Journal: Biochimica et biophysica acta. General subjects

Article Title: Extracellular matrix stiffness regulates degradation of MST2 via SCF βTrCP .

doi: 10.1016/j.bbagen.2022.130238

Figure Lengend Snippet: Fig. 4. Perturbation of ILK and the actomyosin cytoskeleton regulates MST2 levels: A) Immunoblots of MCF10A cells treated with an Integrin-Linked Kinase (ILK) inhibitor (CPD022) for 1, 3 and 6 h. AKT phosphorylation was detected to assess the efficiency of ILK inhibition. β-actin was used as loading control. B) Immunoblot for MST2 of MCF10A cells treated with FAK inhibitor (FAKi 14) for 1, 3 and 6 h. FAK phosphorylation was detected to monitor its inhibition. β-actin was used as loading control. C) Immunoblots of MCF10A cells treated with CPD022 or MLN4294 in combination with MnCl2 for 6 h. Both inhibition of CUL1 neddylation and ILK reversed the effect of integrin hyperactivation. β-actin was used as loading controls. Note that the band corresponding to CUL1 neddylation (indicated by an arrow) disappeared in the presence of MLN4924 indicating that the inhibition was efficient. D) Co-IP revealed that the interaction of endogenous MST2 and βTrCP decreases after ILK inhibition, despite integrin hyperactivation. MCF10A cells were treated with DMSO (ct), CPD022, MnCl2 or CPD022 concomitantly with MnCl2 for 6 h. Co-IP was performed with an MST2 antibody or a control IgG, followed by incubation with protein A/G-coated beads. βTrCP and MST2 were only detected in the MST2 immunoprecipitants. MST2 was detected to confirm the Co-IP. Arrows indicate bands corresponding to IgG that were detected by the secondary antibody. E) Immunoblot for MST2 of MCF10A cells treated with a myosin ATPase activity inhibitor (Blebbistatin, 2.5 μM) for 1, 3 and 6 h. β-actin was used as loading control. (A–E) Fold changes of protein levels and protein phosphorylation (phosphorylated/total) are shown under their respective immunoblots. F) Schematic depicting the pathway of MST2 degradation induced by ECM stiffness. In a stiff ECM, hyperactive integrin signaling results in ILK activation and actomyosin contraction leading to ubiquitylation of MST2 by SCFβTrCP and consequent MST2 degradation via proteasome 26S. This schematic was generated with Biorender.

Article Snippet: To screen the pathways mediating MST2 degradation, MCF10A cells were treated for 1, 3 and 6 h with the inhibitors for ILK (1 μM CPD022, Calbiochem, #407331), FAK (5 μM FAK inhibitor 14, Tocris # 3414), PI3K (30 μM LY294002, Gibco # PHZ1144), AKT (20 μM MK2206, Cayman Biochemicals #1032350-13-2) and myosin ATPase activity inhibitor (5 μM Blebbistatin, Tocris #1852).

Techniques: Western Blot, Phospho-proteomics, Inhibition, Control, Co-Immunoprecipitation Assay, Incubation, Activity Assay, Activation Assay, Generated