besides full length PKN2 only the chimera PKN2/1 was able to rescue the migration defect on PKN2 depletion in these bladder cells; this was despite the expression of higher levels of other chimeras. The selective ability of PKN2 to rescue over PKN1, and of the PKN2/1 chimera to rescue compared to others, is indicative of specific regulatory inputs acting through the PKN2 regulatory domain to effect its action in migration in this cell model.
The evidence here indicates that although expression patterns seem to reflect the ability of PKN isoforms to influence migration, the patterns themselves are overlaid by the exertion of regulatory inputs that display specificity – in 5637 cells these inputs are principally PKN2 directed. This heterogeneity of regulation is consistent with the findings that there are distinct lipid and Rho family GTPase inputs to this family of kinases. The kinase activity of PKN1 for example is highly stimulated by phosphatidylinositol(4,5)bisphosphate, phosphatidylinositol(3,4,5)trisphosphate, lysophosphatidic acid and arachidonic acid 
. PKN2 as well as PKN3 are less sensitive to arachidonic acid than PKN1 
. With respect to GTPases, PKN1 was shown to bind Rac and RhoA 
where the HR1a motif binds both Rac and RhoA with high affinity and the HR1b motif binds Rac with much higher affinity than RhoA 
. PKN1 HR1b is also able to bind RhoA GDP 
. PKN2 binds and responds to Rac as does PKN1, but the regulation of the kinase by Rho GTPases is still controversial. Quilliam and colleagues 
found that RhoA binds PKN2 in a GTP dependent manner, but Vincent and Settleman reported that PKN2 binds Rho GDP and Rho GTP with similar kinetics 
. The extreme C-terminus of PKN2 is also implicated in Rho GTP binding 
, although this specific amino acid sequence seems dispensible for our rescue experiments, since the PKN2/1 chimera will partially rescue. PKN2 and PKN3 contain a proline rich region before the catalytic domain and binding of adaptor proteins known to interact with PKN2 such as Nck or Grb4 has been mapped to this proline rich region 
. These interactions are not conserved with PKN1. These distinctions in upstream regulators are consistent with the conclusion drawn here that cell-specific regulatory domain inputs determine PKN action in migration.
The single orthologue of the mammalian PKN proteins has been shown to be essential for the development of the Drosophila embryo. Drosophila PKN-/-
are embryonic lethal due to defects in dorsal closure 
, a process which involves migration of the lateral epidermal flanks and dynamic changes in cell shape to close a hole in the dorsal epidermis occupied by the amnioserosa. The dorsal closure process has been linked to migration of epithelial cells in other organisms and in a similar manner Rho GTPases have been shown to play an important role 
. Resonating with the results described in this paper, Betson & Settlement who analyzed the ability of PKN mutants to rescue the dorsal closure defect in Drosophila also found a requirement for the N-terminal domain of PKN in particular to respond to Rho signalling 
. Interestingly, the kinase domain of PKN could be replaced by that of Protein Kinase C53E which is similar to mammalian PKCα 
; as observed here, these related AGC kinase domains seem more interchangeable than their regulatory domains.
Migration of 5637 bladder cells was examined previously by Koivunen and colleagues, who demonstrated that cell migration of this epithelial cell line was strongly inhibited by the application of the conventional PKC inhibitor Gö6976. This inhibitor caused translocation of β1 and β4 integrins as well as an increased number of adherens junctions and desmosomes. Together with the data from 
these findings suggest that PKN and PKCα/β might be linked to the same signalling cascade controlling cell migration.
Migration of cells can occur through individual cell movement in a manner often referred to as amoeboid migration or in a more extended morphology, as well as in a form of collective migration; different requirements are associated with distinct movement behaviours 
. Collective migration provides the active and passive translocation of mobile and non-mobile cells due to groups of cells being held together by cell adhesion molecules such as E-cadherin. Interestingly, PKN2 has been implicated in the regulation of cell-cell adhesion 
. Upon Rho stimulation PKN2 either directly or indirectly activates Fyn tyrosine kinase, which in turn phosphorylates β-catenin that binds to E-cadherin and leads to its translocation to cell-cell contacts and increased cell adhesion. The bladder tumour 5637 cells migrate in sheets in the wound assays employed here. It is conceivable that depletion of PKN2 could cause disturbances in cell-cell adhesion and hence slow down migration of these cells in wound healing assays. In adhesion assays of PKN1 and PKN2 depleted cells on different substrates such as collagen and fibronectin, we found that the ability and number of cells adhering to these substrates was no different to luciferase siRNA control cells (data not shown). However, by using immunoflourescene, we noted that focal adhesions seemed larger in cells lacking PKN1 or PKN2 than in control cells (unpublished). Knock-down of the PKN1 downstream target phospholipase D in HeLa cells, also results in increased focal adhesions 
. Furthermore, 5637 cells when stimulated with proepithelin have been shown to migrate through engagement of a paxillin, FAK and ERK pathway 
. PKN1 was shown to bind the actin bundling protein α-actinin in a phosphatidylinositol 4,5bisphosphate dependent manner 
. α-actinin helps to shape dorsal and ventral stress fibers which are anchored with one or both ends in focal adhesions 
. Disruption of the PKN-actinin interaction could result in defective focal adhesions and impair cell motility. PKN isoforms are also implicated as upstream kinases of mitogen activated kinases such as p38 (
). It is possible therefore, that cells depleted of PKN1 and PKN2 have modified focal adhesions that make them adhere stronger to the substrate and might result in a delayed detraction of the cell body during cell migration.
In conclusion, PKN3 is not alone in the PKN family in playing a role in migration. In 5637 cells PKN2 depletion alone inhibits 2D and 3D migration. As the case grows for PKN isoforms as targets for intervention in invasive disease, consideration will need to be given to targeting multiple isoforms in some if not all situations.