MLK3 is required for mitogen activation of B-Raf, ERK and cell proliferation

Abstract

The ERK group of mitogen-activated protein kinases (MAPKs) is essential for cell proliferation stimulated by mitogens, oncogenic ras and raf (ref. 1). All MAPKs are activated by MAP3K/MEK/MAPK core pathways and the Raf proto-oncoproteins, especially B-Raf, are ERK-specific MAP3Ks (refs 1-3). Mixed lineage kinase-3 (MLK3) is a MAP3K that was thought to be a cytokine-activated, and comparatively selective, regulator of the JNK group of MAPKs (refs 1, 4-6). Here we report that silencing of mlk3 by RNAi suppressed mitogen and cytokine activation not only of JNK but of ERK and p38 as well. Silencing mlk3 also blocked mitogen-stimulated phosphorylation of B-Raf at Thr 598 and Ser 601, a step required for B-Raf activation. Furthermore, silencing mlk3 prevented serum-stimulated cell proliferation and the proliferation of tumour cells bearing either oncogenic Ki-Ras or loss-of-function neurofibromatosis-1 (NF1) or NF2 mutations. The proliferation of tumour cells containing activating B-raf or raf-1 mutations was unaffected by silencing mlk3. Our results define an unexpected role for MLK3 in mitogen regulation of B-Raf, ERK and cell proliferation.

Inhibition of mixed-lineage kinase (MLK) activity during G2-phase disrupts microtubule formation and mitotic progression in HeLa cells

“…In contrast, CEP-11004 treatment inhibited MLK3 induced ERK activation (Fig. 4C), which is consistent with a role for MLK3 in activating the ERK pathway in tumor cells [14]. This further supports the opposing roles for MLK3 in promoting proliferation of non-neuronal tumor cells but cell death of neuronal cells.…”

“…MLK3 function in cell death has been largely attributed to its role in activating the stress activated protein kinases, which include JNK and p38 [1]. However, recent studies also show that MLK3 can mediate cell proliferation through activation of the B-Raf and ERK pathway [14]. To identify mechanisms by which MLK proteins may affect cell proliferation, we examined the effects of MLK inhibition during G2-phase on subsequent progression through mitosis.…”

“…For example, overexpression of MLK3 has been reported to induce NIH 3T3 cell transformation and growth on soft agar through a mechanism involving MEK1 and ERK activation [13]. Similarly, growth factors may utilize MLK3 to activate B-Raf and the ERK pathway in proliferating tumor cells [14]. MLK3 activity may regulate cell proliferation by affecting centrosome organization and microtubule stability during mitosis through a mechanism that is JNK-independent [15].…”

MAP3Ks as central regulators of cell fate during development

“…However, MLK3 knockout mice have reduced JNK activation in response to TNF␣ (Brancho et al, 2005). B-raf/ERK activation by mitogens is also regulated by MLK3 (Chadee and Kyriakis, 2004), but this is apparently dispensable during development. Of the known MLKs, MLK3 and MLK7 are detected in the adult heart (Bloem et al, 2001).…”

Mixed Lineage Kinase Phosphorylates Transcription Factor E47 and Inhibits TrkB Expression to Link Neuronal Death and Survival Pathways

“…Nonetheless, mammalian MLK3 is localized to the centrosome, and its activity is enhanced during G 2 /M phase transition, when the JNK pathway remains inactive (23). It has been reported that silencing MLK3 can block mitogen-stimulated B-Raf activation and prevent serum-or Ki-Ras-stimulated cell proliferation (24). In addition, MLK2 has been involved in modulation of NeuroD and Alien transcription factors (25,26).…”