Characterization of in vivo keratin 19 phosphorylation on tyrosine-391

Qin Zhou, Natasha T. Snider, Jian Liao, Daniel H. Li, Anita Hong, Nam-on Ku, Christine A. Cartwright, M. Bishr Omary

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Article numbere13538
JournalPloS one
Volume5
Issue number10
DOIs
Publication statusPublished - 2010 Nov 17

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Keratin-19
Phosphorylation
keratin
Keratins
Tyrosine
tyrosine
phosphorylation
polypeptides
Peptides
src-Family Kinases
phosphotransferases (kinases)
Phosphoric Monoester Hydrolases
Type I Keratin
mice
intermediate filament proteins
Mutagenesis
Antibodies
antibodies
Site-Directed Mutagenesis
site-directed mutagenesis

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Zhou, Q., Snider, N. T., Liao, J., Li, D. H., Hong, A., Ku, N., ... Bishr Omary, M. (2010). Characterization of in vivo keratin 19 phosphorylation on tyrosine-391. PloS one, 5(10), [e13538]. https://doi.org/10.1371/journal.pone.0013538
Zhou, Qin ; Snider, Natasha T. ; Liao, Jian ; Li, Daniel H. ; Hong, Anita ; Ku, Nam-on ; Cartwright, Christine A. ; Bishr Omary, M. / Characterization of in vivo keratin 19 phosphorylation on tyrosine-391. In: PloS one. 2010 ; Vol. 5, No. 10.
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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.",
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Zhou, Q, Snider, NT, Liao, J, Li, DH, Hong, A, Ku, N, Cartwright, CA & Bishr Omary, M 2010, 'Characterization of in vivo keratin 19 phosphorylation on tyrosine-391', PloS one, vol. 5, no. 10, e13538. https://doi.org/10.1371/journal.pone.0013538

Characterization of in vivo keratin 19 phosphorylation on tyrosine-391. / Zhou, Qin; Snider, Natasha T.; Liao, Jian; Li, Daniel H.; Hong, Anita; Ku, Nam-on; Cartwright, Christine A.; Bishr Omary, M.

In: PloS one, Vol. 5, No. 10, e13538, 17.11.2010.

Research output: Contribution to journalArticle

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T1 - Characterization of in vivo keratin 19 phosphorylation on tyrosine-391

AU - Zhou, Qin

AU - Snider, Natasha T.

AU - Liao, Jian

AU - Li, Daniel H.

AU - Hong, Anita

AU - Ku, Nam-on

AU - Cartwright, Christine A.

AU - Bishr Omary, M.

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N2 - 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.

AB - 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.

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