We screened 95 kinase inhibitors whether they affect cAMP-dependent proteolysis of GATA-6 or not. Among them 7 inhibitors inhibited the proteolysis at the concentration range of μM around their IC50. They are inhibitors for protein kinase A (H-89 and 4- cyano-3-methylisoquinoline), c-Jun N-terminal kinase (SP600125), phosphatidylinositol 3-kinase (Wort- mannin and LY-294002), casein kinase II (TBB) and cyclin dependent kinase (Cdk1/2 inhibitor III). It is of interest how these kinases play roles in the degradation process of GATA-6 since this transcription factor is essential for development and tissue-specific gene expression of mammals. Inhibitors identified in this study would be helpful to study molecular mechanisms of phenomena in which GATA-6 participates.
The sequence-specific DNA-binding protein GATA-6 [1,2] is essential for early developmental processes and tissue-specific gene expression of mammals [
When GATA-6 was fixed on the cytoplasmic side of endoplasmic reticulum membrane as a fusion protein with the carboxy-terminal membrane domain of SREBP, it was degraded similarly to that expressed in the nucleus, suggesting that the degradation of GATA-6 occurs at least in the cytoplasm [
We could obtain mutant clones in which GATA-6 is not degraded even in the presence of dbcAMP although mutation sites were unidentified [
In this study, we compared 95 kinase inhibitors [
The SCADS inhibitor kit III was provided from Screening Committee of Anticancer Drugs supported by Grantin-Aid for Scientific Research on Priority Area “Cancer” from The Ministry of Education, Culture, Sports, Science and Technology, Japan [
CHO-K1 cells (1.5 × 106 per well, in a 100 mm dish) stably expressing rat GATA-6 (tc1-17a cells) [
Cells treated with both dbcAMP and a kinase inhibitor for 24 h were washed with phosphate-buffered saline [10 mM sodium phosphate buffer (pH 7.2), 137 mM NaCl, 3 mM KCl] (PBS), and scraped into 1 ml PBS with rubber policeman. Cells were precipitated in micro-centrifuge (3000 rpm) for 2 min at 4˚C, and then suspended in 200 µL Buffer A [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-KOH (pH 7.6), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 0.2 mM phenylmethanesulfonyl fluoride (PMSF)]. After incubation for 10 min at 4˚C, the suspension was mixed with vortex mixer, and centrifuged (3000 rpm) for 10 min at 4˚C. The nuclear pellet was suspended for 30 min at 4˚C in 50 µl Buffer C [20 mM HEPES-KOH (pH 7.6), 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM DTT, 0.2 mM PMSF, 25% (v/v) glycerol]. The suspension was centrifuged (15,000 rpm) for 2 min at 4˚C, and the supernatant was stored at –80˚C until use [
Proteins (10 µg) were subjected to sodium dodecylsulfate (SDS)-polyacrylamide gel-electrophoresis [7.5% (w/v) separation gel] [
We have previously reported that the activation of Akinase by cAMP caused the degradation of GATA-6 by proteasomes [
Among 95 kinase inhibitors [
On the other hand, inhibitors for tyrosine kinases did not have any effects. Only weak effects could be detected for several inhibitors (sample No. 28, 29, 30, 43 and 65 shown in
Next we examined the effective concentration of 6 kinase inhibitors (Cdk1/2 inhibitor III, TBB, SP600125, LY-294002, Wortmannin, and 4-cyano-3-methylisoquinoline) on the inhibition of GATA-6 degradation. As shown in
We screened 95 kinase inhibitors whether they could inhibit cAMP-dependent proteolysis of GATA-6, and found that 7 of them strongly and moderately inhibited the induced proteolysis. Two (H-89 and 4-cyano-3- methylisoquinoline) are inhibitors of A-kinase, indicating that our previous observation [
Other five effective inhibitors are those of JNK (SP600125), PI3K (Wortmannin and LY-294002), CKII (TBB) and CDK (Cdk1/2 inhibitor III). Titration experiments of these inhibitors demonstrated that they inhibited the proteolysis at the concentration around their IC50 [
the GATA-6 degradation pathway. We further demonstrated that JNK not only induces proteolysis of GATA-6 by proteasomes but also stimulates export of GATA-6 from nucleus to cytoplasm [
GATA-6 is essential for developmental process of endoderm [
The positive effects of inhibitors for JNK, PI3K, CKII, and CDK suggest that these protein kinases could be functionally interacted. These kinases are known to be essential for stress responses such as ultraviolet, heat, and hypotonicity, cell cycle regulation, differentiation, growth and so on [22-26]. It is reported that JNK directly phosphorylates some transcription factors, and regulates
stability of the target transcription factors through ubiquitin-proteasome system [32,33]. Furthermore, DNA binding activities of Jun and c-Myc transcription factors are controlled via phosphorylation process by CKII [34, 35]. Thus it would be of interest how these kinases communicate with the signaling pathway for cAMPdependent proteolysis. These lines of experiments are now in progress and the results will be reported in near future.
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 grants from MEXT for Strategic Medical Science Research Centers, 2010-2014 (The MIAST Project) and JSPS KAKENHI Grant Number 24701032 to H.U.