A Novel Role for Mixed Lineage Kinase 3 (MLK3) in B-Raf Activation and Cell proliferation

Abstract

Once the issue is complete and page numbers have been assigned, the citation will change accordingly. KEY WORDS MLK3, cell proliferation, MAP kinase, B-Raf, EGF ABBREVIATIONS CRIB A Novel Role for Mixed Lineage Kinase 3 (MLK3) in B-Raf Activation and Cell Proliferation ABSTRACTThe extracellular signal-regulated kinase (ERK) group of MAPKs is essential for cell proliferation, including that stimulated by mitogens, oncogenic ras and raf. The Raf kinases (especially B-Raf) are ERK-specific, mitogen-activated MAP3Ks. Mixed lineage kinase-3 (MLK3) is a MAP3K previously thought to be a selective regulator of the JNK group of MAPKs. Surprisingly, we found that silencing of mlk3 by RNAi suppresses mitogen and cytokine activation not only of JNK but of ERK and p38 as well. Silencing mlk3 also blocks mitogen-stimulated phosphorylation of B-Raf at Thr598 and Ser601-a step required for B-Raf activation. Finally, silencing mlk3 prevents serum-stimulated cell proliferation and the proliferation of tumor cells bearing either oncogenic Ki-Ras or loss of function neurofibromatosis-1 (NF1) or NF2 mutations. The proliferation of tumor cells with activating mutations in B-raf or raf-1 are unaffected by silencing mlk3. These results define a new role for MLK3 in B-Raf activation, ERK signaling and cell proliferation. Accordingly, targeting MLK3 could be beneficial to the treatment of tumors with activated receptor tyrosine kinase or ras mutations, and to the treatment of NF1 or NF2 tumors.Mammalian mitogen-activated protein kinase (MAPK) signaling cascades function to integrate and transmit signals from the cell surface to the nucleus resulting in the regulation of specific gene expression. A common feature of MAPK signaling is a three-tiered core signaling module in which MAPKs are activated by Tyr and Thr phosphorylation that is catalyzed by MAPK kinases (MKKs) which are in turn phosphorylated on Ser and Thr residues by MAPK kinase kinases (MAP3Ks). 1 To date, many MAP3Ks have been identified that respond to a wide range of extracellular stimuli and relay specific signals to a relatively small number of downstream MKKs. The mechanism of regulation of the MAP3Ks by upstream activators and extracellular stimuli remains an area of intense study and the biological function of many of these enzymes have yet to be elucidated.MLK3 (mixed lineage kinase 3) is a MAP3K that was shown to specifically activate the MKKs, MKK4 and MKK3, which in turn activate the Jun N-terminal kinase (JNK) and p38 pathways, respectively. 2,3 Overexpression of MLK3 has also been reported to activate NF-κB. 4 Extracellular stimuli that activate MLK3 include tumor necrosis factor alpha (TNFα), ceramide and engagement of the T-cell receptor by costimulation with anti-CD3 and anti-CD28 antibodies. 4,5 Upstream activators of MLK3 include Cdc42 and germinal center kinase. 6,7 MLK3 contains an amino-terminal Src-homology 3 (SH3) domain, a kinase domain, a leucine zipper and a Cdc42/Rac interactive binding (CRIB) motif. MLK3 can dimerize through its leucine zippers an...

MLK3 regulates bone development downstream of the faciogenital dysplasia protein FGD1 in mice

“…In particular, this study establishes MLK3 alongside TAK1 as a MAP3K relevant to osteoblast differentiation in vivo. Whereas TAK1 only regulates p38 MAPK in osteoblasts, MLK3 contributes to both p38 and ERK activation, validating previous in vitro studies (20,21,41). Given that both MLK3 and TAK1 contribute to p38 activation, it will be of particular interest to examine how these roles are coordinately regulated and what, if any, overlap exists between the 2 MAP3K in vivo.…”

A Role for Mixed Lineage Kinases in Regulating Transcription Factor CCAAT/Enhancer-binding Protein-β-dependent Gene Expression in Response to Interferon-γ

“…siRNA sequences that can specifically knock down the expression of human and mouse MLK3 genes were based on the sequences described recently (28). The following double-stranded oligonucleotides bearing the target sequences were synthesized and cloned into the SalI and XbaI sites of the targeting vector pSuppressor-Neo (Imgenex, Inc.): mouse MLK3, 5Ј-TCGAGGGGCAGCGATGTCTGGA-GCTTGAGTACTGAAGCTCCAGACATCGCTGCCCTTTTT-3Ј; and human MLK3, 5Ј-TCGAGGGGCAGTGACGTCTGGAGTTTGAGTAC-TGAAACTCCAGACGTCACTGCCCTTTTT-3Ј.…”

“…The following double-stranded oligonucleotides bearing the target sequences were synthesized and cloned into the SalI and XbaI sites of the targeting vector pSuppressor-Neo (Imgenex, Inc.): mouse MLK3, 5Ј-TCGAGGGGCAGCGATGTCTGGA-GCTTGAGTACTGAAGCTCCAGACATCGCTGCCCTTTTT-3Ј; and human MLK3, 5Ј-TCGAGGGGCAGTGACGTCTGGAGTTTGAGTAC-TGAAACTCCAGACGTCACTGCCCTTTTT-3Ј. Because of the differences in the primary sequences of mouse and human MLK3 mRNAs (underlined bases), these siRNAs target only the cognate mRNA for degradation in the cells (28).…”

Mixed Lineage Kinase MLK4 Is Activated in Colorectal Cancers Where It Synergistically Cooperates with Activated RAS Signaling in Driving Tumorigenesis

“…8) followed sequentially by a kinase domain, a leucine-zipper region and a Cdc42/Rac-interactive binding (CRIB) motif. The carboxyl terminus of all MLKs is proline-rich but diverges significantly among different members of the family, suggesting that this region serves different regulatory functions (9,10).…”