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. 2011 Dec 25;44(2):165-9.
doi: 10.1038/ng.1041.

Frequent somatic mutations in MAP3K5 and MAP3K9 in metastatic melanoma identified by exome sequencing

Mitchell S Stark  1 , Susan L Woods, Michael G Gartside, Vanessa F Bonazzi, Ken Dutton-Regester, Lauren G Aoude, Donald Chow, Chris Sereduk, Natalie M Niemi, Nanyun Tang, Jonathan J Ellis, Jeffrey Reid, Victoria Zismann, Sonika Tyagi, Donna Muzny, Irene Newsham, YuanQing Wu, Jane M Palmer, Thomas Pollak, David Youngkin, Bradford R Brooks, Catherine Lanagan, Christopher W Schmidt, Bostjan Kobe, Jeffrey P MacKeigan, Hongwei Yin, Kevin M Brown, Richard Gibbs, Jeffrey Trent, Nicholas K Hayward
Affiliations

Frequent somatic mutations in MAP3K5 and MAP3K9 in metastatic melanoma identified by exome sequencing

Mitchell S Stark et al. Nat Genet. .

Abstract

We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.

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Figures

Figure 1
Figure 1
Schema depicting MAP3K5 and MAP3K9 domain structure, phosphorylation sites (below each gene) and the position of validated somatic non-synonymous mutations identified by whole-exome sequencing (above each gene).
Figure 2
Figure 2
Mutations found in melanoma are predicted to affect the function or regulation of MAP3K5 and MAP3K9 kinase domains. (a) The top panel shows MAP3K5 kinase domain (PDB ID 2CLQ). The kinase domain was aligned with PDB ID 2PHK (not shown) to show approximate binding positions of ATP (yellow) and a substrate (light green). Bottom left panel shows wild-type I780 (green) and its surroundings; bottom right panel shows mutant F780and its surroundings (green). (b) The top left panel shows MAP3K9 (PDB ID 3DTC) kinase domain. The top right panel is the same domain rotated by 180 degrees to show the location of all residues of interest. The kinase domain has been aligned with PDB ID 2PHK (not shown) to show approximate binding positions of ATP (yellow) and a substrate (green). The middle panels show the wild-type residues in green (R160, P263 and E319) and their surroundings; the bottom panels show the mutant residues in green (C160, L263 and K319). The disruption of hydrogen bonds can be seen for R160C and E319K. MAP3K9 D176N is in a disordered loop and the MAP3K5 E663 residue is not resolved in the crystal structure; therefore, they do not appear in the diagrams. Only side chains within 5Å of the residue of interest are depicted.
Figure 3
Figure 3
Melanoma-associated mutations decrease the kinase activity of MAP3K9 and MAP3K5. HEK293T cells were transfected with wild type and mutant MAP3K9 (K171A and W333X) or MAP3K5 (E663K and I780F) to assess the effect of mutation on kinase activity in-vitro. Autophosphorylation of MAP3K9 (a) or MAP3K5 (b), and phosphorylation of the kinase substrate myelin basic protein (MBP), were measured by 32P-γATP incorporation and quantitated using PhosphorImager. Error bars represent standard deviations; *p<0.05, **p<0.01, ***p<0.001 by two-tailed Student’s t-test.
Figure 4
Figure 4
Mutation of MAP3K5 or MAP3K9 results in decreased phospho-MEK/ERK and phospho-JNK compared to wild type proteins, indicating altered downstream signaling through multiple pathways. HEK293T cell lines were transfected with expression plasmids for EGFP, wild-type or mutant MAP3K5 (a) or EGFP, wild-type or mutant MAP3K9 (b) for 48 h before analysis of protein expression and MAPK signaling by Western blot using the indicated antibodies. P-indicates the phosphorylated protein is being detected. Results are representative of three independent repeats of this experiment.
Figure 5
Figure 5
Reduced MAP3K9 level protects melanoma cells from chemotherapeutic treatment. (a) Melanoma cell lines (UACC903 and UACC647) were transfected with siRNA targeting GFP (Control) or three independent siRNAs targeting MAP3K9 (siRNA 1-3) for 120 h. Expression of MAP3K9 determined by qRT-PCR normalized to GAPDH and relative to control siRNA is shown. Experiment performed in triplicate; error bars denote standard deviation. All three independent siRNAs resulted in decreased expression of MAP3K9. (b) UACC903 (i) and UACC647 (ii) cells were transfected as in a. for 24 h before treatment with the indicated dose of temozolomide (TMZ). Cell viability determined after 5 days indicated that reduced MAP3K9 expression in the presence of TMZ resulted in a resistant phenotype.
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