Characterization of the major physiologic phosphorylation site of human keratin 19 and its role in filament organization

Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simple-type epithelia, Little is known rega rding K19 regulation or function, and the only other type I keratin that ha s been studied in terms of regulation is keratin 18 (K18), We characterized K19 phosphorylation as a handle to study its function. In vivo, serine is the major phosphorylated residue, and phosphopeptide mapping of (PO4)-P-32- labeled K19 generates one major phosphopeptide. Edman degradation suggested that the radiolabeled phosphopeptide represents K19 Ser-10 and/or Ser-35 p hosphorylation, Mutation of Ser-10 or Ser-35 followed by transfection confi rmed that Ser-35 is the major K19 phosphorylation site. Transfection of Ser -35 --> Ala K19 showed a filament assembly defect as compared with normal o r with Ser-10 --> Ala K19, Comparison of K18 and K19 phosphorylation featur es in interphase cells showed that both are phosphorylated primarily at a s ingle site, preferentially in the soluble versus the insoluble keratin frac tions. K19 has higher basal phosphorylation, whereas K18 phosphorylation is far more sensitive to phosphatase type I and IIA inhibition. Our results d emonstrate that Ser-35 is the major K19 interphase phosphorylation site and that it plays a role in keratin filament assembly. K19 and K18 phosphoryla tions share some features but also have distinct properties that suggest di fferent regulation of type I keratins within the same cells.