The pancreas ductal adenocarcinoma (PDAC) is one of the most common types of cancer, accounting for a large number of cancer related deaths. Rapid tumour metastasis is a major problem in pancreatic cancer, and little is known on the molecular events governing this process, however, features like changes in cell shape, modulation of cell-to-cell adhesion, enhanced cell motility and matrix degrading potential seem to be important. In the literature the Protein kinase D (PKD) family of serine/threonine kinases, which consists of 3 structurally related isoforms, PKD1/PKCμ, PKD2 and PKD3/PKCnu has been implicated in the regulation of some of these processes, yet the molecular mechanisms involved largely remained unclear. That's why this study, using PDAC cell lines, focused on the function of PKD in the metastatic progression of cancer cells. In this work, PKD was found to be strongly expressed and also active in Panc89 cells. Focusing on this cell line, PKD was shown to colocalise with F-Actin and respective markers, indicating active Actin remodeling. For example, Arp3, a member of the Actin-related Arp2/3 protein complex, which is responsible for de novo Actin nucleation and dendritic branching of Actin filaments, colocalised with PKD. Further, PKD colocalised with a subcellular pool of Vinculin at the edge of membrane protrusions, again indicating active Actin turnover, possibly at nascent focal complexes, however PKD was not localised to Vinculin-positive mature focal adhesions to the substratum. In addition, PKD localised with Cortactin, which is enriched within lamellipodia and membrane ruffles. It is implicated in the stabilisation of F-Actin branch points and therefore exhibits important functions at the cortical F-Actin cytoskeleton, which are directly linked to cell migration. Surprisingly, PKD was also found to directly bind to F-Actin in vitro. The binding domain was mapped to 46 amino acids in the N-terminal region of PKD. An alignment of the respective PKD sequence indicated highly conserved motifs, both amongst PKD isoforms and also between human, mouse, and Drosophila species, pointing to a general feature of PKDs. Apart from the binding to F-Actin, PKD and Cortactin also interacted biochemically and PKD was shown to phosphorylate Cortactin in vitro at S298, as well as at additional unspecified sites. Unfortunately an in vivo phosphorylation of Cortactin could not be demonstrated up to now. Employing stable Panc89 cell lines, PKD1 impaired 3D cell migration, while for PKD1KD expressing cells, migration was enhanced. These effects can either be explained by a model implicating Cortactin phosphorylation by PKD in the regulation of F-Actin turnover and rigidity, or by the phosphorylation of yet unknown PKD substrates at the respective F-Actin-rich structures, negatively regulating cell migration. Since mutation of the potential Cortactin phosphorylation site Ser298 to an alanine residue also increased cell migration in stable Panc89 cells, Cortactin might be a potential target of PKD in the regulation of F-Actin dynamics.
PKD has also been implicated in the regulation of Ca2+-dependent cell-to-cell adhesion. PKD2KD and, to lesser extent, PKD1KD, strongly enhanced cell-to-cell adhesion of stable Panc89 cells, whereas E-Cadherin expression in the respective parental cell lines was reduced. At least in the case of PKD2KD cells, aggregation was only partially dependent on E-Cadherin, pointing to the expression of additional Cadherin isoforms. In the PKD2 expressing cell line, aggregation was very weak, in some assays even resembling the vector control. This phenotype could be correlated with processed E-Cadherin fragments in the supernatant of PKD2 expressing cells, which have been implicated in the literature with the inhibition of cell-to-cell adhesion of cancer cell lines, thereby also increasing their invasive potential. Initial results demonstrated that the E-Cadherin fragments from the supernatant of stable PKD2 expressing Panc89 cells were processed by a serine-protease, possibly Plasmin or cationic Trypsin. How the PKD2 isoform is implicated in this process remains to be investigated further. Yet, these findings are in line with data obtained from expression profiling experiments with the respective stable Panc89 cells, indicating that the urokinase-like Plasminogen-activator-receptor (PLAUR) is strongly up-regulated in PKD2 expressing cells, triggering the activation of the uPA-Plasminogen-Plasmin-cascade, which has been demonstrated to be involved in the processing of E-Cadherin.
During the course of this work novel aspects concerning the role of PKD in cell migration and cellular adhesion were revealed. The localisation of PKD at the F-Actin cytoskeleton and its in vitro binding to F-Actin implicate possible functions for the PKD protein family in the regulation of F-Actin dynamics, which might influence cell adhesion and motility.