Characterization of in vivo keratin 19 phosphorylation on tyrosine-391.
BACKGROUND
Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simple-type 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.


RESULTS
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
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.
Department of Medicine, Stanford University School of Medicine, Palo Alto, California, United States of America.
Qin
Q
Zhou
Natasha T
NT
Snider
Jian
J
Liao
Daniel H
DH
Li
Anita
A
Hong
Nam-On
NO
Ku
Christine A
CA
Cartwright
M Bishr
MB
Omary
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D053539:Keratin-19 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D014443:Tyrosine / Q000378:metabolism*
D053539:Keratin-19; D014443:Tyrosine