title: Functional Implications of O-GlcNAcylation-dependent Phosphorylation at a Proximal Site on Keratin 18.
abstract: Keratins 8/18 (K8/18) are phosphoglycoproteins and form the major intermediate filament network of simple epithelia. The three O-GlcNAcylation (Ser(29), Ser(30), and Ser(48)) and two phosphorylation (Ser(33) and Ser(52)) serine sites on K18 are well characterized. Both of these modifications have been reported to increase K18 solubility and regulate its filament organization. In this report, we investigated the site-specific interplay between these two modifications in regulating the functional properties of K18, like solubility, stability, and filament organization. An immortalized hepatocyte cell line (HHL-17) stably expressing site-specific single, double, and triple O-GlcNAc and phosphomutants of K18 were used to identify the site(s) critical for regulating these functions. Keratin 18 mutants where O-GlcNAcylation at Ser(30) was abolished (K18-S30A) exhibited reduced phosphorylation induced solubility, increased stability, defective filament architecture, and slower migration. Interestingly, K18-S30A mutants also showed loss of phosphorylation at Ser(33), a modification known to regulate the solubility of K18. Further to this, the K18 phosphomutant (K18-S33A) mimicked K18-S30A in its stability, filament organization, and cell migration. These results indicate that O-GlcNAcylation at Ser(30) promotes phosphorylation at Ser(33) to regulate the functional properties of K18 and also impact cellular processes like migration. O-GlcNAcylation and phosphorylation on the same or adjacent sites on most proteins antagonize each other in regulating protein functions. Here we report a novel, positive interplay between O-GlcNAcylation and phosphorylation at adjacent sites on K18 to regulate its fundamental properties.
authors.affiliation: From the Advanced Centre for Treatment, Research, and Education in Cancer (ACTREC), Tata Memorial Centre, Sector 22, Kharghar, Navi Mumbai 410210, India.
authors.forename: Poonam S
authors.initials: PS
authors.lastname: Kakade
authors.affiliation: From the Advanced Centre for Treatment, Research, and Education in Cancer (ACTREC), Tata Memorial Centre, Sector 22, Kharghar, Navi Mumbai 410210, India s.budnar@imb.uq.edu.au srikanth.budnar@gmail.com.
authors.forename: Srikanth
authors.initials: S
authors.lastname: Budnar
authors.affiliation: From the Advanced Centre for Treatment, Research, and Education in Cancer (ACTREC), Tata Memorial Centre, Sector 22, Kharghar, Navi Mumbai 410210, India.
authors.forename: Rajiv D
authors.initials: RD
authors.lastname: Kalraiya
authors.affiliation: From the Advanced Centre for Treatment, Research, and Education in Cancer (ACTREC), Tata Memorial Centre, Sector 22, Kharghar, Navi Mumbai 410210, India mvaidya@actrec.gov.in.
authors.forename: Milind M
authors.initials: MM
authors.lastname: Vaidya
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000215:Acylation; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002465:Cell Movement / Q000235:genetics; D005353:Fibronectins / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D053538:Keratin-18 / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D020125:Mutation, Missense; D010766:Phosphorylation; D012694:Serine / Q000235:genetics / Q000378:metabolism*
chemical_list: D005353:Fibronectins; C488421:KRT18 protein, human; D053538:Keratin-18; D012694:Serine; D000117:Acetylglucosamine
keywords: O-GlcNAcylation; cytoskeleton; intermediate filament; keratin; migration; phosphorylation; protein degradation