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P23/Tmp21 associates with protein kinase Cdelta (PKCdelta) and modulates its apoptotic function.

ABSTRACT: There is emerging evidence that C1 domains, motifs originally identified in PKC isozymes and responsible for binding of phorbol esters and diacylglycerol, interact with the Golgi/endoplasmic reticulum protein p23 (Tmp21). In this study, we investigated whether PKC?, a kinase widely implicated in apoptosis and inhibition of cell cycle progression, associates with p23 and determined the potential functional implications of this interaction. Using a yeast two-hybrid approach, we found that the PKC? C1b domain associates with p23 and identified two key residues (Asp(245) and Met(266)) implicated in this interaction. Interestingly, silencing p23 from LNCaP prostate cancer cells using RNAi markedly enhanced PKC?-dependent apoptosis and activation of PKC? downstream effectors ROCK and JNK by phorbol 12-myristate 13-acetate. Moreover, translocation of PKC? to the plasma membrane by phorbol 12-myristate 13-acetate was enhanced in p23-depleted LNCaP cells. Notably, a PKC? mutant that failed to interact with p23 triggered a strong apoptotic response when expressed in LNCaP cells. In summary, our data compellingly support the concept that C1 domains have dual roles both in lipid and protein associations and provide strong evidence that p23 acts as an anchoring protein that retains PKC? at the perinuclear region, thus limiting the availability of this kinase for activation in response to stimuli.

SUBMITTER: Wang H

PROVIDER: S-EPMC3091192 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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p23/Tmp21 associates with protein kinase Cdelta (PKCdelta) and modulates its apoptotic function.

Wang HongBin H Xiao Liqing L Kazanietz Marcelo G MG

The Journal of biological chemistry 20110316 18

There is emerging evidence that C1 domains, motifs originally identified in PKC isozymes and responsible for binding of phorbol esters and diacylglycerol, interact with the Golgi/endoplasmic reticulum protein p23 (Tmp21). In this study, we investigated whether PKCδ, a kinase widely implicated in apoptosis and inhibition of cell cycle progression, associates with p23 and determined the potential functional implications of this interaction. Using a yeast two-hybrid approach, we found that the PKCδ ...[more]

PMID: 21454541

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Project description:We previously demonstrated that protein kinase C? (PKC?; PKC delta) is an oxidative stress-sensitive kinase that plays a causal role in apoptotic cell death in neuronal cells. Although PKC? activation has been extensively studied, relatively little is known about the molecular mechanisms controlling PKC? expression. To characterize the regulation of PKC? expression, we cloned an ?2-kbp 5'-promoter segment of the mouse Prkcd gene. Deletion analysis indicated that the noncoding exon 1 region contained multiple Sp sites, including four GC boxes and one CACCC box, which directed the highest levels of transcription in neuronal cells. In addition, an upstream regulatory region containing adjacent repressive and anti-repressive elements with opposing regulatory activities was identified within the region -712 to -560. Detailed mutagenesis studies revealed that each Sp site made a positive contribution to PKC? promoter expression. Overexpression of Sp family proteins markedly stimulated PKC? promoter activity without any synergistic transactivating effect. Furthermore, experiments in Sp-deficient SL2 cells indicated long isoform Sp3 as the essential activator of PKC? transcription. Importantly, both PKC? promoter activity and endogenous PKC? expression in NIE115 cells and primary striatal cultures were inhibited by mithramycin A. The results from chromatin immunoprecipitation and gel shift assays further confirmed the functional binding of Sp proteins to the PKC? promoter. Additionally, we demonstrated that overexpression of p300 or CREB-binding protein increases the PKC? promoter activity. This stimulatory effect requires intact Sp-binding sites and is independent of p300 histone acetyltransferase activity. Finally, modulation of Sp transcriptional activity or protein level profoundly altered the cell death induced by oxidative insult, demonstrating the functional significance of Sp-dependent PKC? gene expression. Collectively, our findings may have implications for development of new translational strategies against oxidative damage.

Dataset Information

P23/Tmp21 associates with protein kinase Cdelta (PKCdelta) and modulates its apoptotic function.

Publications

p23/Tmp21 associates with protein kinase Cdelta (PKCdelta) and modulates its apoptotic function.

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