Elsevier

Cellular Signalling

Volume 20, Issue 7, July 2008, Pages 1275-1283
Cellular Signalling

βγ subunits of Gi/o suppress EGF-induced ERK5 phosphorylation, whereas ERK1/2 phosphorylation is enhanced

https://doi.org/10.1016/j.cellsig.2008.02.016Get rights and content

Abstract

Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation and gene expression. ERK5 is twice the size of ERK1/2, the amino-terminal half contains the kinase domain that shares the homology with ERK1/2 and TEY activation motif, whereas the carboxy-terminal half is unique. In this study, we examined the cross-talk mechanism between G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases, focusing on ERK1/2 and 5. The pretreatment of rat pheochromocytoma cells (PC12) with pertussis toxin (PTX) specifically enhanced epidermal growth factor (EGF)-induced ERK5 phosphorylation. In addition, lysophosphatidic acid (LPA) attenuated the EGF-induced ERK5 phosphorylation in LPA1 receptor- and Gi/o-dependent manners. On the other hand, LPA alone activated ERK1/2 via Gβγ subunits and Ras and potentiated EGF-induced ERK1/2 phosphorylation at late time points. These results suggest Gi/o negatively regulates ERK5, while it positively regulates ERK1/2. LPA did not affect cAMP levels after EGF treatment, and the reagents promoting cAMP production such as forskolin and cholera toxin also attenuated the EGF-induced ERK5 phosphorylation, indicating that the inhibitory effect of LPA on ERK5 inhibition via Gi/o is not due to inhibition of adenylyl cyclase by Gαi/o. However, the inhibitory effect of LPA on ERK5 was abolished in PC12 cells stably overexpressing C-terminus of GPCR kinase2 (GRK2), and overexpression of Gβ1 and γ2 subunits also suppressed ERK5 phosphorylation by EGF. In response to LPA, Gβγ subunits interacted with EGF receptor in a time-dependent manner. These results strongly suggest that LPA negatively regulates the EGF-induced ERK5 phosphorylation through Gβγ subunits.

Introduction

Extracellular signal-regulated kinases (ERKs) or mitogen-activated protein kinases (MAPKs) are involved in proliferation, differentiation, migration and gene expression. ERK1/2 are activated by variety of stimuli, and the signaling pathway leading to ERK1/2 activation has been well characterized [1], [2], [3]. ERK5 is approximately double the size of ERK1/2. The kinase domain is encoded in its amino-terminal half and shares approximately 50% of homology with ERK1/2, while its unique carboxy-terminus encodes two proline-rich regions, a nuclear export domain and a nuclear localization domain [4], [5]. The threonine and tyrosine residues on ERK5 are phosphorylated by MEK5, but not MEK1/2. Importantly, MEK inhibitors used frequently such as PD98059 and U0126 also blocks MEK5 [6], indicating that cellular responses that have been considered as a result of ERK1/2 activation may result from ERK5 activation.

Several physiological roles of ERK5 have been reported [4], [5], [7]. For example, ERK5 regulates S-phase entry by epidermal growth factor (EGF) in Hela cells [8]. In dorsal root ganglia, ERK5 is activated retrogradely by nerve growth factor (NGF), and prevents apoptosis [9]. In developing cortical neurons, ERK5 plays a critical role in brain-derived neurotrophic factor (BDNF)-promoted survival [10]. Neuronal differentiation is reduced by ERK5 knock-down using antisense morpholino oligonucleotides in Xenopus laevis [11]. Also, ERK5 is required for generation of neurons from cortical progenitors [12]. In animal models, ERK5 gene knockout is lethal at E9.5–10.5 due to cardiovascular defects, indicating the involvement of ERK5 in heart development [13]. In pathological conditions, ERK5 is involved in tumor development and hypertrophy [5]. Thus, ERK5 plays essential physiological roles in addition to ERK1/2.

In rat pheochromocytoma cells (PC12), EGF and NGF activate ERK5 via the small G-protein Ras [6]. But, it has been shown that BDNF activates ERK5 through Rap1 in cortical neurons [14]. In the mink lung epithelial cell line, interaction of Lck-associated adapter (Lad) with MEKK2 is involved in activation of ERK5 [15], while in bone marrow-derived mast cells, protein kinase C mediates FcɛR1-induced ERK5 activation and cytokine production [16]. G-protein-coupled receptor (GPCR) agonists such as carbachol and thrombin also activate ERK5 via Gαq/11 and Gα12/13 families of heterotrimeric G-proteins [3], [17]. The ERK5 activation by carbachol and thrombin is not blocked by dominant-negative mutants of Ras and Rho nor C3 toxin that inactivates Rho-mediated functions [17]. In vascular smooth muscle cells, angiotensin II promotes ERK5 activation [18]. In contrast, prostaglandin E2 or forskolin, both of which promote cAMP production, attenuates ERK5 phosphorylation induced by EGF via PKA [19].

