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Mixed lineage kinase 3 gene mutations in mismatch repair deficient gastrointestinal tumours.

Velho S, Oliveira C, Paredes J, Sousa S, Leite M, Matos P, Milanezi F, Ribeiro AS, Mendes N, Licastro D, Karhu A, Oliveira MJ, Ligtenberg M, Hamelin R, Carneiro F, Lindblom A, Peltomaki P, Castedo S, Schwartz S, Jordan P, Aaltonen LA, Hofstra RM, Suriano G, Stupka E, Fialho AM, Seruca R - Hum. Mol. Genet. (2009)

Bottom Line: Mixed lineage kinase 3 (MLK3) is a serine/threonine kinase, regulating MAPkinase signalling, in which cancer-associated mutations have never been reported.MLK3 mutations were significantly associated with MSI phenotype in primary tumours (P = 0.0005), occurring in 21% of the MSI carcinomas.Further, we demonstrated that MLK3 missense mutations found in MSI gastrointestinal carcinomas are functionally relevant.

View Article: PubMed Central - PubMed

Affiliation: IPATIMUP-Institute of Molecular Pathology and Immunology of the University of Porto, 4200-465 Porto, Portugal.

ABSTRACT
Mixed lineage kinase 3 (MLK3) is a serine/threonine kinase, regulating MAPkinase signalling, in which cancer-associated mutations have never been reported. In this study, 174 primary gastrointestinal cancers (48 hereditary and 126 sporadic forms) and 7 colorectal cancer cell lines were screened for MLK3 mutations. MLK3 mutations were significantly associated with MSI phenotype in primary tumours (P = 0.0005), occurring in 21% of the MSI carcinomas. Most MLK3 somatic mutations identified were of the missense type (62.5%) and more than 80% of them affected evolutionarily conserved residues. A predictive 3D model points to the functional relevance of MLK3 missense mutations, which cluster in the kinase domain. Further, the model shows that most of the altered residues in the kinase domain probably affect MLK3 scaffold properties, instead of its kinase activity. MLK3 missense mutations showed transforming capacity in vitro and cells expressing the mutant gene were able to develop locally invasive tumours, when subcutaneously injected in nude mice. Interestingly, in primary tumours, MLK3 mutations occurred in KRAS and/or BRAF wild-type carcinomas, although not being mutually exclusive genetic events. In conclusion, we have demonstrated for the first time the presence of MLK3 mutations in cancer and its association to mismatch repair deficiency. Further, we demonstrated that MLK3 missense mutations found in MSI gastrointestinal carcinomas are functionally relevant.

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Related in: MedlinePlus

3D model to predict how MLK3 mutations could interfere with the MLK3 protein function. (A) Predictive 3D model of the MLK3 kinase domain, constructed using the crystal structure from MLK1 (Pdb: 3dtc; 70% amino acid identity with MLK3) as a template (17). Kinase active site, nucleotide binding loop, hinge region, the N- and C-termini lobes and the six missense mutations identified in this study are highlighted. (a1–a6) Magnified views of the different MLK3 mutations. Prediction of the impact of the six amino acid substitutions is described in the Discussion section. Dotted yellow lines represent potential H-bonds. (B) Prediction of mutations effects on the MLK3 kinase domain stability, based on the site directed mutator (SDM) program (37). SS element, secondary structure element (H, helix, C, coil, L, loop); HB, hydrogen bond; OS, overall stability.
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DDP536F2: 3D model to predict how MLK3 mutations could interfere with the MLK3 protein function. (A) Predictive 3D model of the MLK3 kinase domain, constructed using the crystal structure from MLK1 (Pdb: 3dtc; 70% amino acid identity with MLK3) as a template (17). Kinase active site, nucleotide binding loop, hinge region, the N- and C-termini lobes and the six missense mutations identified in this study are highlighted. (a1–a6) Magnified views of the different MLK3 mutations. Prediction of the impact of the six amino acid substitutions is described in the Discussion section. Dotted yellow lines represent potential H-bonds. (B) Prediction of mutations effects on the MLK3 kinase domain stability, based on the site directed mutator (SDM) program (37). SS element, secondary structure element (H, helix, C, coil, L, loop); HB, hydrogen bond; OS, overall stability.

Mentions: Within the MLK3 kinase domain, we mapped six single-amino acid changes (A165S, R240C, P252H, A296T, A352R and A356V). These mutations were spread along the kinase domain and did not occur at specific and highly conserved functional amino acid positions of the kinase catalytic core. Based on our predictive model, all these missense mutations affect surface-exposed residues, causing overall destabilization of the molecule (Fig. 2A and B). Mutants A165S, P252H and A352R are likely to induce the formation of new intramolecular hydrogen bonds with neighbouring residues, producing conformational changes in the protein backbone (Fig. 2a1, a3, a5). R240C mutation occurs at a coil region that runs parallel to an alpha helix in the C-terminal lobe of the MLK3 kinase domain (Fig. 2a2). The replacement of a larger residue (R) by a smaller one (C) creates higher dissociation energy, resulting in a decrease of the steric hindrance. The A at position 296 is located at an exposed loop of the MLK3 kinase domain. The substitution of a hydrophobic A residue by and hydrophilic T residue (A296T) is predicted to destabilize this area of the protein, impacting most probably the scaffold properties of MLK3 (Fig. 2a4). The A residue at position 356 is part of an Alanine-rich helix of MLK3. The substitution of A residue by a V (A356V) does not alter the polarity at this site, as both are non-polar aliphatic; nevertheless, a Valine occupies more space than an Alanine and, therefore, the helix is likely to assume a different shape/deformation and/or suffer a destabilizing effect (Fig. 2a6).


