The E3 Ligase CHIP Mediates Ubiquitination and Degradation of Mixed-Lineage Kinase 3

Abstract

Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that has regulatory roles in diverse biological processes such as proliferation, migration, differentiation, invasion, and neuronal cell apoptosis (1-9). MLK3 activates the c-Jun N-terminal kinase (JNK) and p38 MAPK signaling pathways, and in certain cellular contexts, MLK3 has a kinase-independent function in the activation of extracellular signal-regulated kinase (ERK) MAPK signaling (1, 10-12). Aberrant levels of total and/or activated MLK3 protein have been observed in ovarian and breast cancer cells in comparison to nonneoplastic ovarian and breast epithelial cells (5, 6). Moreover, a critical function for MLK3 in breast and ovarian cancer cell invasion was recently identified (5, 6). The regulation of MLK3 by phosphorylation, dimerization, and interaction with Rho GTPases has been the focus of numerous studies; however, the mechanism(s) that controls the level of total MLK3 protein in cells remains poorly understood (13-16).We previously identified that in SKOV3 ovarian cancer cells, the proinflammatory cytokines tumor necrosis factor alpha (TNF-␣) and interleukin-1␤ (IL-1␤) induced the activation of MLK3 and that TNF-␣ also stimulated MLK3 ubiquitination (17). TNF-␣-dependent activation of MLK3 is facilitated by TNF receptor-associated factor 2 (TRAF2) and TRAF6, which interact with MLK3 and are recruited to the TNF receptor (17,18). Recently, it was demonstrated that TRAF6 promotes K63-linked polyubiquitination of MLK3 in vitro (19). Furthermore, MLK3 activation in response to IL-1␤ is dependent on TRAF6 binding and K63-linked polyubiquitination (17,19). MLK3 also undergoes K48-linked polyubiquitination; however, the role of this modification in MLK3 protein function and turnover has not been elucidated (17).MLK3 interacts with the chaperone protein Hsp90 and the cochaperone protein p50cdc37, which participate in the folding and stabilization of signaling proteins involved in proliferation, apoptosis, and survival (20, 21). The dissociation of Hsp90 from its client proteins can be triggered by specific stimuli or by exposure to Hsp90 ATPase inhibitors such as geldanamycin (GA) (22,23). Treatment of cancer cell lines with GA causes a reduction in the level of endogenous MLK3 protein and an inhibition of JNK signaling, which suggests that the Hsp90-MLK3 interaction is important for MLK3 function and stability (21,24). The disruption of Hsp90 chaperone-client interactions can lead to ubiquitination and proteasomal degradation of the client proteins via Hsp70-dependent recruitment of the carboxyl terminus of Hsc70-interacting protein (CHIP) E3 ubiquitin ligase (20,22,23). CHIP is a Ubox E3 protein that mediates cytosolic protein polyubiquitination and targets Hsp70-bound proteins for degradation by the 26S proteasome, thereby coupling the chaperone and ubiquitin-proteasome systems (25)(26)(27)(28). In this study, we investigated the role of CHIP in the ubiquitination and degradation of MLK3 in response to GA, hea...

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

“…To determine if MLK4β protein is also regulated by osmotic and heat stresses, SKOV3 ovarian cancer cells were treated with 250 mM sorbitol (osmotic stress) or cultured at 42 °C over an 8 h time course (Fig. 1A) [21]. In sorbitol-treated SKOV3 cells, endogenous MLK4β was significantly reduced at 2, 4, 6 and 8 h in comparison to untreated cells, whereas endogenous MLK3 was significantly reduced at 0.3, 1, 2, 4, 6 and 8 h, in comparison to untreated cells (Figs.…”

“…Cells were either left untreated, treated with vehicle, or treated with a final concentration of 10 μM GA (InvivoGen, San Diego, CA, USA), 50 μM cycloheximide (CHX) (Thermo Fisher Scientific, Rockford, IL, USA), 250 mM sorbitol, or cultured at 42 °C for the indicated time periods, as previously described [21]. The cells were harvested immediately after treatments and immunoprecipitations were performed or whole cell extracts were prepared.…”

“…Mammalian FLAG-CHIP, GST-MLK4β and His-MLK3 proteins were expressed in BL21 E. coli cells and purified as performed previously [21]. …”

“…A Customized E2 Screening Kit (UBPBio, Aurora, CO, USA) provided the purified E2 enzymes, UBE1 enzyme, human ubiquitin, and these components were mixed with His-CHIP and GST-MLK4β to carry out the ubiquitination reactions according to the manufacturer’s instructions [21]. …”

“…Hsp70 recruits an E3 ubiquitin ligase, carboxy-terminus of Hsc70-interacting protein (CHIP), that ubiquitinates and marks target proteins for degradation by the 26S proteasome [17]. We have previously shown that MLK3 undergoes CHIP-mediated polyubiquitination and proteasomal degradation in response to GA, heat shock and osmotic shock [21]. In light of recent findings that MLK4 influences cellular responses important for tumor development, we sought to gain insight into the modulation of MLK4β protein in ovarian cancer cells.…”