Protein Kinase C Negatively Regulates Akt Activity and Modifies UVC-induced Apoptosis in Mouse Keratinocytes

Luowei Li,Keeran Sampat,Nancy Hu,Julia Zakari,Stuart H Yuspa

doi:10.1074/jbc.m512167200

Abstract

Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCdelta and PKCepsilon from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCdelta and PKCepsilon were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCalpha enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCdelta and PKCepsilon provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell survival pathways in response to external stimuli.

Highlights

Akt/protein kinase B (PKB),2 a serine/threonine kinase, is an important regulator of cell proliferation and survival
These results suggested that activation of the protein kinase C (PKC) signaling pathway negatively regulates basal and stimulated Akt activity and could potentially modify the cell survival pathway in keratinocytes
Our present study has indicated that activation of PKC reduces both phosphorylation of Akt on Ser-473 and Akt catalytic activity in mouse keratinocytes under both basal and IGF-1-stimulated conditions

Summary

Introduction

Akt/PKB,2 a serine/threonine kinase, is an important regulator of cell proliferation and survival. When keratinocytes were pretreated with TPA (250 nM) for 15 min, IGF-1- and EGF-induced Akt phosphorylation (Fig. 2B) on Ser-473 was substantially reduced. Constitutive and IGF-1-induced Akt kinase activities were inhibited by TPA pretreatment of keratinocytes when tested in an in vitro kinase assay from total cell lysates (Fig. 2C).

Results

Conclusion