Detection of malignancy-associated phosphoproteome changes in human colorectal cancer induced by cell surface binding of growth-inhibitory galectin-4

IUBMB Life. 2019 Mar;71(3):364-375. doi: 10.1002/iub.1987. Epub 2018 Dec 14.

Abstract

Emerging evidence on efficient tumor growth regulation by endogenous lectins directs interest to determine on a proof-of-principle level the range of information on alterations provided by full-scale analysis using phosphoproteomics. In our pilot study, we tested galectin-4 (gal-4) that is a growth inhibitor for colon cancer cells (CRC), here working with the LS 180 line. In order to cover monitoring of short- and long-term effects stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analyses were conducted on LS 180 cell preparations collected 1 and 72 h after adding gal-4 to the culture medium. After short-term treatment, 981 phosphosites, all of them S/T based, were detected by phosphoproteomics. Changes higher than 1.5-fold were seen for eight sites in seven proteins. Most affected were the BET1 homolog (BET1), whose level of phosphorylation at S50 was about threefold reduced, and centromere protein F (CENPF), extent of phosphorylation at S3119 doubling in gal-4-treated cells. Phosphoproteome analysis after 72 h of treatment revealed marked changes at 33 S/T-based phosphosites from 29 proteins. Prominent increase of phosphorylation was observed for cofilin-1 at position S3. Extent of phosphorylation of the glutamine transporter SLC1A5 at position S503 was decreased by a factor of 3. Altered phosphorylation of BET1, CENPF, and cofilin-1 as well as a significant effect of gal-4 treatment on glutamine uptake by cells were substantiated by independent methods in the Vaco 432, Colo 205, CX 1, and HCT 116 cell lines. With the example of gal-4 which functions as a tumor suppressor in CRC cells, we were able to prove that cell surface binding of the lectin not only markedly influences the cell proteome, but also has a bearing on malignancy-associated intracellular protein phosphorylation. These results underscore the potential of this approach to give further work on elucidating the details of signaling underlying galectin-triggered growth inhibition a clear direction. © 2018 IUBMB Life, 71(3):364-375, 2019.

Keywords: Glutamine transporter; colorectal cancer; galectin-4; growth-inhibition; phosphoproteome; tumor suppressor.

MeSH terms

  • Amino Acid Transport System ASC / genetics
  • Amino Acid Transport System ASC / metabolism
  • Antineoplastic Agents / pharmacology*
  • Biological Transport / drug effects
  • Carbon Isotopes
  • Cell Line, Tumor
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism
  • Cofilin 1 / genetics
  • Cofilin 1 / metabolism
  • Galectin 4 / pharmacology*
  • Glutamine / metabolism
  • HCT116 Cells
  • Humans
  • Isotope Labeling
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism
  • Minor Histocompatibility Antigens / genetics
  • Minor Histocompatibility Antigens / metabolism
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism*
  • Nitrogen Isotopes
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Phosphorylation / drug effects
  • Protein Processing, Post-Translational / drug effects*
  • Proteome / genetics
  • Proteome / metabolism*
  • Qc-SNARE Proteins / genetics
  • Qc-SNARE Proteins / metabolism
  • Recombinant Proteins / pharmacology

Substances

  • Amino Acid Transport System ASC
  • Antineoplastic Agents
  • BET1L protein, human
  • CFL1 protein, human
  • Carbon Isotopes
  • Chromosomal Proteins, Non-Histone
  • Cofilin 1
  • Galectin 4
  • Microfilament Proteins
  • Minor Histocompatibility Antigens
  • Neoplasm Proteins
  • Nitrogen Isotopes
  • Phosphoproteins
  • Proteome
  • Qc-SNARE Proteins
  • Recombinant Proteins
  • SLC1A5 protein, human
  • centromere protein F
  • Glutamine