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Characterization of in vivo keratin 19 phosphorylation on tyrosine-391.

ABSTRACT: 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.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.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.

SUBMITTER: Zhou Q

PROVIDER: S-EPMC2963603 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Characterization of in vivo keratin 19 phosphorylation on tyrosine-391.

Zhou Qin Q Snider Natasha T NT Liao Jian J Li Daniel H DH Hong Anita A Ku Nam-On NO Cartwright Christine A CA Omary M Bishr MB

PloS one 20101025 10

<h4>Background</h4>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.<h4>Methodology/principal findings</h4>In this study, biochemical, molecular and immunological approaches were undertaken in order to identify ...[more]

PMID: 21049038

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Project description:PurposeTo evaluate the expression patterns of cytokeratin (K) 12, 13, and 19 in normal epithelium of the human ocular surface to determine whether K13 could be used as a marker for conjunctival epithelium.MethodsTotal RNA was isolated from the human conjunctiva and central cornea. Those transcripts that had threefolds or higher expression levels in the conjunctiva than the cornea were identified using microarray technique. Expression levels of three known signature genes and of two conjunctival genes, K13 and K19 were confirmed by using quantitative real-time PCR (qRT-PCR). Protein expression of K12, K13, and K19 was confirmed by immunostaining with specific antibodies on histologic sections of human sclerocornea that contained the conjunctiva, limbus, and cornea and on impression cytology (IC) specimens of the cornea and conjunctiva from normal donors. Double staining of K13/K12 and K19/K12 on histologic sections and IC specimens was performed.ResultsThere were 337 transcripts that were preferentially expressed in the conjunctiva. K13 and K19 were among the top twenty transcripts in the conjunctiva and this preferential expression pattern of K13 and K19 was confirmed by qRT-PCR. Immunohistochemical studies showed that K13 was expressed at the posterior limbal epithelium and conjunctival epithelium but was totally absent in the cornea. K12 was expressed in the corneal and anterior limbal epithelia except for the basal layer and was absent from the conjunctiva. In contrast, K19 was detected in the corneal, limbal and conjunctival epithelia. Immunostaining of the IC specimens showed K12(+) epithelial cells in the corneal region, K13(+) cells in the conjunctival area, and K19(+) cells in the corneal and conjunctival specimens. Expression of K13 and K12 on the ocular surface was mutually exclusive on both the histologic and IC samples using double immunostaining.ConclusionsK13 is more specific to the conjunctival epithelial cells than K19 and potentially could be used as a marker to identify conjunctival epithelial cells in limbal stem cell deficiency.

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Characterization of in vivo keratin 19 phosphorylation on tyrosine-391.

Publications

Characterization of in vivo keratin 19 phosphorylation on tyrosine-391.

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