cAMP-dependent proteolysis of GATA-6 is linked to JNK-signaling pathway

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Abstract

A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6 by proteasomes around its IC50. We further examined the effects of SP600125 on the degradation of GATA-6 in detail, since an activator of JNK (anisomycin) is available. Interestingly, anisomycin immediately stimulated the export of nuclear GATA-6 into the cytoplasm, and then the cytoplasmic content of GATA-6 decreased slowly through degradation by proteasomes. Such an effect of anisomycin was inhibited by SP600125, indicating that the observed phenomenon might be linked to the JNK signaling pathway. The inhibitory effect of SP600125 could not be ascribed to the inhibition of PKA, since phosphorylation of CREB occurred in the presence of dbcAMP and SP600125. The nuclear export of GATA-6 was inhibited by leptomycin B, suggesting that CRM1-mediated export could be activated by anisomycin. Furthermore, it seems likely that the JNK activated by anisomycin may stimulate not only the nuclear export of GATA-6 through CRM1 but also the degradation of GATA-6 by cytoplasmic proteasomes. In contrast, A-kinase might activate only the latter process through JNK.

Highlights

► A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6. ► Effect of a JNK activator anisomycin on the proteolysis was examined. ► Anisomycin stimulated the export of nuclear GATA-6 into the cytoplasm. ► JNK activated the CRM1 mediated nuclear export of GATA-6. ► JNK further stimulated slowly the degradation of GATA-6 by cytoplasmic proteasomes.

Introduction

Transcription factor GATA-6, originally named gastric GATA-GT1 [1], [2], is essential for the development of and tissue-specific gene expression in mammals [3]. GATA-6 stably expressed in CHO-K1 cells is degraded by proteasomes upon elevation of the cytoplasmic cAMP concentration [4]. It has been suggested by inhibitor studies that this signaling pathway is mediated by PKA, but not by protein kinase C, MAPK (MAPK/ERK kinase and p38), Ca2+/calmodulin-dependent protein kinase II, or Src kinase [5]. A similar degradation pathway has been demonstrated for the proteolysis of Sp1 in NRK cells [6], [7]. Although the phenomenon of cAMP-dependent proteolysis was evidently found in mammalian cultured cells, its physiological role has not been unveiled.

The degradation of GATA-6 occurs at least in the cytoplasm, as we reported that GATA-6 fixed on the cytoplasmic face of the endoplasmic reticulum membrane is degraded similarly to that expressed in the nucleus [8]. We also obtained mutant clones in which GATA-6 is not degraded even in the presence of dbcAMP [9]. Although it has been suggested that PKA cross-talks with other intracellular signaling pathways [10], nothing is known about the component molecules that participate in the signaling pathway for GATA-6 degradation except cAMP and PKA.

In this study, we examined whether or not the JNK signaling pathway participates in the cAMP-dependent proteolysis of GATA-6 since a JNK inhibitor, SP600125 [11], inhibited the proteolysis. JNK is a member of the MAPK group of signaling proteins, and is known to phosphorylate and activate some activator protein-1 transcription factors and other cellular factors that regulate gene expression and cellular growth [12]. Since the activation of JNK seems to be closely related to the pathogeneses of human diseases such as inflammatory, vascular, neurodegenerative, metabolic and oncological diseases, drug discovery targeting JNK has been of keen interest [13].

Interestingly, we found that anisomycin, an activator of JNK [14], [15], stimulates the nuclear export of GATA-6 through CRM1 rapidly, and then further enhances the proteolytic degradation of GATA-6 by proteasomes. Such a novel finding will be discussed from the viewpoints of both the intra-cellular localization of GATA-6 and GATA-mediated gene regulation.

Section snippets

Materials

FBS was obtained from Gibco BRL. Ham F-12 and dbcAMP were purchased from Sigma. The SCADS inhibitor kit III was provided by the Screening Committee on Anticancer Drugs supported by a Grant-in-Aid for Scientific Research on Priority Area “Cancer” from The Ministry of Education, Culture, Sports, Science and Technology, Japan [16]. HRP-linked donkey anti-rabbit Ig was purchased from Amersham Biosciences. Leptomycin B and MTT were obtained from Calbiochem and Nacalai Tesque, respectively. SP600125

Knockdown of CRM1 mRNA

To construct a vector expressing CRM1 siRNA, a synthetic oligonucleotide cassette (5′-GATCCGGAGCCCAGCAAAGAATGGTTCAAGAGACCATTCTTTGCTGGGCTCCTTA-3′ and 5′-AGCTTAAGGAGCCCAGCAAAGAATGGTCTCTTGAACCATTCTTTGCTGGGCTCCG-3′) encoding siRNA targeting CRM1 [18] was inserted between the BamHI and HindIII sites of the pSilencer 2.0-U6 vector (Ambion). The resulting vector, pSilencer 2.0-U6-CRM1, was introduced transiently into tc1–17a cells in the presence of Lipofectamine 2000 (Invitrogen) [19]. The molecular

Immunoblotting Analysis of GATA-6

Cells treated with both dbcAMP (2 mM) and a kinase inhibitor (1 μM) for 24 h were collected to prepare a nuclear extract by the published method [5]. Briefly, cells were washed with PBS and then scraped into 1 ml PBS with a rubber policeman. The cells were precipitated in a micro-centrifuge (3000 rpm) for 2 min at 4 °C, and then suspended in 200 μL Buffer A [10 mM HEPES-KOH (pH 7.6), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 0.2 mM PMSF]. After incubation for 10 min at 4 °C, the suspension was

Effect of JNK inhibitor SP600125 on cAMP-dependent proteolysis of GATA-6

We previously reported that the activation of PKA by dbcAMP and cholera toxin resulted in the degradation of GATA-6 by proteasomes [4], [5]. However, nothing is known about the component molecules communicating between cAMP and proteasomes except for PKA. We anticipate cross-talk between PKA and other protein kinases that we have not examined. Thus, we examined the effects of kinase inhibitors (SCADS inhibitor kit III) [16] on cAMP-dependent proteolysis of GATA-6 using tc1–17a cells, which is a

Discussion

JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6 [4] at concentration around its IC50 without inhibition of phosphorylation of CREB by PKA (Fig. 1). Thus, JNK could functionally interact with PKA and participate in the GATA-6 degradation pathway. Possibly PKA could phosphorylate JNK directly or indirectly, and stimulate cytoplasmic degradation of GATA-6 by proteasomes [8].

Interestingly JNK activator anisomycin stimulated the export of nuclear-localized GATA-6 through

Conflict of interest

The authors have no financial conflicts of interest.

Acknowledgments

We thank the Screening Committee of Anticancer Drugs supported by a Grant-in-Aid for Scientific Research on Priority Area “Cancer” from MEXT for the SCADS inhibitor kit III. This research was supported in part by a grant from MEXT for Strategic Medical Science Research Centers, 2010–2014 (The MIAST Project).

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