GPCRs are one of the most important drug targets. In fact, 30% of marketed small-molecule drugs target GPCRs [20]. Therefore, it is important to examine their down-stream signaling pathways to gain an understanding the action of drugs. Also, as most growth factors, receptor tyrosine kinases (RTKs) and their down-stream effectors are categorized as proto-oncogenes, alternations in RTK signaling are closely associated with the development of many cancers. To date, many reports have been shown the cross-talk between GPCRs and RTKs. For examples, GPCR agonists transactivates RTKs via either secretion of EGF-like substances through matrix metalloproteases or tyrosine phosphorylation of RTKs by tyrosine kinases such as Src [21], [22]. In other words, GPCRs can utilize RTK signaling pathways to activate their down-stream effectors such as ERKs and Akt. Conversely, RTKs also utilize GPCR signaling pathways. For example, Gi/o is partially involved in phosphorylation of ERK1/2 and Akt by NGF in PC12 cells [23], [24], [25]. In addition, receptor of insulin-like growth factor (IGF) I utilizes Gαi for mitogenic signaling, whereas insulin receptor does Gαq/11 for metabolic actions [26]. Thus, it is important to investigate the cross-talk mechanism to understand the physiological roles of GPCRs and RTKs.

In the process of examining the cross-talk mechanism between GPCRs and RTKs, we found that βγ subunits of Gi/o suppressed the EGF-induced ERK5 phosphorylation, while ERK1/2 phosphorylation was rather enhanced by Gi/o in PC12 cells. Therefore, the role of heterotrimeric G-protein, Gi/o in ERK signaling was investigated in detail in this study.

Section snippets

Materials

EGF was purchased from PeproTech EC. (London, UK). NGF, lysophosphatidic acid (LPA), d-luciferin, H89, Ro201724, Ki16425 and anti-flag M2 agarose were purchased from Sigma-Aldrich (St. Louis, MO). Pertussis toxin (PTX) and cholera toxin (CTX) were purchased from List Biological laboratories Inc. (Campbell, CA). Forskolin was purchased from Wako Pure Chemicals (Tokyo, Japan). CGS21680 was purchased from Tocris Cookson Ltd. (Bristol, UK). The RNA extraction kit was from Roche Diagnostics

LPA inhibits EGF and NGF-induced ERK5 phosphorylation via Gi/o

To examine the role of Gi/o in the growth factor signaling, PC12 cells were cultured for 48 h in the presence or absence of PTX (100 ng/ml), which blocks the interaction of Gi/o with their corresponding receptors by ADP ribosylation. After serum starvation overnight, the cells were stimulated with NGF (100 ng/ml) for 5 and 40 min or EGF (100 ng/ml) for 5 min. Since PC12 cells express LPA1 receptor (or edge2) [24] which couples with Gi/o or G12, LPA (10 μM) was also used as a control to confirm

Discussion

In the present study, we investigated the cross-talk mechanism between GPCRs and RTKs in PC12 cells, focusing on ERK5. Whereas LPA alone caused phosphorylation of ERK1/2 via βγ subunits of Gi/o and Ras, the EGF-induced ERK5 phosphorylation was blocked by LPA via LPA1 receptor and βγ subunits of Gi/o. The putative signaling cascade is shown in Fig. 9. The effect of LPA was not specific to EGF as a similar effect was observed for NGF (Fig. 2D). Also, the effect of LPA on ERK5 was observed in Cos7

Acknowledgements

This work was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science (No. 18790039 to Y.O. and No. 18057002 and 19659011 to N.N.). We thank Dr. Philip Stork (Vollum Institute, Oregon Health Sciences University, Portland, OR) and Dr. Yung Wong (Hong Kong University of Science and Technology, Hong Kong, China) for providing DNA plasmids. We thank Dr. Tara Dillon (Vollum Institute, Oregon Health Sciences University) for critical scientific discussion. We thank Dr.

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