Biochemical and Biophysical Research Communications
Cobalt chloride-mediated protein kinase Cα (PKCα) phosphorylation induces hypoxia-inducible factor 1α (HIF1α) in the nucleus of gastric cancer cell
Introduction
Hypoxia induces a number of proteins which enable cells to adapt to low-oxygen environment. The master transcriptional regulator in hypoxic cells is a heterodimeric protein hypoxia-inducible factor 1 (HIF1). HIF1 binds to the hypoxia-response element (HRE) of the target gene to induce transcription. It comprises of α and β subunits. The β subunit is constitutively expressed but HIF1α is only detected in hypoxic cells or in cells that are under oxidative stress [1]. Prolyl hydroxylation of HIF1α in the normoxic cells catalyzed by prolyl hydroxylases (PHDs) leads to its binding with the pVHL-E3 ubiquitin ligase complex following degradation. Hypoxia inhibits prolyl hydroxylation of HIF1α and stabilizes it [2]. Cobalt chloride (CoCl2.6H2O) induces biochemical responses similar to hypoxia [3].
Blood circulation is inefficient in solid tumors and as a result, hypoxic regions are common at the core of solid tumors [4]. Prolonged hypoxia induces apoptosis but cells that can survive hypoxic assault, emerge as more drug-resistant and metastatic [5], [6], [7]. Besides HIF1, the PKC family of Ser/Thr kinases are also induced by hypoxia that can regulate various cellular functions including cell growth, differentiation and apoptosis [8]. Tissue ischemia or hypoxic stress leads to the activation of various protein kinase C (PKC) family members. PKCs protect against hypoxic injury and are implicated in ischemia-related diseases [9]. The PKC family has ten isoforms grouped in three subfamilies based on their differences in structure and catalytic domains and on their ability to respond to the cofactors Ca++ and diacylglycerol (DAG). PKCα is a conventional or classical isoform and requires both Ca++ and DAG for activation [10].
PKCα promotes invasiveness of various cancers including gastric cancer [11], [12]. But contradicting reports on PKCα downregulation in several cancers [11] and PKCα-mediated apoptosis induction in 12-O-tetradecanoyl phorbol-13-acetate-treated human gastric cancer cell lines [13] also exist. Cellular localization of PKCs determines their function and the effect is tissue or cell line-specific. For example, nuclear translocation of PKCδ in C5 cells induces apoptosis whereas its mitochondrial translocation is required for apoptosis in SP1 cells [14], [15], [16]. Phosphorylation and dephosphorylation influence PKC-mediated cellular signaling and impart specificities to PKC binding to distinct substrates as well [17]. Subcellular fractionation shows that phosphorylated active PKCα can translocate to the nucleus [18]. So, subcellular localization of PKCα might be the key factor determining its role in cancer progression. However, its expression status and role in hypoxic gastric cancer cells have not yet been studied.
This study identifies that PKCα phosphorylation at T638 is induced in the nucleus of GECs after treatment with CoCl2. Moreover, this study establishes that T638-phosphorylated PKCα induces expression of HIF1α after CoCl2 treatment in GECs. As human gastric neoplasia samples also showed highly-increased expression of P-PKCα as compared to noncancerous samples, P-PKCα might be useful as a molecular marker to study gastric cancer progression. Since fluctuations in blood flow during solid tumor development creates hypoxic state, our results implicate that P-PKCα might be a regulator of HIF1α activity in aggressively growing solid tumors.
Section snippets
Cell culture and treatment
Cell lines and reagents used in this work are listed in supplementary materials.
Plasmids, site-directed mutagenesis and transfection
Wild type (WT) PKCα and its dominant negative (DN) mutant K368R (mutated in the ATP binding cassette and is kinase function-dead) were gifted by Bernard Weinstein (Addgene plasmids # 21232 and #21235, respectively) [19]. PKCα T638A mutant construct was generated from the WT PKCα construct using QuikChange site directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). Mutagenesis primers and transfection
Short treatment with CoCl2 induces nuclear P-PKCα expression
Expression of PKCα and P-PKCα was assessed in normoxic (or control) and CoCl2-treated AGS cells. Comparison of western blots (n = 3) of whole cell lysates prepared from control or 30 min, 1 h and 3 h CoCl2-treated (200 μM) AGS cells did not show any change in expression of PKCα and P-PKCα (Fig. 1A). CoCl2 at 200 μM was not cytotoxic to AGS cells (data not shown). Expression of HIF1α time-dependently increased with CoCl2 treatment. Western blot results of the nuclear fractions identified
Discussion
Signaling events in hypoxia determine cancer progression, metastasis, drug resistance properties and overall treatment outcome in solid cancer. Research spanning the past two decades has implicated PKCα in various solid cancers [25], [26]. However, the role of PKCα in CoCl2-treated GECs has not been studied at all. We show that CoCl2 induces P-PKCα expression at T638 residue in the nuclei of GECs. Moreover, we report that CoCl2-induced P-PKCα enhances HIF1α expression and transcriptional
Conflict of interest
The authors declare no conflict of interests.
Acknowledgment
This work was partly supported by a grant to Asima Bhattacharyya (Sanction No. SR/FT/LS-38/2010), Science and Engineering Research Board (SERB), Govt. of India and institutional funding from NISER, Bhubaneswar. We acknowledge Mr. Alok Kumar Jena for his assistance with confocal imaging.
References (37)
- et al.
Cobalt chloride induces neuronal differentiation of human mesenchymal stem cells through upregulation of microRNA-124a
Biochem. Biophys. Res. Commun.
(2014) - et al.
Protein kinase C as a stress sensor
Cell. Signal
(2007) - et al.
Classical PKC isoforms in cancer
Pharmacol. Res.
(2007) - et al.
Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes
J. Biol. Chem.
(2003) - et al.
Phosphorylation of threonine 638 critically controls the dephosphorylation and inactivation of protein kinase Calpha
Curr. Biol.
(1996) - et al.
Phosphorylation of Activation Transcription Factor-2 at Serine 121 by Protein Kinase C Controls c-Jun-mediated activation of transcription
J. Biol. Chem.
(2009) - et al.
Nuclear protein kinase C
Biochim. Biophys. Acta
(2006) - et al.
Erk1/2-dependent phosphorylation of PKCalpha at threonine 638 in hippocampal 5-HT(1A) receptor-mediated signaling
Biochem. Biophys. Res. Commun.
(2010) - et al.
Phosphorylation of protein kinase C-alpha on serine 657 controls the accumulation of active enzyme and contributes to its phosphatase-resistant state
J. Biol. Chem.
(1997) - et al.
Protein kinase C is regulated in vivo by three functionally distinct phosphorylations
Curr. Biol.
(1995)
Oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases
Physiol. Rev.
The updated biology of hypoxia-inducible factor
EMBO J.
The unique physiology of solid tumors: opportunities (and problems) for cancer therapy
Cancer Res.
Targeting hypoxia cell signaling for cancer therapy
Cancer Metastasis Rev.
Hypoxia and metastasis
Clin. Cancer Res.
Targeting hypoxic tumour cells to overcome metastasis
BMC cancer
Protein kinase C alpha (PKC alpha): regulation and biological function
J. Biochem.
Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions
Chem. Rev.
Cited by (0)
- 1
Contributed equally.