Functions of the Brk Tyrosine Kinase in the Gastrointestinal Tract

描述(申请人提供)：我们努力识别调节肠道上皮细胞分化的基因，从而发现了BRK(乳腺肿瘤激酶，也称为PTK6和SIK)。BRK是一种非肉豆蔻酸化的、细胞内上皮细胞特异性的酪氨酸激酶，在乳腺肿瘤中表达，已被认为与肿瘤信号转导有关。然而，我们发现BRK在正常肠道中表达水平最高，在那里它定位于未分裂的、分化的上皮细胞。为了阐明BRK在体内的功能，我们中断了小鼠的BRK基因，发现BRK的缺失促进了小肠的生长和延缓了肠道细胞的分化。此外，我们还发现肠上皮隐窝细胞对γ-射线的反应诱导了BRK。我们检测到全身照射后BRK-/-小鼠的细胞凋亡受损，表明在隐窝中诱导BRK有助于细胞凋亡。此外，我们的初步数据表明，BRK-/-小鼠比野生型小鼠更容易感染结肠癌致癌物偶氮甲烷。我们假设BRK调节肠道组织的动态平衡，并在肠道中作为肿瘤抑制因子，与其在乳腺癌中的作用相反。为了明确BRK在调节肠上皮细胞生长、分化和凋亡中的作用，我们建议：1)利用工程细胞系、野生型和BRK缺陷小鼠，确定BRK是否通过负调控Akt来调节生长和凋亡。在初步研究中，我们在BRK-/-小鼠的肠道中检测到Akt的激活，Akt是生长和生存信号的关键正向调节因子，也是BRK的底物；2)利用新的细胞培养和动物模型，探索BRK调节肠道上皮细胞分化的潜在机制；3)评估BRK在肠道肿瘤发生中的作用。我们将使用各种不同的小鼠结肠癌模型系统和工程细胞系来确定BRK是否具有肿瘤抑制功能。我们的数据表明，BRK在正常肠道和乳腺癌中具有不同的功能，这可能取决于它在细胞内的定位和对细胞内不同信号分子的访问。我们的研究旨在确定BRK酪氨酸激酶的正常生理功能，这对于了解其对肠道组织稳态和肠癌发生的潜在贡献将是重要的。

DESCRIPTION (provided by applicant): Our efforts to identify genes that regulate intestinal epithelial cell differentiation led to the discovery of Brk (Breast tumor kinase, also called PTK6 and Sik). Brk is a non-myristoylated, intracellular epithelial-specific tyrosine kinase that is expressed in breast tumors where it has been proposed to contribute to oncogenic signaling. However we discovered that Brk is expressed at highest levels in the normal intestine, where it is localized to nondividing, differentiated epithelial cells. To elucidate functions of Brk in vivo, we disrupted the mouse Brk gene, and found that loss of Brk enhanced growth and delayed enterocyte differentiation in the small intestine. In addition, we discovered that Brk is induced in intestinal epithelial crypt cells in response to ?-irradiation. We detected impaired apoptosis in the Brk-/- mouse after total body irradiation, indicating that induction of Brk in the crypts contributes to apoptosis. In addition our preliminary data suggest that Brk-/- mice are more susceptible to the colon carcinogen azoxymethane than wild type mice. We hypothesize that Brk regulates intestinal tissue homeostasis, and acts as a tumor suppressor in the intestinal tract, in contrast to its role in breast cancer. To define the roles of Brk in regulation of growth, differentiation, and apoptosis in intestinal epithelial cells, we propose: 1) To determine if Brk regulates growth and apoptosis by negatively regulating Akt using engineered cell lines, and wild type and Brk-deficient mice. In preliminary studies, we detected increased activation of Akt, a key positive regulator of growth and survival signaling and a substrate of Brk in intestines of Brk- /- mice; 2) To explore mechanisms underlying the ability of Brk to regulate differentiation of intestinal epithelial cells, using novel cell culture and animal models, and 3) To evaluate contributions of Brk to tumorigenesis in the intestine. We will determine if Brk has tumor suppressor functions using a variety of different mouse colon cancer model systems and engineered cell lines. Our data suggest that Brk has different functions in normal intestine and in breast cancer, which may be dependent on its intracellular localization and access to different signaling molecules within the cell. Our studies are directed at determining the normal physiological functions of the Brk tyrosine kinase, which will be important for understanding its potential contributions to intestinal tissue homeostasis and the development of intestinal cancers.