Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca²⁺/calmodulin-dependent protein kinase III, functions in downregulating peptide chain elongation through inactivation of EEF2 (eukaryotic translation elongation factor 2). Currently, there is a limited amount of information on the promotion of autophagic survival by EEF2K in breast and glioblastoma cell lines. However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood. In this study, contrary to the reported autophagy-promoting activity of EEF2K in certain cancer cells, EEF2K is shown to negatively regulate autophagy in human colon cancer cells as indicated by the increase of LC3-II levels, the accumulation of LC3 dots per cell, and the promotion of autophagic flux in EEF2K knockdown cells. EEF2K negatively regulates cell viability, clonogenicity, cell proliferation, and cell size in colon cancer cells. Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206. In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation. Knockdown of AMPK or ULK1 significantly abrogates EEF2K silencing-induced increase of LC3-II levels, accumulation of LC3 dots per cell as well as cell proliferation in colon cancer cells. In conclusion, silencing of EEF2K promotes autophagic survival via activation of the AMPK-ULK1 pathway in colon cancer cells. This finding suggests that upregulation of EEF2K activity may constitute a novel approach for the treatment of human colon cancer.     

Regulation of growth hormone mRNA synthesis by 3,5,3'-triiodo-L-thyronine in cultured growth hormone-producing rat pituitary tumor cells (GC cells). Dissociation between nuclear iodothyronine receptor concentration and growth hormone mRNA synthesis during the deoxyribonucleic acid synthesis phase of the cell cycle

3,5,3'-Triiodo-L-thyronine (T3) regulates the growth rate and GH production of cultured GC cells, a rat pituitary tumor cell line. We have previously demonstrated a parallel increase in cellular content of DNA and nuclear T3 and glucocorticoid receptors during the DNA synthesis (S) phase of the GC cell growth cycle. To determine the relationship between the increase in nuclear hormone receptors and GH production in S-phase cultures, we measured the synthesis rate of GH by pulse-labeling with [3H]leucine and immunoprecipitation as well as the relative concentration of GH mRNA by dot hybridization employing formaldehyde-treated cytoplasm and GH cDNA. Total protein synthesis was similar in S-phase and asynchronous cultures. However, in comparison to asynchronous cultures, S-phase cells had an increased GH synthesis rate, p less than 0.005 (from 13,430 +/- 609 to 19,150 +/- 1160 cpm/10(6) cells/2 h) and increased GH mRNA, p less than 0.001 (from 7.2 +/- 1.2 to 14.5 +/- 1.5 relative A units). The S-phase-associated augmentation in GH production did not appear to result from a decrease in ADP-ribosylation induced by 2 mM thymidine treatment which was utilized for the S-phase synchronization. To determine whether increased GH mRNA and GH synthesis in S-phase was associated with an increase in synthesis of GH mRNA, we measured the incorporation of [3H]uridine into GH mRNA by incubating partially synchronized S-phase cells with [3H]uridine and isolating 3H-labeled GH mRNA by hybridization to GH cDNA immobilized on nitrocellulose filters. Total RNA synthesis was similar in asynchronous, S-phase and G1 cell populations. However, the mean incorporation of [3H]uridine into GH mRNA of S-phase cultures was decreased to 52, 59, and 61% (counts/min of GH mRNA/10(6) cells), 49, 59, and 65% (ppm of total RNA), and 64 and 69% (ppm of poly(A)+ RNA) of asynchronous cultures. Our studies show further that the decrease in [3H]uridine incorporation into GH mRNA did not result from a cell cycle specific change in efficiency of hybridization or exclusively to an S-phase associated increased rate of degradation of GH mRNA. Thus, despite increased nuclear T3 and glucocorticoid receptors and, increased GH mRNA and GH synthesis, the synthesis rate of GH mRNA appears decreased in S-phase GC cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Zinc(II) inhibits the release of thyroid and glucocorticoid receptors from chromatin of cultured GC cells

