Cooperation between PKC-α and PKC-ε in the regulation of JNK activation in human lung cancer cells☆
Introduction
Protein kinase C (PKC) is a family of at least 11 structurally related phospholipid-dependent serine/threonine kinases that play crucial roles in transducing signals, and it has been implicated in the regulation of diversified cellular functions, including proliferation, differentiation, and apoptosis [1], [2]. The PKC family can be divided into three subgroups based on differences in their structures and biochemical properties: the classic isoforms (cPKC-α, βI, βII, and γ), which are Ca2+ and phorbol ester/diacylglycerol (DAG)-dependent; the novel isoforms (nPKC-δ, ε, θ, and η), which are phorbol ester/DAG-dependent but Ca2+-independent; and the atypical isoforms (aPKC-ζ and ι/λ), which are Ca2+ and phorbol ester/DAG-independent. The heterogeneous expression profiles of PKC isoforms in tissues strongly suggest that each isoform has a unique individual role. Their different subcellular localization and isoform-specific cofactor and activator requirements indicate that each isoform could be differentially regulated [2], [3]. Although the cellular roles of the different PKC isoforms remain unclear, accumulated evidence has shown that individual PKC isoforms may play distinct roles in response to various stimuli.
Phorbol esters and their related derivatives are potent activators of PKC, and are the most widely used tumor promoting agents in animal models of carcinogenesis. In cell culture systems, phorbol esters can mimic distinct intracellular signaling events triggered by activated growth-factor receptors, resulting in diverse effects ranging from proliferation and cell survival to differentiation and cell death [4]. It is well established that PKC is a key mediator of phorbol ester actions. PKC activation by phorbol esters or diacylglycerol activates at least two mitogen-activated protein kinase (MAPK) pathways, the extracellular signal-regulated kinase (ERK) pathway and the c-Jun N-terminal kinase (JNK) pathway. The ERK pathway, which consists of the Raf kinase, ERK kinase (MEK1/2), and ERK1/2, is ubiquitously expressed in mammalian cells. Acute treatment with phorbol esters induces a rapid activation of the ERK pathway in most cell types [5], presumably through stimulation of Raf-1 activity. Both classic and novel PKCs respond to phorbol esters and have been shown to phosphorylate and activate Raf-1 in vitro and in vivo [6], [7]. Hamilton et al. [8] reported recently that PKC-ε, a member of the novel PKC subgroup, forms a latent complex with Raf-1 and N-Ras, and its activation by phorbol ester directly regulates Raf-1 activity. Together with other studies, it is predicted that classic and/or novel PKCs may act at the level of, or upstream from, the Raf-1 kinase in mediating phorbol ester-induced ERK activation.
Unlike ERKs that are primarily activated by mitogens, JNKs are potently and preferentially activated by cellular stress and by inflammatory cytokines [9]. Consistent with this notion, studies have showed that phorbol esters have little effect on JNK activation in diverse cell types [10], [11]. However, some studies demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) could effectively activate JNK in human myeloid leukemia cells [12], [13], suggesting that JNK activation by phorbol ester may be cell type-specific. Recent studies suggest that the induction of JNK activity may depend on the expression of specific PKC isoforms in a cell. For example, it was reported [13] that in human myeloid leukemia cells JNK activation by TPA was dependent on the expression of the PKC-β isoform. Ectopic expression of PKC-β in PKC-β-deficient cells restored the response to TPA and the induction of JNK activity. Additionally, novel PKCs (θ or δ) were shown to be critical in PMA-mediated JNK and NF-κB activation in B cells [14]. However, how cell-specific factors (or cell context-dependent mechanisms) contribute to JNK activation remains largely unexplored.
In the present study, we found that phorbol 12-myristate 13-acetate (PMA) induced JNK activation only in non-small cell lung cancer (NSCLC) cells, but not in small cell lung cancer (SCLC) cells, whereas ERK activation was detected in both cell types. We demonstrate that the mechanism(s) that control this cell type-specific JNK activation involve the cooperative action between the PKC-α and PKC-ε isoforms. PMA-induced JNK activation appears to be mediated, at least, by two distinct signaling pathways, the MEK-dependent and the Rac1/Cdc42-dependent pathways. PKC-ε was found to be important for both JNK and ERK activation, while PKC-α appears to solely control the signaling to JNK in response to PMA. Our studies suggest that a cell context-dependent signaling network regulates JNK signal specificity, which may be important in mediating distinct PMA-induced effects in the NSCLC subtype of lung cancer cells.
Section snippets
Materials
Mouse monoclonal antibodies against specific PKC isoforms were purchased from BD Transduction Laboratories (Lexington, KY). Rabbit polyclonal antibodies against phospho-JNK (Thr183/Tyr185) and phospho-MEK1/2 (Ser217/221) were purchased from Cell Signaling (Beverly, MA). Anti-JNK1 (C-17), anti-ERK1 (C-16), and anti-phospho-ERK1/2 (Tyr204) (E-4) were from Santa Cruz Biotechnology (Santa Cruz, CA). PKC inhibitors (GF109203X, Gö6976, and rottlerin) and MEK inhibitor U0126 were purchased from
PMA induces JNK activation in NSCLC cells, but not in SCLC cells
PMA has been implicated in stimulating the proliferation of SCLC cells [18], [19], while it induced differentiation and/or growth inhibition in NSCLC cells [20], [21]. To understand the signaling events that mediate cell type-specific effects of PMA, we investigated the activation of JNK and ERK pathways by PMA in lung cancer cells. A panel of lung cancer cell lines including three SCLC and five NSCLC was used. These cells were first serum-starved by culturing in serum-free medium for 24 h and
Discussion
Several recent studies have demonstrated a link between cell-specific induction of JNK activity and PMA-dependent cellular response such as differentiation. Of interest, it was reported that PMA induced distinct cellular effects in human lung cancer cells in a phenotype-specific manner. While PMA stimulation resulted in proliferation and the activation of the ERK pathway in SCLC cells, it caused growth inhibition and/or differentiation in NSCLC cells. We showed that PMA induced activation of
Acknowledgements
We are grateful to Dr. A.P. Fields for kindly providing cDNA clones of PKC-α and PKC-ε, to Dr. R.J. Davis for JNK1 expression vector, and to Dr. M. Karin for expression constructs for Rac1N17 and Cdc42N17. We also thank Greg Tyler for general technical and administrative assistance, and Mary Wall for her secretarial assistance.
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This work was supported in part by the NIH Grant R01-CA88815.
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Present address: Department of Thoracic Head and Neck Medical Oncology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard., Houston, TX 77030, USA.