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...

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.

The mechanism of pathogenesis associated with APOL1 polymorphisms and risk for non-diabetic chronic kidney disease (CKD) is not fully understood. Prior studies have minimized a causal role for the circulating APOL1 protein, thus efforts to understand kidney pathogenesis have focused on APOL1 expressed in renal cells. Of the kidney cells reported to express APOL1, the proximal tubule expression patterns are inconsistent in published reports, and whether APOL1 is synthesized by the proximal tubule or possibly APOL1 protein in the blood is filtered and reabsorbed by the proximal tubule remains unclear. Using both protein and mRNA in situ methods, the kidney expression pattern of APOL1 was examined in normal human and APOL1 bacterial artificial chromosome transgenic mice with and without proteinuria. APOL1 protein and mRNA was detected in podocytes and endothelial cells, but not in tubular epithelia. In the setting of proteinuria, plasma APOL1 protein did not appear to be filtered or reabsorbed by the proximal tubule. A side-by-side examination of commercial antibodies used in prior studies suggest the original reports of APOL1 in proximal tubules likely reflects antibody non-specificity. As such, APOL1 expression in podocytes and endothelia should remain the focus for mechanistic studies in the APOL1-mediated kidney diseases.

The renal podocyte is central to the filtration function of the kidney that is dependent on maintaining both highly organized, branched cell structures forming foot processes, and a unique cell‐cell junction, the slit diaphragm. Our recent studies investigating the developmental formation of the slit diaphragm identified a novel claudin family tetraspannin, TM4SF10, which is a binding partner for ADAP (also known as Fyn binding protein Fyb). To investigate the role of ADAP in podocyte function in relation to Fyn and TM4SF10, we examined ADAP knockout (KO) mice and podocytes. ADAP KO mice developed glomerular pathology that began as hyalinosis and progressed to glomerulosclerosis, with aged male animals developing low levels of albuminuria. Podocyte cell lines established from the KO mice had slower attachment kinetics compared to wild‐type cells, although this did not affect the total number of attached cells nor the ability to form focal contacts. After attachment, the ADAP KO cells did not attain typical podocyte morphology, lacking the elaborate cell protrusions typical of wild‐type podocytes, with the actin cytoskeleton forming circumferential stress fibers. The absence of ADAP did not alter Fyn levels nor were there differences between KO and wild‐type podocytes in the reduction of Fyn activating phosphorylation events with puromycin aminonucleoside treatment. In the setting of endogenous TM4SF10 overexpression, the absence of ADAP altered the formation of cell‐cell contacts containing TM4SF10. These studies suggest ADAP does not alter Fyn activity in podocytes, but appears to mediate downstream effects of Fyn controlled by TM4SF10 involving actin cytoskeleton organization.