O-GlcNAcylation determines the solubility, filament organization, and stability of keratins 8 and 18.
Keratins 8 and 18 (K8/18) are intermediate filament proteins expressed specifically in simple epithelial tissues. Dynamic equilibrium of these phosphoglycoproteins in the soluble and filament pool is an important determinant of their cellular functions, and it is known to be regulated by site-specific phosphorylation. However, little is known about the role of dynamic O-GlcNAcylation on this keratin pair. Here, by comparing immortalized (Chang) and transformed hepatocyte (HepG2) cell lines, we have demonstrated that O-GlcNAcylation of K8/18 exhibits a positive correlation with their solubility (Nonidet P-40 extractability). Heat stress, which increases K8/18 solubility, resulted in a simultaneous increase in O-GlcNAc on these proteins. Conversely, increasing O-GlcNAc levels were associated with a concurrent increase in their solubility. This was also associated with a notable decrease in total cellular levels of K8/18. Unaltered levels of transcripts and the reduced half-life of K8 and K18 indicated their decreased stability on increasing O-GlcNAcylation. On the contrary, the K18 glycosylation mutant (K18 S29A/S30A/S48A) was notably more stable than the wild type K18 in Chang cells. The K18-O-GlcNAc mutant accumulated as aggregates upon stable expression, which possibly altered endogenous filament architecture. These results strongly indicate the involvement of O-GlcNAc on K8/18 in regulating their solubility and stability, which may have a bearing on the functions of these keratins.
Srikanth
Budnar
B
Advanced Centre for Treatment Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India.
Vaidya
Milind M
MM
Kalraiya
Rajiv D
RD
D000117:Acetylglucosamine / Q000737:chemistry*; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D006031:Glycosylation; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D007382:Intermediate Filaments / Q000737:chemistry; D053538:Keratin-18 / Q000737:chemistry*; D053533:Keratin-8 / Q000737:chemistry*; D009154:Mutation; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry; D011487:Protein Conformation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012995:Solubility
D053538:Keratin-18; D053533:Keratin-8; D046988:Proteasome Endopeptidase Complex; D000117:Acetylglucosamine