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

Zhou Q, Snider NT, Liao J, Li DH, Hong A, Ku NO, Cartwright CA, Omary MB - PLoS ONE (2010)

Bottom Line: 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.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.Human K19 tyrosine 391 is phosphorylated, potentially by Src kinase, and is the first well-defined tyrosine phosphorylation site of any keratin protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Stanford University School of Medicine, Palo Alto, California, United States of America.

ABSTRACT

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.

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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Related in: MedlinePlus

Human K19 is phosphorylated on ‘tail’ domain tyrosine-391.BHK-21 cells were co-transfected with wild type (WT) K8 and one of four K19 constructs (WT, Y4F, Y391F, or Y4,391F). The transfected cells were treated with 1 mM PV for 90 min, then harvested and lysed. A. Whole cell lysates were initially analyzed by 1D gel electrophoresis followed immunoblotting using mAb 4.62 (which recognizes total K19) to confirm expression. B. For the 2D gel analysis (equal K19 protein loading), isoelectric focusing (IEF) was done in the first dimension followed by SDS page in the second dimension. Immunoblots for total K19 (4.62 mAb) and pan-pY (mAb PY20) are shown and the corresponding phospho-isoforms are labeled as ‘1’ and ‘2’.
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pone-0013538-g001: Human K19 is phosphorylated on ‘tail’ domain tyrosine-391.BHK-21 cells were co-transfected with wild type (WT) K8 and one of four K19 constructs (WT, Y4F, Y391F, or Y4,391F). The transfected cells were treated with 1 mM PV for 90 min, then harvested and lysed. A. Whole cell lysates were initially analyzed by 1D gel electrophoresis followed immunoblotting using mAb 4.62 (which recognizes total K19) to confirm expression. B. For the 2D gel analysis (equal K19 protein loading), isoelectric focusing (IEF) was done in the first dimension followed by SDS page in the second dimension. Immunoblots for total K19 (4.62 mAb) and pan-pY (mAb PY20) are shown and the corresponding phospho-isoforms are labeled as ‘1’ and ‘2’.

Mentions: Based on the prediction scores that were assigned to the tyrosine residues (Table S1), site-directed mutagenesis was performed on the K19 cDNA to convert Tyr4→Phe (K19 Y4F), Tyr391→Phe (K19 Y391F), and Tyr4,391→Phe (K19 Y4/391F). Following sequencing analysis to confirm the mutation results, BHK-21 cells were transfected with the cDNA of K19 wild type (K19 WT) or one of the mutants. Additionally, the cDNA of wild type human K8 (K8 WT) was co-transfected in each case to allow for stabilization of the transfected keratins as occurs with the normal co-expression of type I and type II keratins [28]. After a 90-min treatment of the transfected cells with pervanadate (PV), an irreversible protein-tyrosine phosphatase inhibitor, whole cell lysates were prepared and subjected to one (1D) or two dimensional (2D) gel electrophoresis and immune blotting using anti-K19 or anti-pan phosphotyrosine (pY) antibodies. Protein expression was confirmed (Figure 1A) and equal amounts of K19 were subjected to 2D analysis.


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

Zhou Q, Snider NT, Liao J, Li DH, Hong A, Ku NO, Cartwright CA, Omary MB - PLoS ONE (2010)

Human K19 is phosphorylated on ‘tail’ domain tyrosine-391.BHK-21 cells were co-transfected with wild type (WT) K8 and one of four K19 constructs (WT, Y4F, Y391F, or Y4,391F). The transfected cells were treated with 1 mM PV for 90 min, then harvested and lysed. A. Whole cell lysates were initially analyzed by 1D gel electrophoresis followed immunoblotting using mAb 4.62 (which recognizes total K19) to confirm expression. B. For the 2D gel analysis (equal K19 protein loading), isoelectric focusing (IEF) was done in the first dimension followed by SDS page in the second dimension. Immunoblots for total K19 (4.62 mAb) and pan-pY (mAb PY20) are shown and the corresponding phospho-isoforms are labeled as ‘1’ and ‘2’.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2963603&req=5

pone-0013538-g001: Human K19 is phosphorylated on ‘tail’ domain tyrosine-391.BHK-21 cells were co-transfected with wild type (WT) K8 and one of four K19 constructs (WT, Y4F, Y391F, or Y4,391F). The transfected cells were treated with 1 mM PV for 90 min, then harvested and lysed. A. Whole cell lysates were initially analyzed by 1D gel electrophoresis followed immunoblotting using mAb 4.62 (which recognizes total K19) to confirm expression. B. For the 2D gel analysis (equal K19 protein loading), isoelectric focusing (IEF) was done in the first dimension followed by SDS page in the second dimension. Immunoblots for total K19 (4.62 mAb) and pan-pY (mAb PY20) are shown and the corresponding phospho-isoforms are labeled as ‘1’ and ‘2’.
Mentions: Based on the prediction scores that were assigned to the tyrosine residues (Table S1), site-directed mutagenesis was performed on the K19 cDNA to convert Tyr4→Phe (K19 Y4F), Tyr391→Phe (K19 Y391F), and Tyr4,391→Phe (K19 Y4/391F). Following sequencing analysis to confirm the mutation results, BHK-21 cells were transfected with the cDNA of K19 wild type (K19 WT) or one of the mutants. Additionally, the cDNA of wild type human K8 (K8 WT) was co-transfected in each case to allow for stabilization of the transfected keratins as occurs with the normal co-expression of type I and type II keratins [28]. After a 90-min treatment of the transfected cells with pervanadate (PV), an irreversible protein-tyrosine phosphatase inhibitor, whole cell lysates were prepared and subjected to one (1D) or two dimensional (2D) gel electrophoresis and immune blotting using anti-K19 or anti-pan phosphotyrosine (pY) antibodies. Protein expression was confirmed (Figure 1A) and equal amounts of K19 were subjected to 2D analysis.

Bottom Line: 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.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.Human K19 tyrosine 391 is phosphorylated, potentially by Src kinase, and is the first well-defined tyrosine phosphorylation site of any keratin protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Stanford University School of Medicine, Palo Alto, California, United States of America.

ABSTRACT

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.

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.

Show MeSH
Related in: MedlinePlus