Date of Award

2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biochemistry Program

Abstract

Protein kinase C delta (PKCδ) is an essential component of the intrinsic apoptotic program. Following DNA damage, such as exposure to UV radiation, PKCδ is cleaved in a caspase-dependent manner, generating a constitutively active catalytic fragment (PKCδ-cat) which is necessary and sufficient for keratinocyte (KC) apoptosis. We found that in addition to inducing apoptosis, expression of PKCδ-cat caused a pronounced G2/M cell cycle arrest in both primary human KCs and immortalized HaCaT cells. Consistent with a G2/M arrest, PKCδ-cat induced phosphorylation of Cdk1 (Tyr15), a critical event in the G2/M checkpoint. Treatment with the ATM/ATR inhibitor caffeine was unable to prevent PKCδ-cat induced G2/M arrest, suggesting that PKCδ-cat is functioning downstream of ATM/ATR in the G2/M checkpoint. To better understand the role of PKCδ and PKCδ-cat in the cell cycle response to DNA damage, we exposed wild type and PKCδ null MEFs to UV radiation. Wild type MEFs underwent a pronounced G2/M arrest, Cdk1 phosphorylation, and induction of apoptosis following UV exposure, while PKCδ null MEFs were resistant to these effects. Expression of PKCδ-GFP, but not caspase-resistant or kinase inactive PKCδ, was able to restore G2/M checkpoint integrity in PKCδ null MEFs. The function of PKCδ in the DNA damage-induced G2/M cell cycle checkpoint may be a critical component of its tumor suppressor function. In light of recent reports suggesting the importance of nuclear localized PKCδ in the apoptotic response, we examined changes in PKCδ sub-cellular localization following exposure to UV radiation. Using a PKCδ-GFP fusion protein we determined that nuclear PKCδ was present after UV exposure, and that it formed foci coinciding with regions of intense DAPI staining, suggesting localization to condensed chromatin. We have previously shown that expression of the PKCδ catalytic fragment induced phosphorylation of Histone H3 (Ser10), an important event for mitotic chromatin condensation. Interestingly, PKCδ-cat induction of P~H3(S10) was not prevented by inhibition of the mitotic H3 kinase Aurora B, and was induced throughout all phases of the cell cycle, supporting the idea that this event is distinct from the P~H3(S10) induction during mitosis. In vitro phosphorylation studies using recombinant proteins demonstrated that PKCδ-cat is capable of directly phosphorylating H3 on Ser10 raising the possibility that PKCδ may function as a Histone H3 kinase in the cell. Using confocal microscopy we found that UV-induced PKCδ-GFP foci co-localized with regions of positive P~H3(S10) staining. To avoid any artifacts associated with over-expression of the PKCδ-GFP fusion protein, we confirmed the formation of endogenous PKCδ foci after UV radiation in normal keratinocytes. This novel chromatin modifying activity of PKCδ may be an important component of its tumor suppressor function for UV induced skin cancers in the human epidermis.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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