title: A conserved rod domain phosphotyrosine that is targeted by the phosphatase PTP1B promotes keratin 8 protein insolubility and filament organization.
abstract: Post-translational modifications are important functional determinants for intermediate filament (IF) proteins. Phosphorylation of IF proteins regulates filament organization, solubility, and cell-protective functions. Most known IF protein phosphorylation sites are serines localized in the variable "head" and "tail" domain regions. By contrast, little is known about site-specific tyrosine phosphorylation or its implications on IF protein function. We used available proteomic data from large scale studies to narrow down potential phospho-tyrosine sites on the simple epithelial IF protein keratin 8 (K8). Validation of the predicted sites using a pan-phosphotyrosine and a site-specific antibody, which we generated, revealed that the highly conserved Tyr-267 in the K8 "rod" domain was basally phosphorylated. The charge at this site was critically important, as demonstrated by altered filament organization of site-directed mutants, Y267F and Y267D, the latter exhibiting significantly diminished solubility. Pharmacological inhibition of the protein-tyrosine phosphatase PTP1B increased K8 Tyr-267 phosphorylation, decreased solubility, and increased K8 filament bundling, whereas PTP1B overexpression had the opposite effects. Furthermore, there was significant co-localization between K8 and a "substrate-trapping" mutant of PTP1B (D181A). Because K8 Tyr-267 is conserved in many IFs (QYE motif), we tested the effect of the paralogous Tyr in glial fibrillary acidic protein (GFAP), which is mutated in Alexander disease (Y242D). Similar to K8, Y242D GFAP exhibited highly irregular filament organization and diminished solubility. Our results implicate the rod domain QYE motif tyrosine as an important determinant of IF assembly and solubility properties that can be dynamically modulated by phosphorylation.
authors.affiliation: From the Departments of Molecular and Integrative Physiology and.
authors.forename: Natasha T
authors.initials: NT
authors.lastname: Snider
authors.forename: Haewon
authors.initials: H
authors.lastname: Park
authors.forename: M Bishr
authors.initials: MB
authors.lastname: Omary
mesh_terms: D038261:Alexander Disease / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D006224:Cricetinae; D005904:Glial Fibrillary Acidic Protein; D056945:Hep G2 Cells; D006801:Humans; D053533:Keratin-8 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020125:Mutation, Missense; D041681:NIH 3T3 Cells; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D019000:Phosphotyrosine / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1 / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
chemical_list: D005904:Glial Fibrillary Acidic Protein; C426203:KRT8 protein, human; D053533:Keratin-8; C486595:Krt8 protein, mouse; D009419:Nerve Tissue Proteins; C528900:glial fibrillary astrocytic protein, mouse; D019000:Phosphotyrosine; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; C516609:Ptpn1 protein, mouse
keywords: Alexander Disease; Cytoskeleton; GFAP; Intermediate Filaments; Keratin; Phosphotyrosine; Post-translational Modification; Protein-tyrosine Phosphatase (Tyrosine Phosphatase); Tyrosine Phosphatase