Figure 5. MK2 positively regulates autophagy.

(A) Effects of wild-type, dominant negative (K/R) and constitutively active (EE) MK2 on endogenous Beclin 1 S90 phosphorylation, MK2/MK3 activation (levels of p-HSP27) and autophagy (levels of p62 degradation and LC3-II conversion) in HeLa cells grown in normal medium (starvation−) or HBSS for 2 hr (starvation+). Actin is shown as a loading control. (B) Effects of wild-type, dominative negative (K/R) and constitutively active (EE) MK2 on autophagy (GFP-LC3 puncta numbers in the presence or absence of 100 nM bafilomycin A1) in HeLa cells grown in normal medium (starvation−) or HBSS for 3 hr (starvation+). Bars are mean + SEM of triplicate samples (>50 cells analyzed per sample). Similar results were observed in three independent experiments. **p < 0.01, *p < 0.05, NS, not significant; one-way ANOVA for indicated comparisons. (C) Beclin 1 S90 phosphorylation and autophagy (levels of p62 degradation and LC3-II conversion) in MK2^−/−/MK3^−/− MEFs and MK2^−/−/MK3^−/− MEFs stably transformed with wild-type MK2. Cells were grown in normal medium (starvation−) or HBSS for 2 hr (starvation+). (D) Quantitation of GFP-LC3 puncta (autophagosomes) in MK2^−/−/MK3^−/− MEFs and MK2^−/−/MK3^−/− MEFs stably transformed with wild-type MK2 during growth in normal media or HBSS (starvation) for 3 hr in the presence or absence of 100 nM bafilomycin A1. Bars are mean + SEM of triplicate samples (>50 cells analyzed per sample). Similar results were observed in three independent experiments. ***p < 0.001, NS, not significant; one-way ANOVA. p < 0.001 for the magnitude of change between normal and starvation conditions in MK2^−/−/MK3^−/− MEFs vs that in in MK2^−/−/MK3^−/− + MK2 MEFs; two-way ANOVA. See also Figure 5—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.05289.011

Figure 5—figure supplement 1. Dominant p38α inhibits starvation-induced MK2 activation and Beclin 1 S90 phosphorylation.

(A) Effects of dominant-negative (DN) JNK1 and DN p38α on Beclin 1 S90 phosphorylation, MK2 activation (MK2 band shift and p-HSP27) and autophagy (p62 degradation and LC3-II conversion) in HeLa cells grown in normal media (starvation−) or HBSS for 2 hr (starvation+). (B) Constitutively active MK2 (MK2 EE) blocks DN p38α suppression of starvation-induced Beclin 1 S90 phosphorylation. HeLa cells were co-transfected with plasmids expressing Flag-DN p38α and either empty vector or MK2 EE and grown in normal media (starvation−) or HBSS for 2 hr (starvation+).

DOI: http://dx.doi.org/10.7554/eLife.05289.012