Elevated expression of protein kinase Cδ induces cell scattering upon serum deprivation

Abstract

SummaryTumor metastasis might be evoked in response to microenvironmental stress, such as a shortage of oxygen. Although the cellular response to hypoxia has been well established, we know little about how tumors adapt themselves to deprivation of growth factor. Protein kinase Cd (PKCd), a stress-sensitive protein kinase, has been implicated in tumor progression. In this study, we demonstrate that elevated expression of PKCd in Madin-Darby canine kidney cells induces a scatter response upon serum starvation, a condition that mimics growth-factor deprivation. Serum starvation stimulates the catalytic activity and Y311 phosphorylation of PKCd through reactive oxygen species (ROS) and the Src family kinases. Mutation of PKCd at Y311 and Y322, both of which are phosphorylation sites for Src, impairs its activation and ability to promote cell scattering upon serum deprivation. Once activated by ROS, PKCd itself activates ROS production at least partially through NADPH oxidase. In addition, the c-Jun N-terminal kinase is identified as a crucial downstream mediator of ROS and PKCd for induction of cell scattering upon serum deprivation. We demonstrate that the C1B domain of PKCd is essential not only for its localization at the Golgi complex, but also for its activation and ability to induce cell scattering upon serum deprivation. Finally, depletion of PKCd in human bladder carcinoma T24 cells restores their cell-cell contacts, which thereby reverses a scattered growth pattern to an epithelial-like growth pattern. Collectively, our results suggest that elevated expression of PKCd might facilitate the scattering of cells in order to escape stress induced by growth-factor deprivation. (B)An equal amount of whole cell lysate from neomycin-resistant control MDCK cells (neo) and those stably expressing HA-PKCd or HA-FAK was analyzed by immunoblotting with anti-HA. (C)MDCK cells as described for A were allowed to grow as cell colonies in medium supplemented with 10% serum. Subsequently, they were maintained in medium with (+) or without (-) 10% serum and monitored by time-lapse video-microscopy. Representative micrographs at 0, 24, 30 and 36 hours are shown. Scale bars: 150mm. (D)Cell colonies formed by MDCK cells stably expressing HA-PKCd or HA-FAK were maintained in medium with (+) or without (-) 10% serum and monitored by time-lapse video-microscopy. Representative micrographs at 0, 24, 30 and 36 hours are shown. Scale bars: 150mm. (E)MDCK cells stably expressing GFP-PKCd were allowed to grow as colonies and then maintained in medium containing different amounts of serum. A cell colony was judged as a 'scattered' one when one third of the cells in it had lost contact with their neighbors and exhibited a fibroblast-like phenotype. The percentage of scattered colonies in total counted colonies (n>200) was measured. Values (means ± s.d.) are from three independent experiments. (F)MDCK cells stably expressing HA-PKCd were allowed to grow as colonies and then maintained in medium containing different amount of serum. Th...

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