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The neuroblastoma ALK(I1250T) mutation is a kinase-dead RTK in vitro and in vivo
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
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2011 (English)In: Translational Oncology, ISSN 1944-7124, E-ISSN 1936-5233, Vol. 4, no 4, 258-265 p.Article in journal (Other academic) Published
Abstract [en]

Activating mutations in the kinase domain of anaplastic lymphoma kinase (ALK) have recently been shown to be an important determinant in the genetics of the childhood tumor neuroblastoma. Here we discuss an in-depth analysis of one of the reported gain-of-function ALK mutations—ALKI1250T—identified in the germ line DNA of one patient. Our analyses were performed in cell culture-based systems and subsequently confirmed in a Drosophila model. The results presented here indicate that the germ line ALKI1250T mutation is most probably not a determinant for tumor initiation or progression and, in contrast, seems to generate a kinase-dead mutation in the ALK receptor tyrosine kinase (RTK). Consistent with this, stimulation with agonist ALK antibodies fails to lead to stimulation of ALKI1250T and we were unable to detect tyrosine phosphorylation under any circumstances. In agreement, ALKI1250T is unable to activate downstream signaling pathways or to mediate neurite outgrowth, in contrast to the activated wild-type ALK receptor or the activating ALKF1174S mutant. Identical results were obtained when the ALKI1250T mutant was expressed in a Drosophila model, confirming the lack of activity of this mutant ALK RTK. We suggest that the ALKI1250T mutation leads to a kinase-dead ALK RTK, in stark contrast to assumed gain-of-function status, with significant implications for patients reported to carry this particular ALK mutation.

Place, publisher, year, edition, pages
2011. Vol. 4, no 4, 258-265 p.
Keyword [en]
ALK, neuroblastoma, gain of function, kinase dead
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-36979DOI: 10.1593/tlo.11139ISI: 000294253200008PubMedID: 21804922OAI: oai:DiVA.org:umu-36979DiVA: diva2:356947
Projects
Exploiting Drosophila as a model system for studying Anaplastic Lymphoma Kinase in vivo
Available from: 2010-10-14 Created: 2010-10-14 Last updated: 2017-01-17Bibliographically approved
In thesis
1. Exploiting Drosophila as a model system for studying anaplastic lymphoma kinase in vivo
Open this publication in new window or tab >>Exploiting Drosophila as a model system for studying anaplastic lymphoma kinase in vivo
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Anaplastic Lymphoma Kinase (ALK) is a Receptor Tyrosine Kinase (RTK) and an oncogene associated with several human diseases, but its normal function in humans and other vertebrates is unclear. Drosophila melanogaster has an ALK homolog, demonstrating that the RTK has been conserved throughout evolution. This makes Drosophila a suitable model organism for studying not only Drosophila ALK function, but also to study mammalian forms of ALK. In Drosophila the ligand Jeb activates ALK, initiating signaling crucial for visceral mesoderm development. The activating ligand for mammalian ALK is unclear, and for this reason Drosophila was employed in a cross-species approach to investigate whether Drosophila Jeb can activate mouse ALK. Jeb is unable to activate mouse ALK, and therefore mouse ALK is unable to substitute for and rescue the Drosophila ALK mutant phenotype. This suggests that there has been significant evolution in the ALK-ligand relationship between the mouse and Drosophila.

In humans ALK has recently been shown to be involved in the development of neuroblastoma, a cancer tumor in children. I have developed a Drosophila model for examining human gain of function ALK mutants found in neuroblastoma patients. The various ALK variants have acquired point mutations in the kinase domain that have been predicted to activate the RTK in a constitutive and ligand independent manner. When expressed in the fly eye, active human ALK mutants result in a rough eye phenotype, while inactive wild type ALK does not, due to the lack of an activating ligand in the fly. In this way  several of the ALK mutations identified in neuroblastoma patients could be confirmed to be activated in a ligand independent manner. Moreover, a novel ALK mutant; ALKF1174S, was discovered in a neuroblastoma patient and was in the Drosophila model shown to be a gain of function mutation, and a previously predicted gain of function mutation; ALKI1250T, was shown to be a kinase dead mutation. This fly model can also be used for testing ALK selective inhibitors, for identifying activating ligands for human ALK and for identifying conserved components of the ALK signaling pathway.

Gut musculature development in Drosophila is dependent on ALK signaling, while somatic muscle development is not. Proteins of the Wasp-Scar signaling network regulate Arp2/3-complex mediated actin polymerization, and I have investigated their function in visceral and somatic muscle fusion. I found that Verprolin and other members of this protein family are essential for somatic but not visceral muscle development. Despite fusion defects in both tissues in Verprolin and other examined mutants, gut development proceeds, suggesting that fusion is not crucial for visceral mesoderm development. Hence the actin polymerization machinery functions in both somatic and visceral muscle fusion, but this process only appears to be essential in somatic muscle development.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2010. 58 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1376
Keyword
Anaplastic lymphoma kinase, Receptor tyrosine kinase, Jeb, neuroblastoma, actin polymerization, Wasp, Scar, Vrp1, Arp2/3
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-36991 (URN)978-91-7459-090-6 (ISBN)
Public defence
2010-11-05, Major Groove, Byggnad 6L, Umeå universitet, Umeå, 09:00 (English)
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Projects
Exploiting Drosophila as a model system for studying Anaplastic Lymphoma Kinase in vivo
Available from: 2010-10-15 Created: 2010-10-14 Last updated: 2010-10-15Bibliographically approved

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Schönherr, ChristinaRuuth, KristinaEriksson, ThereseYamazaki, YasuoKamaraj, SattuPalmer, RuthHallberg, Bengt

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