NBP, a zebrafish homolog of human Kank3, is a novel Numb interactor essential for epidermal integrity and neurulation
Boggetti B., Jasik J., Takamiya M., Strähle U., Reugels A. M., and Campos-Ortega J. A.
Institut für Entwickl ungsbiologie, University of Cologne, 50923 Cologne, Germany
Department of Molecular Genetics, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, 06466 Gatersleben, Germany
Institute of Cellular and Molecular Botany, University of Bonn, 53115 Bonn, Germany
Institute for Toxicology and Genetics, Forschungszentrum Karlsruhe, Postfach 3640, 76021 Karlsruhe, Germany
Developmental Biology 365 (2012) 164-174.
Numb is an adaptor protein implicated in diverse basic cellular processes. Numb was originally identified as a gene required for normal development of the peripheral nervous system in Drosophila. In recent years, many studies in mammals have shown the importance of Numbs in different fields such as developmental neurobiology, cancer biology and degenerative diseases. Although the exact biochemical role of Numbs has yet to be discovered, increasing amounts of evidence show that Numbs may act as adaptors facilitating the formation of multi-protein complexes (1).
In this study, a new interactor of Numb is isolated in zebrafish that is an ortholog of human Kank3. Kank3 belongs to the kidney ankyrin repeat-containing protein family defined in 2009 by Kakinuma et. al. Kank1 was originally found as a tumor supressor of renal cell carcinoma and its deletion was connected with cerebral palsy (2). The authors reveal that zebrafish Numbs Binding Protein (NBP) interacts with Numbs through a novel motif well conserved in all vertebrates Kanks. They also demonstrate that NBP is a protein with nonredundant function during zebrafish embryogenesis as its deficiency causes serious embryo defects and lethality within 48 hours. They also discover a role of NBP in establishing and/or maintaining cell adhesion and polarity.
Yeast two-hybrid assay — Used to screen zebrafish cDNA library for either Numb1 or Numb3. Also used for quantitative analysis of protein binding and for identification of interaction domains
Domain and Protein Structure Analysis — NBP amino acid sequence was compared to the protein NCBI database(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Full-length protein sequences with structures resembling NBP were aligned by ClustalW XXL (http://www.ch.embnet.org/software/ClustalW-XXL.html).
Creation of knockdown embryos and EGTA treatment — NBP and Numbs knockdown embryo lines were generated using the antisense morpholino oligonucleotides (MO). NBP MO and Numb MO were injected into one-four-cell stage embryos to create single or double knockdown morphants. For EGTA treatment, embryos were kept with the chorion and briefly washed with calcium-magnesium free medium followed by 1 hour incubation in 20 mM EGTA.
Biomolecular fluorescence complementation analysis (BiFC), GFP technology and immunohystochemistry — Cappes mRNAs for BiFC and GFP technologies were amplified in vitro. RNA samples were injected into one-four-cell stage wild-type embryos. For immunohistochemistry, embryos from the BiFC assay were sectioned and probed with primary antibodies, washed, then treated for appropriate secondary antibodies.
- NBP interacts with Numbs through a well conserved region among Kank proteins
- Numb proteins and NBP bind in vitro and in living embryos
- The expression pattern of NBP is developmentally regulated
- NBP is polarly localized
- NBP is required for embryo developement and surviving
- Numb and NBP interact on genetic level
NBP and Numbs morphants show defects in development.
NBP is required for neural tube development.
The authors demonstrate that NBP, the zebrafish Kank 3, directly interacts with Numbs. The NBP expression experiments and embryo lethality of the NBP zebrafish morphants directly implies the importance of this non-redundant gene in embryo development. The authors also characterize another domain responsible for binding NBP to Numbs, which is conserved among all Kanks and may bind other proteins containing PTB domains.
Review/Strengths & Weaknesses:
Although the authors did an outstanding job with their experimental assay and displaying their data, they were not truly able to answer their initial question about what exactly does NBP do. Although they made some revelations and have promising theories, the precise biochemical function of NBP remains a mystery. The authors also did not develop any additional findings regarding Kanks, only mentioning that, in general, Kanks is rather poorly investigated and warrants more research as to its specific biological role.
- Gulino, A., Di Marcotullio, L., Screpanti, I., 2010. “The multiple functions of Numb.” Exp. Cell Res. 316, 900–906.
- Sarkar, S., Roy, B.C., Hatano, N., Aoyagi, T., Gohji, K., Kiyama, R., 2002. “A novel ankyrin repeat-containing gene (Kank) located at 9p24 is a growth suppressor of renal cell carcinoma.” J. Biol. Chem. 277, 36585 –36591.