title: Alteration of O-GlcNAcylation affects serine phosphorylation and regulates gene expression and activity of pyruvate kinase M2 in colorectal cancer cells.
abstract: O-GlcNAcylation is a dynamic post-translational modification that has extensive crosstalk with phosphorylation either at the same or adjacent sites of various proteins. We have previously reported that O-GlcNAcylation level was increased in primary breast and colorectal cancer, but the interplay of the two modifications remains unclear. Therefore, we explored crosstalk of the modifications by RNA interference against O-GlcNAc transferase (OGT) in colorectal cancer cells. Two-dimensional immunoblotting and mass spectrometric analysis showed that the levels of O-GlcNAc and serine phosphorylation of many proteins including serine hydroxymethyltransferase, cytokeratin-8, pyruvate kinase M2 (PKM2), heterogeneous nuclear ribonucleoprotein L, and lamin-B1, were reduced in siOGT cells compared to siScramble cells. In HT29 cells, immunoprecipitated PKM2 revealed decreased O-GlcNAc and serine phosphorylation levels after siOGT knockdown, but increased levels after treatment with Thiamet-G, an inhibitor of O-GlcNAcase (OGA). In addition, when global O-GlcNAcylation was enhanced by treating cells with Thiamet-G, PKM2 expression level was upregulated, but PKM2-specific activity was decreased. On the other hand, in OGT knockdown cells, PKM2 expression level was downregulated, but PKM2-specific activity was increased. Moreover, the metastatic colorectal cancer cells, SW620, had more O-GlcNAc-PKM2 and showed lower PKM2-specific activity compared to the non-metastatic colorectal cancer SW480 cells. These results suggested roles of O-GlcNAcylation in modulating serine phosphorylation, as well as in regulating PKM2 activity and expression. Interfering levels of O-GlcNAcylation of PKM2 may be a novel target in controlling cancer metabolism and tumorigenesis of colorectal cancer.
authors.affiliation: Applied Biological Sciences Program, Chulabhorn Graduate Institute, Bangkok, Thailand.
authors.forename: Parunya
authors.initials: P
authors.lastname: Chaiyawat
authors.affiliation: Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok, Thailand.
authors.forename: Daranee
authors.initials: D
authors.lastname: Chokchaichamnankit
authors.affiliation: Applied Biological Sciences Program, Chulabhorn Graduate Institute, Bangkok, Thailand.
authors.forename: Kriengsak
authors.initials: K
authors.lastname: Lirdprapamongkol
authors.affiliation: Applied Biological Sciences Program, Chulabhorn Graduate Institute, Bangkok, Thailand.
authors.forename: Chantragan
authors.initials: C
authors.lastname: Srisomsap
authors.affiliation: Applied Biological Sciences Program, Chulabhorn Graduate Institute, Bangkok, Thailand.
authors.forename: Jisnuson
authors.initials: J
authors.lastname: Svasti
authors.affiliation: Applied Biological Sciences Program, Chulabhorn Graduate Institute, Bangkok, Thailand.
authors.forename: Voraratt
authors.initials: V
authors.lastname: Champattanachai
mesh_terms: D000215:Acylation / Q000235:genetics; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D019073:HT29 Cells; D006801:Humans; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis / Q000235:genetics*; D009363:Neoplasm Proteins / Q000096:biosynthesis; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D011770:Pyruvate Kinase / Q000096:biosynthesis* / Q000235:genetics; D034622:RNA Interference; D012694:Serine / Q000378:metabolism
chemical_list: D009363:Neoplasm Proteins; D012694:Serine; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D011770:Pyruvate Kinase