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 regarding K19 regulation or function, and the only other type I keratin that has 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 32PO4-labeled K19 generates one major phosphopeptide. Edman degradation suggested that the radiolabeled phosphopeptide represents K19 Ser-10 and/or Ser-35 phosphorylation. Mutation of Ser-10 or Ser-35 followed by transfection confirmed that Ser-35 is the major K19 phosphorylation site. Transfection of Ser-35 --> Ala K19 showed a filament assembly defect as compared with normal or with Ser-10 --> Ala K19. Comparison of K18 and K19 phosphorylation features in interphase cells showed that both are phosphorylated primarily at a single site, preferentially in the soluble versus the insoluble keratin fractions. K19 has higher basal phosphorylation, whereas K18 phosphorylation is far more sensitive to phosphatase type I and IIA inhibition. Our results demonstrate that Ser-35 is the major K19 interphase phosphorylation site and that it plays a role in keratin filament assembly. K19 and K18 phosphorylations share some features but also have distinct properties that suggest different regulation of type I keratins within the same cells.
Zhou
X
X
Veterans Affairs Palo Alto Health Care System and Stanford University Digestive Disease Center, Palo Alto, California 94304, USA.
Liao
J
J
Hu
L
L
Feng
L
L
Omary
M B
MB
D000818:Animals; D006801:Humans; D007382:Intermediate Filaments / Q000378:metabolism*; D007633:Keratins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism
D012694:Serine; D007633:Keratins