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