The active Protein Kinase C Delta catalytic fragment induces a mitotic checkpoint in human keratinocytes

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Tumor suppressor proteins often perform vital functions in the initiation of apoptosis, as well as limiting proliferation by triggering cell cycle checkpoints. The novel protein kinase C isoform, PKCδ, is a key participant in the UV-induced apoptotic cascade in keratinocytes (KCs), and is lost or inactivated in both chemically and UV-induced skin tumors, consistent with a tumor suppressor function. Retroviral expression of the PKCδ catalytic fragment (PKCδ-cat) resulted in a pronounced accumulation of cells with 4N DNA in both normal human KCs as well as the HaCaT cell line. In addition, PKCδ-cat expression was associated with an increase in the number of polyploid cells, indicative of aberrant mitosis. To identify the cell cycle status of PKCδ-cat expressing KCs, retrovirally infected KCs were released from nocodazole-induced metaphase arrest, PKCδ-cat was activated, and cells were monitored for their ability to exit mitosis. While control KCs progressively divided and entered G1, cells expressing PKCδ-cat were unable to successfully complete mitosis. This indicates a role for PKCδ-cat in delaying progression through late stage mitosis, possibly through induction of the spindle assembly checkpoint or interference with cytokinesis. Expression of PKCδ-cat also induced phosphorylation of histone H3 on Ser 10 (P~H3) in normal KCs, consistent with a mitotic arrest. However these cells did not display typical mitotic morphology as they lacked distinct mitotic spindles and contained uncondensed chromatin. Flow cytometric analysis confirmed the presence of P~H3 positive cells containing 2N DNA suggesting that PKCδ-cat induces phosphorylation of H3 outside of its normal mitotic context. Inhibition of the classical mitotic H3 kinase, Aurora B, failed to prevent this induction confirming a role for PKCδ-cat in a non-canonical pathway of H3 phosphorylation. In fact, PKCδ-cat was able to phosphorylate H3 on Ser10 in an in vitro kinase assay suggesting that PKCδ-cat may be directly phosphorylating H3 in keratinocytes. Histone H3 phosphorylation has been reported to occur during mitotic chromatin condensation as well as transcriptional activation. Whether PKCδ-cat induced H3 phosphorylation is associated with either event, or if PKCδ-cat induced P~H3 represents a novel signaling event remains unclear. Taken together, this data provides evidence for cell cycle checkpoint activation by PKCδ-cat. PKCδ-cat induced cell cycle arrest may function in concert with PKCδ-cat pro-apoptotic effects to prevent the survival and proliferation of cells with damaged genomes.