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.
Highlights
Intermediate filament (IF) proteins, including keratin 8 and glial fibrillary acidic protein (GFAP), are regulated by serine phosphorylation, whereas phospho-tyrosine sites are poorly understood
Because keratin 8 (K8) Tyr-267 is conserved in many intermediate filament (IF) (QYE motif), we tested the effect of the paralogous Tyr in glial fibrillary acidic protein (GFAP), which is mutated in Alexander disease (Y242D)
K8 Is Phosphorylated on Conserved Rod Domain Residue Tyr-267—Upon surveying the literature on phosphotyrosine proteomic studies, we found that phospho-peptides of K8 were reported in several independent studies that utilized different antibodies for immune enrichment [27,28,29,30,31,32]
Summary
Intermediate filament (IF) proteins, including keratin 8 and GFAP, are regulated by serine phosphorylation, whereas phospho-tyrosine sites are poorly understood. Results: Keratin 8 phospho-Tyr-267 is dephosphorylated by PTP1B and promotes insolubility and filament organization, as does the paralogous GFAP tyrosine. Post-translational modifications are important functional determinants for intermediate filament (IF) proteins. 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. 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.
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