A conserved rod domain phosphotyrosine that is targeted by the phosphatase PTP1B promotes keratin 8 protein insolubility and filament organization.

[alexander disease]

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 (K 8 ). 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 K 8 "rod " domain was basally phosphorylated . The charge at this site was critically important , as demonstrated by altered filament organization of site-directed mutants , Y 267 F and Y 267 D , the latter exhibiting significantly diminished solubility . Pharmacological inhibition of the protein-tyrosine phosphatase PTP 1 B increased K 8 Tyr-267 phosphorylation , decreased solubility , and increased K 8 filament bundling , whereas PTP 1 B overexpression had the opposite effects . Furthermore , there was significant co -localization between K 8 and a "substrate-trapping " mutant of PTP 1 B (D 181 A ). Because K 8 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 (Y 242 D ). Similar to K 8 , Y 242 D 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 .

Diseases
Validation

Diseases presenting "altered filament organization of site-directed mutants" symptom

alexander disease

You can validate or delete this automatically detected symptom