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- Title
Characterization of In Vivo Keratin 19 Phosphorylation on Tyrosine-391.
- Authors
Qin Zhou; Snider, Natasha T.; Jian Liao; Li, Daniel H.; Anita Hong; Nam-On Ku; Cartwright, Christine A.; Omary, M. Bishr
- Abstract
Background: Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simpletype epithelia. Although K19 is known to be phosphorylated on tyrosine residue(s), conclusive site-specific characterization of these residue(s) and identification potential kinases that may be involved has not been reported. Methodology/Principal Findings: In this study, biochemical, molecular and immunological approaches were undertaken in order to identify and characterize K19 tyrosine phosphorylation. Upon treatment with pervanadate, a tyrosine phosphatase inhibitor, human K19 (hK19) was phosphorylated on tyrosine 391, located in the 'tail' domain of the protein. K19 Y391 phosphorylation was confirmed using site-directed mutagenesis and cell transfection coupled with the generation of a K19 phospho (p)-Y391-specific rabbit antibody. The antibody also recognized mouse phospho-K19 (K19 pY394). This tyrosine residue is not phosphorylated under basal conditions, but becomes phosphorylated in the presence of Src kinase in vitro and in cells expressing constitutively-active Src. Pervanadate treatment in vivo resulted in phosphorylation of K19 Y394 and Y391 in colonic epithelial cells of non-transgenic mice and hK19-overexpressing mice, respectively. Conclusions/Significance: Human K19 tyrosine 391 is phosphorylated, potentially by Src kinase, and is the first well-defined tyrosine phosphorylation site of any keratin protein. The lack of detection of K19 pY391 in the absence of tyrosine phosphatase inhibition suggests that its phosphorylation is highly dynamic.
- Subjects
PHOSPHORYLATION; CHEMICAL reactions; TYROSINE; AMINO acids; EPITHELIAL cells; PHOSPHATASES; ADENOSINE triphosphatase; MUTAGENESIS; LABORATORY mice
- Publication
PLoS ONE, 2010, Vol 5, Issue 10, p1
- ISSN
1932-6203
- Publication type
Article
- DOI
10.1371/journal.pone.0013538