Protein Kinase Cδ Is Required for Survival of Cells Expressing Activated p21RAS

Shuhua Xia,Lora W Forman,Douglas V Faller

doi:10.1074/jbc.m610225200

Shuhua Xia, Lora W Forman + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m610225200

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Abstract

Inhibition of protein kinase C (PKC) activity in transformed cells and tumor cells containing activated p21(RAS) results in apoptosis. To investigate the pro-apoptotic pathway induced by the p21(RAS) oncoprotein, we first identified the specific PKC isozyme necessary to prevent apoptosis in the presence of activated p21(RAS). Dominant-negative mutants of PKC, short interfering RNA vectors, and PKC isozyme-specific chemical inhibitors directed against the PKCdelta isozyme demonstrated that PKCdelta plays a critical role in p21(RAS)-mediated apoptosis. An activating p21(RAS) mutation, or activation of the phosphatidylinositol 3-kinase (PI3K) Ras effector pathway, increased the levels of PKCdelta protein and activity in cells, whereas inhibition of p21(RAS) activity decreased the expression of the PKCdelta protein. Activation of the Akt survival pathway by oncogenic Ras required PKCdelta activity. Akt activity was dramatically decreased after PKCdelta suppression in cells containing activated p21(RAS). Conversely, constitutively activated Akt rescued cells from apoptosis induced by PKCdelta inhibition. Collectively, these findings demonstrate that p21(RAS), through its downstream effector PI3K, induces PKCdelta expression and that this increase in PKCdelta activity, acting through Akt, is required for cell survival. The p21(RAS) effector molecule responsible for the initiation of the apoptotic signal after suppression of PKCdelta activity was also determined to be PI3K. PI3K (p110(C)(AAX), where AA is aliphatic amino acid) was sufficient for induction of apoptosis after PKCdelta inhibition. Thus, the same p21(RAS) effector, PI3K, is responsible for delivering both a pro-apoptotic signal and a survival signal, the latter being mediated by PKCdelta and Akt. Selective suppression of PKCdelta activity and consequent induction of apoptosis is a potential strategy for targeting of tumor cells containing an activated p21(RAS).

Highlights

Brane-bound guanine nucleotide-binding proteins and act as molecular switches, thereby regulating signal transduction pathways for hormones, growth factors, and cytokine receptors [1]
Selective Down-regulation of protein kinase C (PKC)␦ Induces Apoptosis in Cells with Activated p21Ras—To begin determination of whether a specific PKC isozyme is responsible for suppression of Rasmediated apoptosis, six isozyme-specific or nonspecific PKC inhibitors were used to suppress PKC␣, PKC␣/␤, PKC␤1/2, PKC␦, or general PKC activity (Table 1)
PKC␦ Inhibition Induces Mitochondrial Apoptotic Pathways in Cells Expressing an Activated p21Ras—To further characterize the molecular mechanisms of p21RAS-mediated apoptotic pathways, we investigated the influence of mitochondrial apoptotic signaling when PKC␦ activity is suppressed by assay of caspase-3 and caspase-9 activation

Summary

Introduction

Brane-bound guanine nucleotide-binding proteins and act as molecular switches, thereby regulating signal transduction pathways for hormones, growth factors, and cytokine receptors [1]. Our data further demonstrate that unregulated Ras activity, through activation of the downstream effector PI3K, up-regulates PKC␦ expression and subsequently activates Akt, generating an anti-apoptotic effect and protecting against Ras-mediated apoptosis.

Results

Conclusion