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Volume 23
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10.3390/ijms23126414
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Article

Combining Recombinase-Mediated Cassette Exchange Strategy with Quantitative Proteomic and Phosphoproteomic Analyses to Inspect Intracellular Functions of the Tumor Suppressor Galectin-4 in Colorectal Cancer Cells

by
Malwina Michalak
1,2,
Viola Golde
1,
Dominik Helm
3,
Herbert Kaltner
4,
Johannes Gebert
1,2,* and
Jürgen Kopitz
1,2
1
Department of Applied Tumor Biology, Institute of Pathology, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany
2
Clinical Cooperation Unit Applied Tumor Biology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
3
Proteomics Core Facility, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
4
Veterinary Faculty, Institute of Physiological Chemistry, Ludwig-Maximilians-University, 80539 München, Germany
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2022, 23(12), 6414; https://doi.org/10.3390/ijms23126414
Submission received: 5 April 2022 / Revised: 3 June 2022 / Accepted: 6 June 2022 / Published: 8 June 2022
(This article belongs to the Special Issue Proteomics and Its Applications in Cancers)
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Abstract

Galectin-4 (Gal4) has been suggested to function as a tumor suppressor in colorectal cancer (CRC). In order to systematically explore its function in CRC, we established a CRC cell line where Gal4 expression can be regulated via the doxycycline (dox)-inducible expression of a single copy wildtype LGALS4 transgene generated by recombinase-mediated cassette exchange (RMCE). Using this model and applying in-depth proteomic and phosphoproteomic analyses, we systematically screened for intracellular changes induced by Gal4 expression. Overall, 3083 cellular proteins and 2071 phosphosites were identified and quantified, of which 1603 could be matched and normalized to their protein expression levels. A bioinformatic analysis revealed that most of the regulated proteins and phosphosites can be localized in the nucleus and are categorized as nucleic acid-binding proteins. The top candidates whose expression was modulated by Gal4 are PURB, MAPKAPK3, BTF3 and BCAR1, while the prime candidates with altered phosphorylation included ZBTB7A, FOXK1, PURB and CK2beta. In order to validate the (phospho)proteomic data, we confirmed these candidates by a radiometric metabolic-labelling and immunoprecipitation strategy. All candidates exert functions in the transcriptional or translational control, indicating that Gal4 might be involved in these processes by affecting the expression or activity of these proteins.
Keywords: Galectin 4; proteomics; phosphoproteomics; colorectal cancer; Tet-On system Galectin 4; proteomics; phosphoproteomics; colorectal cancer; Tet-On system

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MDPI and ACS Style

Michalak, M.; Golde, V.; Helm, D.; Kaltner, H.; Gebert, J.; Kopitz, J. Combining Recombinase-Mediated Cassette Exchange Strategy with Quantitative Proteomic and Phosphoproteomic Analyses to Inspect Intracellular Functions of the Tumor Suppressor Galectin-4 in Colorectal Cancer Cells. Int. J. Mol. Sci. 2022, 23, 6414. https://doi.org/10.3390/ijms23126414

AMA Style

Michalak M, Golde V, Helm D, Kaltner H, Gebert J, Kopitz J. Combining Recombinase-Mediated Cassette Exchange Strategy with Quantitative Proteomic and Phosphoproteomic Analyses to Inspect Intracellular Functions of the Tumor Suppressor Galectin-4 in Colorectal Cancer Cells. International Journal of Molecular Sciences. 2022; 23(12):6414. https://doi.org/10.3390/ijms23126414

Chicago/Turabian Style

Michalak, Malwina, Viola Golde, Dominik Helm, Herbert Kaltner, Johannes Gebert, and Jürgen Kopitz. 2022. "Combining Recombinase-Mediated Cassette Exchange Strategy with Quantitative Proteomic and Phosphoproteomic Analyses to Inspect Intracellular Functions of the Tumor Suppressor Galectin-4 in Colorectal Cancer Cells" International Journal of Molecular Sciences 23, no. 12: 6414. https://doi.org/10.3390/ijms23126414

APA Style

Michalak, M., Golde, V., Helm, D., Kaltner, H., Gebert, J., & Kopitz, J. (2022). Combining Recombinase-Mediated Cassette Exchange Strategy with Quantitative Proteomic and Phosphoproteomic Analyses to Inspect Intracellular Functions of the Tumor Suppressor Galectin-4 in Colorectal Cancer Cells. International Journal of Molecular Sciences, 23(12), 6414. https://doi.org/10.3390/ijms23126414

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