Osmotic and heat stress-dependent regulation of MLK4? and MLK3 by the CHIP E3 ligase in ovarian cancer cells.

ABSTRACT: 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. MLK4?, another member of the MLK subfamily, is less extensively studied, and the regulation of MLK4? by stress stimuli is not known. In this study, the regulation of MLK4? and MLK3 by osmotic stress, thermostress and heat shock protein 90 (Hsp90) inhibition was investigated in ovarian cancer cells. MLK3 and MLK4? 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 MLK4?. Similar to MLK3, the reduction in MLK4? 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 MLK4? protein, and MLK4? 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 MLK4? elevated the levels of activated MLK3, c-Jun N-terminal kinase (JNK) and p38 MAPKs. Furthermore, MLK3 binds to MLK4?, and this association is regulated by osmotic stress. These results suggest that in the early response to stressful stimuli, MLK4?-MLK3 binding is important for regulating MLK3 activity and MAPK signalling, and after prolonged periods of stress exposure, MLK4? and MLK3 proteins decline via CHIP-dependent degradation. These findings provide insight into how heat and osmotic stresses regulate MLK4? and MLK3, and reveal an important function for MLK4? in modulating MLK3 activity in stress responses.

SUBMITTER: Blessing NA 

PROVIDER: S-EPMC5592140 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature