Functions of the Brk Tyrosine Kinase in the Gastrointestinal Tract

描述（由申请人提供）：我们努力鉴定调节肠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是一种非肉豆肉酰化的细胞内上皮特异性酪氨酸激酶，在乳腺肿瘤中表达，并被认为有助于致癌信号传导。然而，我们发现Brk在正常肠道中表达水平最高，它定位于未分裂的分化上皮细胞。为了阐明Brk在体内的功能，我们破坏了小鼠Brk基因，发现Brk的缺失促进了小肠的生长并延迟了肠细胞的分化。此外，我们发现Brk在肠上皮隐窝细胞中受到γ -辐照的诱导。我们在全身照射后检测到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受到结肠癌致癌物偶氮氧甲烷的影响。我们假设Brk调节肠道组织稳态，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在肠上皮细胞生长、分化和凋亡调控中的作用，我们提出：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调控生长和凋亡。在初步研究中，我们检测到Brk- /-小鼠肠道中Akt激活增加，Akt是生长和生存信号的关键正调节因子，也是Brk的底物；2)利用新型细胞培养和动物模型探索Brk调控肠上皮细胞分化的机制；3)评估Brk在肠肿瘤发生中的作用。我们将使用多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，这可能取决于其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，这对于了解其对肠组织稳态和肠癌发展的潜在贡献至关重要。

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.