Osmotic and heat stress-dependent regulation of MLK4β and MLK3 by the CHIP E3 ligase in ovarian cancer cells

• Published in: Cellular signalling 2017-11, Vol.39, p.66-73
• Main Authors: Blessing, Natalya A., Kasturirangan, Srimathi, Zink, Evan M., Schroyer, April L., Chadee, Deborah N.
• Format: Article
• Language: English
• Subjects: Benzoquinones - pharmacology; Carboxyl-terminus of Hsc70-interacting protein (CHIP); Cell Line, Tumor; Female; Heat-Shock Response; HEK293 Cells; HSC70 Heat-Shock Proteins - metabolism; HSP90 Heat-Shock Proteins - antagonists & inhibitors; Humans; Lactams, Macrocyclic - pharmacology; MAP Kinase Kinase Kinases - genetics; MAP Kinase Kinase Kinases - metabolism; Mitogen activated protein kinase (MAPK); Mitogen-Activated Protein Kinase Kinase Kinase 11; Mixed Lineage Kinase 3 (MLK3); Mixed Lineage Kinase 4 (MLK4); Osmotic Pressure; Ovarian Neoplasms - enzymology; Protein Binding; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; Stress signalling; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitination
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2017.07.021

Mixed Lineage Kinase 3 (MLK3), a member of the MLK subfamily of protein kinases, is a mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) that activates MAPK signalling pathways and regulates cellular responses such as proliferation, invasion and apoptosis. MLK4β, another member of the MLK subfamily, is less extensively studied, and the regulation of MLK4β by stress stimuli is not known. In this study, the regulation of MLK4β and MLK3 by osmotic stress, thermostress and heat shock protein 90 (Hsp90) inhibition was investigated in ovarian cancer cells. MLK3 and MLK4β protein levels declined under conditions of prolonged osmotic stress, heat stress or exposure to the Hsp90 inhibitor geldanamycin (GA); and MLK3 protein declined faster than MLK4β. Similar to MLK3, the reduction in MLK4β protein in cells exposed to heat or osmotic stresses occurred via a mechanism that involves the E3 ligase, carboxy-terminus of Hsc70-interacting protein (CHIP). Both heat shock protein 70 (Hsp70) and CHIP overexpression led to polyubiquitination and a decrease in endogenous MLK4β protein, and MLK4β was ubiquitinated by CHIP in vitro. In untreated cells and cells exposed to osmotic and heat stresses for short time periods, small interfering RNA (siRNA) knockdown of MLK4β elevated the levels of activated MLK3, c-Jun N-terminal kinase (JNK) and p38 MAPKs. Furthermore, MLK3 binds to MLK4β, and this association is regulated by osmotic stress. These results suggest that in the early response to stressful stimuli, MLK4β-MLK3 binding is important for regulating MLK3 activity and MAPK signalling, and after prolonged periods of stress exposure, MLK4β and MLK3 proteins decline via CHIP-dependent degradation. These findings provide insight into how heat and osmotic stresses regulate MLK4β and MLK3, and reveal an important function for MLK4β in modulating MLK3 activity in stress responses. •Osmotic stress, heat, and Hsp90 inhibition reduce MLK4β and MLK3 protein levels.•CHIP ubiquitinates MLK4β.•CHIP mediates osmotic and heat stress-induced decline of MLK4β.•Osmotic stress regulates MLK4β-MLK3 association.•MLK4β negatively regulates MLK3 activity and MAPK signalling pathways.