Characterisation of human MRCK protein kinase and its signalling role in cancer cells: regulation of cell invasion
This thesis describes the characterisation of human MRCKcc (myotonic dystrophy kinase-related Cdc42-binding kinase) and the signalling role of MRCK in tumour cell invasion.
The first results chapter reports the identity and sequence of the human isoform of MRCKa. It is shown that previously reported expression of a sequence known as PK428 in breast tumours with poor prognosis represents MRCKa expression. PK428 is identified as a partial cDNA of the previously uncloned human MRCKa. The cloning of several splice-variants of full-length MRCKa is described. MRCKa expression is also analysed in human tumour samples, but the data are not conclusive enough to establish MRCKa expression as a prognostic marker. Subsequent work details the functional role of MRCKa and MRCK? in mediating invasion of three-dimensional extracellular matrices by tumour cell lines. In tumour cells that exhibit an elongated morphology, abrogation of signalling of both MRCKa/?, by RNA interference, and ROCK (Rhokinase), with the small-molecule inhibitors Y-27632 or HA-1077, results in inhibition of invasion. This is associated with a collapsed cellular morphology and abrogation of phosphorylation of both the myosin targeting subunit (MYPT1) of myosin light chain phosphatase and of myosin light chain (MLC2). Effects on morphology and invasion are recapitulated with blebbistatin, a specific inhibitor of actomyosin contractility. This suggests that MRCK and ROCK have a redundant role in promoting invasion through regulation of myosin activity. In a cell line with a rounded morphology in threedimensional matrices, switching between three morphological states is identified - rounded, elongated and collapsed, in order of decreasing actomyosin contractility. In this cell line, MRCK and ROCK have a redundant role in maintaining the elongated morphology but ROCK alone signals the high level of contractility that drives the rounded morphology.