Couchman G M, Bentley R, Tsao M S, Raszmann K, McLachlan J A, Walmer D K
Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Soc Gynecol Investig. 1997 Mar-Apr;4(2):103-9.
To identify specific tyrosine kinases that are involved in endometrial signaling and to study their in vivo expression in normal and abnormal endometrium. We hypothesized that proteins that are differentially expressed would be more likely to be important in regulated cellular events.
Complementary DNA libraries, constructed from human secretory (n = 5) and proliferative (n = 5) endometrial specimens, were screened with a polyclonal anti-phosphotyrosine antibody. Positive clones were sequenced and screened for differential expression using immunoblotting and Northern analysis of samples from proliferative and secretory endometrium. The expression of one identified clone, lyn, a Src family member, was characterized further with Western and Northern blot analyses and immunolocalization.
One protein identified by the above method was lyn, a member of the src family of protein tyrosine kinases, never before described in the human endometrium. Western blot analysis revealed two forms of lyn protein, p53lyn and p56lyn, that were most abundant in the late secretory phase. Immunohistochemistry demonstrated uniform protein expression by all cells in normal glandular epithelium and suggested a correlation between lyn protein expression and cell differentiation for human endometrial adenocarcinomas, with markedly-elevated levels noted in poorly differentiated adenocarcinomas compared with well-differentiated tumors (n = 3). Northern hybridization confirmed the presence of the expected 3.5-kb lyn transcript in normal and abnormal endometrium.
Our data demonstrate that human cDNA libraries created from different phases of the menstrual cycle can be screened successfully using anti-phosphotyrosine antibodies to identify differentially expressed protein tyrosine kinases. Although p53lyn and p56lyn expression has been thought of as a predominantly lymphoid-specific tyrosine kinase, we show prominent expression of lyn protein and mRNA by normal and malignant epithelium of the human endometrium, suggesting a role in endometrial signaling and human reproduction.
鉴定参与子宫内膜信号传导的特定酪氨酸激酶，并研究它们在正常和异常子宫内膜中的体内表达。我们推测差异表达的蛋白质更有可能在调节细胞事件中发挥重要作用。
用人分泌期（n = 5）和增殖期（n = 5）子宫内膜标本构建互补DNA文库，用多克隆抗磷酸酪氨酸抗体进行筛选。对阳性克隆进行测序，并通过对增殖期和分泌期子宫内膜样本进行免疫印迹和Northern分析来筛选差异表达。通过Western和Northern印迹分析以及免疫定位进一步表征一个鉴定出的克隆lyn（Src家族成员）的表达。
通过上述方法鉴定出的一种蛋白质是lyn，它是蛋白质酪氨酸激酶Src家族的成员，此前从未在人子宫内膜中被描述过。Western印迹分析显示lyn蛋白有两种形式，p53lyn和p56lyn，在分泌晚期最为丰富。免疫组织化学显示正常腺上皮中的所有细胞均有均匀的蛋白表达，并提示lyn蛋白表达与人类子宫内膜腺癌的细胞分化之间存在相关性，与高分化肿瘤相比，低分化腺癌中lyn蛋白水平明显升高（n = 3）。Northern杂交证实正常和异常子宫内膜中存在预期的3.5 kb lyn转录本。
我们的数据表明，使用抗磷酸酪氨酸抗体可以成功筛选从月经周期不同阶段创建的人cDNA文库，以鉴定差异表达的蛋白质酪氨酸激酶。尽管p53lyn和p56lyn的表达被认为主要是淋巴特异性酪氨酸激酶，但我们显示lyn蛋白和mRNA在人子宫内膜的正常和恶性上皮中均有显著表达，提示其在子宫内膜信号传导和人类生殖中发挥作用。
