Department of Medicine, Stanford University School of Medicine, Palo Alto, California, United States of America.
PLoS One. 2010 Oct 25;5(10):e13538. doi: 10.1371/journal.pone.0013538.
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.
角蛋白多肽 19（K19）是一种 I 型中间丝蛋白，在有分层和简单上皮的细胞中表达。虽然已知 K19 在酪氨酸残基（s）上发生磷酸化，但尚未有报道对这些残基（s）进行明确的特异性特征描述，并鉴定可能涉及的潜在激酶。
方法/主要发现：在这项研究中，采用生化、分子和免疫学方法来鉴定和描述 K19 酪氨酸磷酸化。在用过钒酸钠（一种酪氨酸磷酸酶抑制剂）处理后，人 K19（hK19）在位于蛋白质“尾部”结构域的酪氨酸 391 上发生磷酸化。通过定点突变和细胞转染，并生成 K19 磷酸化（p）-Y391 特异性兔抗体，确认了 K19 Y391 磷酸化。该抗体还识别了鼠磷酸化-K19（K19 pY394）。在基础条件下，该酪氨酸残基不发生磷酸化，但在体外 Src 激酶存在下以及在表达组成型激活 Src 的细胞中发生磷酸化。体内用过钒酸钠处理导致非转基因小鼠和 hK19 过表达小鼠的结肠上皮细胞中 K19 Y394 和 Y391 磷酸化。
结论/意义：人 K19 酪氨酸 391 发生磷酸化，可能是由 Src 激酶引起的，这是任何角蛋白蛋白的第一个明确的酪氨酸磷酸化位点。在没有酪氨酸磷酸酶抑制的情况下未检测到 K19 pY391，表明其磷酸化具有高度动态性。
