Manipulation of PKC isozymes by RNA interference and inducible expression of PKC constructs.

Protein kinase C (PKC), a family of serine/threonine kinases, plays an important role in apoptosis. Several members of the PKC family act as substrates for caspases. In addition, PKCs can also regulate caspase activation and cell death by apoptosis. The cleavage of PKCs separates the regulatory domain from the catalytic domain. The full-length, the catalytic domain, and the regulatory domain of PKC family members may have distinct function in apoptosis. Delineating the role of protein kinase C (PKC) isozymes in apoptosis has been challenging because of the lack of selective inhibitors of PKC isozymes and difficulty in generating stable cell lines expressing pro-apoptotic PKC isozymes. In this chapter, we describe the use of RNA interference (siRNA) technology and tetracycline-inducible expression of PKC isozymes to study their function in apoptosis.

Protein kinase C-epsilon protects MCF-7 cells from TNF-mediated cell death by inhibiting Bax translocation.

We have previously shown that protein kinase Cepsilon (PKCepsilon) acts as an antiapoptotic protein and protects breast cancer MCF-7 cells from tumor necrosis factor-alpha (TNF)-mediated apoptosis. In the present study, we have investigated the mechanism by which PKCepsilon inhibits TNF-induced cell death. Overexpression of wild-type PKCepsilon (WT-PKCepsilon) in MCF-7 cells decreased TNF-induced mitochondrial depolarization. Depletion of Bax by small interfering RNA (siRNA) attenuated TNF-induced cell death. Overexpression of PKCepsilon in MCF-7 cells decreased dimerization of Bax and its translocation to the mitochondria. Knockdown of PKCepsilon using siRNA induced Bax dimerization and mitochondrial translocation. PKCepsilon was coimmunoprecipitated with Bax in MCF-7 cells. These results suggest that PKCepsilon mediates its antiapoptotic effect partly by preventing activation and translocation of Bax to the mitochondria.

Protein kinase Cepsilon makes the life and death decision.

Cancer is caused by dysregulation in cellular signaling systems that control cell proliferation, differentiation and cell death. Protein kinase C (PKC), a family of serine/threonine kinases, plays an important role in the growth factor signal transduction pathway. PKCepsilon, however, is the only PKCepsilon isozyme that has been considered as an oncogene. It can contribute to malignancy by enhancing cell proliferation or by inhibiting cell death. This review focuses on how PKCepsilon collaborates with other signaling pathways, such as Ras/Raf/ERK and Akt, to regulate cell survival and cell death. We have also discussed how PKCepsilon mediates its antiapoptotic signal by altering the level or function of pro- and antiapoptotic Bcl-2 family members.

Stimulation of insulin-like growth factor (IGF) binding protein-3 synthesis by IGF-I and transforming growth factor-alpha is mediated by both phosphatidylinositol-3 kinase and mitogen-activated protein kinase pathways in mammary epithelial cells.

IGF binding protein (IGFBP)-3 is an important regulator of mammary epithelial cell (MEC) growth and can enhance the ability of both IGF-I and epidermal growth factor ligands such as TGFalpha to stimulate MEC proliferation. Here we investigate the role of the phosphatidylinositol-3 kinase (PI3K) and MAPK pathways in the regulation of IGFBP-3 expression by IGF-I and TGFalpha in bovine MECs. Both growth factors stimulated DNA synthesis, although IGF-I was the stronger mitogen. IGF-I and TGFalpha also stimulated IGFBP-3 mRNA and protein levels. TGFalpha stimulated rapid, transient activation of Akt that was maximal at 5 min and diminished by 15 min. In contrast, IGF-I-induced Akt activation was maximal between 15 and 90 min and was sustained for 6 h. Although ERK 1/2 was maximally stimulated by TGFalpha between 5 and 15 min, IGF-I did not stimulate discernible activation of ERK 1/2. In addition, TGFalpha but not IGF-I induced rapid phosphorylation of Shc, whereas only IGF-I activated insulin receptor substrate-1. Pretreatment with the PI3K inhibitor LY294002 or knockdown of p85 with small interfering RNA inhibited IGF-I or TGFalpha-stimulated IGFBP-3 expression. Similarly, MAPK kinase-1 inhibitors PD98059 and U0126 each abolished TGFalpha-stimulated increases in IGFBP-3 mRNA levels. In contrast to TGFalpha, IGF-I retained the ability to partially increase IGFBP-3 mRNA levels in the presence of MAPK kinase-1 inhibitors, indicating that IGF-I may activate alternative substrates of the PI3K pathway that are involved in IGFBP-3 regulation. In conclusion, stimulation of IGFBP-3 mRNA levels by mitogens is regulated through both the PI3K and MAPK pathways in bovine MECs.