Calcium Regulation of EGF-Induced ERK5 Activation: Role of Lad1-MEKK2 Interaction

The ERK5 cascade is a MAPK pathway that transmits both mitogenic and stress signals, yet its mechanism of activation is not fully understood. Using intracellular calcium modifiers, we found that ERK5 activation by EGF is inhibited both by the depletion and elevation of intracellular calcium levels. This calcium effect was found to occur upstream of MEKK2, which is the MAP3K of the ERK5 cascade. Co-immunoprecipitation revealed that EGF increases MEKK2 binding to the adaptor protein Lad1, and this interaction was reduced by the intracellular calcium modifiers, indicating that a proper calcium concentration is required for the interactions and transmission of EGF signals to ERK5. In vitro binding assays revealed that the proper calcium concentration is required for a direct binding of MEKK2 to Lad1. The binding of these proteins is not affected by c-Src-mediated phosphorylation on Lad1, but slightly affects the Tyr phosphorylation of MEKK2, suggesting that the interaction with Lad1 is necessary for full Tyr phosphorylation of MEKK2. In addition, we found that changes in calcium levels affect the EGF-induced nuclear translocation of MEKK2 and thereby its effect on the nuclear ERK5 activity. Taken together, these findings suggest that calcium is required for EGF-induced ERK5 activation, and this effect is probably mediated by securing proper interaction of MEKK2 with the upstream adaptor protein Lad1

The stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities

Type I interferons (IFNs) signal for their diverse biological effects by binding a common receptor on target cells, composed of the two transmembrane IFNAR1 and IFNAR2 proteins. We have previously differentially enhanced the antiproliferative activity of IFN by increasing the weak binding affinity of IFN to IFNAR1. In this study, we further explored the affinity interdependencies between the two receptor subunits and the role of IFNAR1 in differential IFN activity. For this purpose, we generated a panel of mutations targeting the IFNAR2 binding site on the background of the IFNalpha2 YNS mutant, which increases the affinity to IFNAR1 by 60-fold, resulting in IFNAR2-to-IFNAR1 binding affinity ratios ranging from 1000:1 to 1:1000. Both the antiproliferative and antiviral potencies of the interferon mutants clearly correlated to the in situ binding IC(50) values, independently of the relative contributions of the individual receptors, thus relating to the integral lifetime of the complex. However, the antiproliferative potency correlated throughout the entire range of affinities, as well as with prolonged IFNAR1 receptor down-regulation, whereas the antiviral potency reached a maximum at binding affinities equivalent to that of wild-type IFNalpha2. Our data suggest that (i) the specific activity of interferon is related to the ternary complex binding affinity and not to affinity toward individual receptor components and (ii) although the antiviral pathway is strongly dependent on pSTAT1 activity, the cytostatic effect requires additional mechanisms that may involve IFNAR1 down-regulation. This differential interferon response is ultimately mediated through distinct gene expression profiling

EM analysis of ULK1 knocked-down cells reveals larger melanosomes.

<p>Cells were treated with (A and C) control or (B) ULK1 siRNA and prepared for EM analysis. For quantification (D) of melanosome number and area, cells were blindly chosen. A series of images were taken within each cell to cover a similar area between all cells at a 49,000X magnification. Over 80 images from total of 15 cells per condition were collected. The number of melanosomes at different stages (shown in control cells C) was manually scored within each cell. (D) Average melanosome size of the melanosomes in each stage in control and ULK1 siRNA treated cells.</p

ULK1 knockdown increases melanin in MNT-1 cells.

<p>(A) MNT-1 cells were treated with a control siRNA, two ULK1-specific siRNAs and a TYR-specific siRNA pool as a positive control for changes in melanin levels. Three days after the second knockdown cells were harvested and their melanin content was analysed by measuring melanin/protein values for each sample. Significant changes relative to control were determined by a one-way ANOVA test based on three individual experiments performed in duplicates. * p<0.05; ** p<0.01; *** p<0.001; data are presented as the mean of values normalized to control within each experiment. (B) Western blot analysis of the siRNA-treated MNT-1 cells verifying the knockdowns. Samples are loaded in duplicates. Tubulin is used as a loading control.</p

The effect of ULK1 depletion on melanin levels is independent of mTORC1 or the ULK1-ATG13-FIP200 complex.

<p>(A) Cells were treated with ULK1, FIP200, ATG13, TYR or control siRNA. Note: the data from one duplex is shown for FIP200 and Atg13, and a second siRNA duplex for each had identical effects. siRNA transfections were performed on day 1 and 3 after plating, and melanin was quantified on day 6. Control and ULK1 knockdown cells were incubated with 100nM Rapamycin (Rapa, +) or DMSO (-) starting from day 1. The data shown represents at least three individual experiments performed in duplicates, and values are normalized to control samples within each experiment. p values for the differences between the treatments and control were determined by a paired student’s t-test. (B) Cells treated as described in (A) were subjected to western blot analysis. mTORC1 activity was monitored by S6K phosphorylation, showing no significant effect after ULK1 depletion. (C) siRNA depletion of cells treated as in (A) was verified by western blot analysis. (D) Changes in basal autophagy were monitored by LC3-I and LC3-II levels, indicating an inhibitory effect for ULK1, ATG13 and FIP200 depletion in these cells. LC3-I levels were quantified by ImageJ and normalised to actin. Data shown is the mean of values normalized to actin from 4 independent experiments. Errors are SEM.</p

Calcium modulates Tyr phosphorylation of MEKK2 but not of Lad1.

<p>HeLa cells transfected with GFP-Lad1 were subject to different treatments as indicated (EGF 20 ng/ml; ionomycin (1 µM) or BAPTA-AM (15 µM) for 15 min; PP2 3 µM). GFP-Lad1 was immunoprecipitated with anti GFP Ab (<b>A, B</b>). Endogenous MEKK2 was immunoprecipitated by anti-MEKK2 Ab (<b>C, D</b>). Their phsophorylation on Tyr residues was detected by pY99 Ab. The experiments in this figure were reproduced 3 times.</p

MEKK2-Lad1 interaction is modulated by calcium.

<p>(<b>A</b>) Exogenously expressed GFP-Lad1 was immunoprecipitated from HeLa cells with indicated treatments. The MEKK2 molecules associated with GFP-Lad1 was detected by anti MEKK2 Ab. The lower panel shows the average quantification of three independent experiments. The error bars represent standard deviation. P values (*: P<0.05; **: P<0.01) were obtained using T-test by comparing the vehicle control group and the iononmycin- or the BAPTA-AM-treated group. (<b>B</b>) MEKK2 was immunoprecipitated from HeLa cells without or with treatment of EGF. It was then incubated with recombinant GST-Lad1 in the presence of various concentrations of calcium. The associated GST-Lad1 was detected by anti Lad1 Ab. The lower panel shows the average quantification of three independent experiments. The error bars represent standard deviation. P values (*: P<0.05) were obtained using T-test within each group, EGF-treated or nontreated, by comparing the interaction at 0.25 µM calcium and that without calcium.</p

Changes in calcium concentrations do not affect Lad1 localization but inhibit nuclear MEKK2 accumulation.

<p>Serum starved HeLa cells were pretreated with vehicle, ionomycin (1 µM) or BAPTA-AM (15 µM) for 15 min and then stimulated with EGF (20 ng/ml) for 10 min. The cells were stained with anti Lad1 or anti MEKK2 Abs and visualized with fluorescent microscopy. This experiment was reproduced 3 times.</p