Mixed lineage kinase 3 gene mutations in mismatch repair deficient gastrointestinal tumours.

Velho S, Oliveira C, Paredes J, Sousa S, Leite M, Matos P, Milanezi F, Ribeiro AS, Mendes N, Licastro D, Karhu A, Oliveira MJ, Ligtenberg M, Hamelin R, Carneiro F, Lindblom A, Peltomaki P, Castedo S, Schwartz S, Jordan P, Aaltonen LA, Hofstra RM, Suriano G, Stupka E, Fialho AM, Seruca R - Hum. Mol. Genet. (2009)

3D model to predict how MLK3 mutations could interfere with the MLK3 protein function. (A) Predictive 3D model of the MLK3 kinase domain, constructed using the crystal structure from MLK1 (Pdb: 3dtc; 70% amino acid identity with MLK3) as a template (17). Kinase active site, nucleotide binding loop, hinge region, the N- and C-termini lobes and the six missense mutations identified in this study are highlighted. (a1–a6) Magnified views of the different MLK3 mutations. Prediction of the impact of the six amino acid substitutions is described in the Discussion section. Dotted yellow lines represent potential H-bonds. (B) Prediction of mutations effects on the MLK3 kinase domain stability, based on the site directed mutator (SDM) program (37). SS element, secondary structure element (H, helix, C, coil, L, loop); HB, hydrogen bond; OS, overall stability.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2807374&req=5

DDP536F2: 3D model to predict how MLK3 mutations could interfere with the MLK3 protein function. (A) Predictive 3D model of the MLK3 kinase domain, constructed using the crystal structure from MLK1 (Pdb: 3dtc; 70% amino acid identity with MLK3) as a template (17). Kinase active site, nucleotide binding loop, hinge region, the N- and C-termini lobes and the six missense mutations identified in this study are highlighted. (a1–a6) Magnified views of the different MLK3 mutations. Prediction of the impact of the six amino acid substitutions is described in the Discussion section. Dotted yellow lines represent potential H-bonds. (B) Prediction of mutations effects on the MLK3 kinase domain stability, based on the site directed mutator (SDM) program (37). SS element, secondary structure element (H, helix, C, coil, L, loop); HB, hydrogen bond; OS, overall stability.
Mentions: Within the MLK3 kinase domain, we mapped six single-amino acid changes (A165S, R240C, P252H, A296T, A352R and A356V). These mutations were spread along the kinase domain and did not occur at specific and highly conserved functional amino acid positions of the kinase catalytic core. Based on our predictive model, all these missense mutations affect surface-exposed residues, causing overall destabilization of the molecule (Fig. 2A and B). Mutants A165S, P252H and A352R are likely to induce the formation of new intramolecular hydrogen bonds with neighbouring residues, producing conformational changes in the protein backbone (Fig. 2a1, a3, a5). R240C mutation occurs at a coil region that runs parallel to an alpha helix in the C-terminal lobe of the MLK3 kinase domain (Fig. 2a2). The replacement of a larger residue (R) by a smaller one (C) creates higher dissociation energy, resulting in a decrease of the steric hindrance. The A at position 296 is located at an exposed loop of the MLK3 kinase domain. The substitution of a hydrophobic A residue by and hydrophilic T residue (A296T) is predicted to destabilize this area of the protein, impacting most probably the scaffold properties of MLK3 (Fig. 2a4). The A residue at position 356 is part of an Alanine-rich helix of MLK3. The substitution of A residue by a V (A356V) does not alter the polarity at this site, as both are non-polar aliphatic; nevertheless, a Valine occupies more space than an Alanine and, therefore, the helix is likely to assume a different shape/deformation and/or suffer a destabilizing effect (Fig. 2a6).

Bottom Line: Mixed lineage kinase 3 (MLK3) is a serine/threonine kinase, regulating MAPkinase signalling, in which cancer-associated mutations have never been reported.MLK3 mutations were significantly associated with MSI phenotype in primary tumours (P = 0.0005), occurring in 21% of the MSI carcinomas.Further, we demonstrated that MLK3 missense mutations found in MSI gastrointestinal carcinomas are functionally relevant.

View Article: PubMed Central - PubMed

Affiliation: IPATIMUP-Institute of Molecular Pathology and Immunology of the University of Porto, 4200-465 Porto, Portugal.

ABSTRACT
Mixed lineage kinase 3 (MLK3) is a serine/threonine kinase, regulating MAPkinase signalling, in which cancer-associated mutations have never been reported. In this study, 174 primary gastrointestinal cancers (48 hereditary and 126 sporadic forms) and 7 colorectal cancer cell lines were screened for MLK3 mutations. MLK3 mutations were significantly associated with MSI phenotype in primary tumours (P = 0.0005), occurring in 21% of the MSI carcinomas. Most MLK3 somatic mutations identified were of the missense type (62.5%) and more than 80% of them affected evolutionarily conserved residues. A predictive 3D model points to the functional relevance of MLK3 missense mutations, which cluster in the kinase domain. Further, the model shows that most of the altered residues in the kinase domain probably affect MLK3 scaffold properties, instead of its kinase activity. MLK3 missense mutations showed transforming capacity in vitro and cells expressing the mutant gene were able to develop locally invasive tumours, when subcutaneously injected in nude mice. Interestingly, in primary tumours, MLK3 mutations occurred in KRAS and/or BRAF wild-type carcinomas, although not being mutually exclusive genetic events. In conclusion, we have demonstrated for the first time the presence of MLK3 mutations in cancer and its association to mismatch repair deficiency. Further, we demonstrated that MLK3 missense mutations found in MSI gastrointestinal carcinomas are functionally relevant.

Show MeSH
Related in: MedlinePlus