The effect of Zn(II) on the association of thyroid and glucocorticoid hormone receptors with chromatin was studied in chromatin from cultured GC cells. Chromatin was incubated at 0-4 degrees C in 20 mM Tris, pH 7.4. When buffers contained 0.15 M NaCl, the release of T3 receptors from chromatin was time-dependent; 50% of T3 receptors were released after 30 min incubation. Receptor release appeared relatively specific since less than 10% of chromatin protein and DNA, and less than 13% of chromatin zinc were released under these conditions. Addition of Zn(II) inhibited receptor release; one-half maximal inhibition occurred at 1 microM ZnCl2. Cd(II) and to a lesser extent Co(II) had similar but smaller effects. Addition of EDTA prevented this effect of Zn(II); EDTA alone enhanced receptor release. Zn(II) also inhibited the release of glucocorticoid receptors from chromatin in similar incubations. Our findings suggest that Zn(II) increases the association of hormone receptors with chromatin and, thereby, may influence receptor function.     

Induction of amino acid transport by L-triiodothyronine in cultured growth hormone-producing rat pituitary tumor cells (GC cells)

The action of L-triiodothyronine (T3) on amino acid transport in the GC clonal strain of rat pituitary cells was investigated by measurement of the uptake of the nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB). The uptake of AIB by GC cells appeared to require energy and Na+ and displayed Michaelis-Menten kinetics. In comparison to cultures maintained in the absence of T3, T3 addition resulted in an increase in AIB uptake which seemed due to an increase in the initial rate of AIB transport. T3 addition resulted in increased AIB accumulation at later time points as well. T3 induction of AIB transport did not occur until 3.5 h after addition of T3, and this effect was blocked by cycloheximide. Maximal induction occurred 48 to 72 h later. One-half maximal induction occurred 24 to 48 h after addition of T3. No detectable changes either in AIB uptake or intracellular water space, measured by uptake of the nonmetabolizable sugar, 3-O-methyl-D-glucose, were noted for the first 120 min after addition of T3. Induction of AIB transport occurred at 0.05 nM T3 (total medium concentration) and one-half maximal induction occurred at 0.17 nM T3. The relative potencies of four iodothyronine analogues for AIB transport were in accord with their reported activities in nuclear T3 receptor binding assays. These data suggest that induction of AIB transport by T3 may be mediated by the nuclear T3 receptor and may reflect the pleiotrophic response of GC cells to thyroid hormone.     

Dimerization of cGMP-dependent protein kinase 1alpha and the myosin-binding subunit of myosin phosphatase: role of leucine zipper domains

Nitric oxide (NO) and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase (cGK)-mediated activation of myosin phosphatase, which dephosphorylates myosin light chains. We recently found that cGMP-dependent protein kinase 1alpha binds directly to the myosin-binding subunit (MBS) of myosin phosphatase via the leucine/isoleucine zipper of cGK. We have now studied the role of the leucine zipper domain of MBS in dimerization with cGK and the leucine/isoleucine zipper and leucine zipper domains of both proteins in homodimerization. Mutagenesis of the MBS leucine zipper domain disrupts cGKIalpha-MBS dimerization. Mutagenesis of the MBS leucine zipper eliminates MBS homodimerization, while similar disruption of the cGKIalpha leucine/isoleucine zipper does not prevent formation of cGK dimers. The MBS leucine zipper domain is phosphorylated by cGK, but this does not have any apparent effect on heterodimer formation between the two proteins. MBS LZ mutants that are unable to bind cGK were poor substrates for cGK. These data support the theory that the MBS leucine zipper domain is necessary and sufficient to mediate both MBS homodimerization and binding of the protein to cGK. In contrast, the leucine/isoleucine zipper of cGK is required for binding to MBS, but not for cGK homodimerization. These data support that the MBS and cGK leucine zipper domains mediate the interaction between these two proteins. The contribution of these domains to both homodimerization and their specific interaction with each other suggest that additional regulatory mechanisms involving these domains may exist.