Mapping the Phospho-dependent ALK Interactome to Identify Novel Components in ALK Signaling

Farzaneh Aboualizadeh; Zhong Yao; Jikui Guan; Luka Drecun; Shivanthy Pathmanathan; Jamie Snider; Ganesh Umapathy; Max Kotlyar; Igor Jurisica; Ruth Palmer; Igor Stagljar

doi:10.1016/j.jmb.2021.167283

Mapping the Phospho-dependent ALK Interactome to Identify Novel Components in ALK Signaling

J Mol Biol. 2021 Nov 19;433(23):167283. doi: 10.1016/j.jmb.2021.167283. Epub 2021 Oct 1.

Authors

Affiliations

¹ Donnelly Centre, University of Toronto, Ontario, Canada.
² Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg SE-40530, Sweden.
³ Krembil Research Institute, University Health Network, Ontario, Canada.
⁴ Krembil Research Institute, University Health Network, Ontario, Canada; Department of Medical Biophysics, University of Toronto, Ontario, Canada; Department of Computer Science, University of Toronto, Ontario, Canada; Institute of Neuroimmunology, Slovak Academy of Sciences, Bratislava, Slovak Republic.
⁵ Donnelly Centre, University of Toronto, Ontario, Canada; Department of Biochemistry, University of Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Ontario, Canada; Mediterranean Institute for Life Sciences, Meštrovićevo Šetalište 45, Split, Croatia; School of Medicine, University of Split, Split, Croatia. Electronic address: https://twitter.com/stagljar.

PMID: 34606829
DOI: 10.1016/j.jmb.2021.167283

Abstract

Protein-protein interactions (PPIs) play essential roles in Anaplastic Lymphoma Kinase (ALK) signaling. Systematic characterization of ALK interactors helps elucidate novel ALK signaling mechanisms and may aid in the identification of novel therapeutics targeting related diseases. In this study, we used the Mammalian Membrane Two-Hybrid (MaMTH) system to map the phospho-dependent ALK interactome. By screening a library of 86 SH2 domain-containing full length proteins, 30 novel ALK interactors were identified. Many of their interactions are correlated to ALK phosphorylation activity: oncogenic ALK mutations potentiate the interactions and ALK inhibitors attenuate the interactions. Among the novel interactors, NCK2 was further verified in neuroblastoma cells using co-immunoprecipitation. Modulation of ALK activity by addition of inhibitors lead to concomitant changes in the tyrosine phosphorylation status of NCK2 in neuroblastoma cells, strongly supporting the functionality of the ALK/NCK2 interaction. Our study provides a resource list of potential novel ALK signaling components for further study.

Keywords: ALK; MaMTH; SH2; protein–protein interaction; receptor tyrosine kinases.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Anaplastic Lymphoma Kinase / metabolism*
Carrier Proteins / metabolism*
Cell Line, Tumor
Humans
Phosphorylation
Protein Binding
Protein Interaction Domains and Motifs
Protein Interaction Mapping* / methods
Signal Transduction*

Substances

Carrier Proteins
ALK protein, human
Anaplastic Lymphoma Kinase

Grants and funding

420989/CIHR/Canada