Regulation of Akt and glucose transport by prolactin

描述(申请人提供)：哺乳对新生动物的存活非常重要。催乳素通过调节乳腺上皮细胞向分泌上皮细胞的分化，在这一过程中起着关键作用。许多关于催乳素信号转导的研究都集中在它在调节牛奶蛋白基因转录中的作用。然而，由于牛奶含有25%-30%的脂肪和约3%的乳糖，控制葡萄糖供应和脂肪生物合成的途径可能对最佳牛奶产量至关重要。我们以前的研究表明，丝氨酸/苏氨酸蛋白激酶Akt，在抑制乳腺退行性变中的细胞凋亡中起着关键作用。我们假设，催乳素调节葡萄糖转运和脂肪生物合成，Akt蛋白激酶是催乳素介导的葡萄糖转运激活所必需的。在这项资助中，我们建议定位催乳素受体的区域(S)，这是激活Akt和葡萄糖运输所必需的。我们将重点放在磷酸化的酪氨酸残基上，因为信号分子与这些位点的结合通常调节它们的激活，特别是含有SH2结构域的分子。我们将使用一种嵌合的催乳素受体，其激活可以由化学二聚体控制。这种嵌合受体将在乳腺上皮细胞中表达，以绘制调控葡萄糖运输和Akt激活的区域。将产生在乳腺中表达嵌合催乳素受体的转基因小鼠，以测试特定受体突变的功能。这些研究将在脑下垂体分泌催乳素受阻的情况下进行，从而阻止内源性催乳素受体的激活。在Src-/-小鼠中，乳腺发育发生了改变，但在Fyn-/-小鼠中没有改变。来自Src-/-和Fyn-/-小鼠的原代乳腺上皮细胞将被用来确定这些激酶在调节Akt激活和葡萄糖运输中的作用。我们还将测试特定的信号分子(c-Cb1、Cbl-B、IRS-1、Gab2、Gab3、SHP 1和SHP2)在调节PI3-Kinase/Akt通路和葡萄糖运输中的作用。最后，我们将确定催乳素增加乳腺上皮细胞葡萄糖转运的机制。到目前为止，调控乳腺上皮细胞葡萄糖转运的机制仍然是个谜。这些研究应该提供有关催乳素在乳腺中的其他作用的重要信息，并确定催乳素调节这些不同过程的机制。

DESCRIPTION (provided by applicant): Lactation is of great importance to survival of newborn animals. Prolactin plays a critical role in this process by regulating the differentiation of mammary epithelial cells into secretory epithelial cells. Much of the research on prolactin signal transduction has focused upon its role in regulating the transcription of milk protein genes. However, since milk is 25 - 30% fat and about 3% lactose, pathways that control glucose availability and lipid biosynthesis are likely to be critical for optimal milk production. Our previous studies indicate that the serine/threonine protein kinase Akt, plays a critical role in suppressing apoptosis in the involuting mammary gland. We hypothesize that prolactin regulates glucose transport and lipid biosynthesis and the Akt protein kinase is required for prolactin-mediated activation of glucose transport. In this grant we propose to map the region(s) of the prolactin receptor that are required for activation of Akt and glucose transport. We will focus on phosphorylated tyrosine residues because the binding of signaling molecules to these sites often regulates their activation, particularly SH2 domain containing molecules. We will use a chimeric prolactin receptor whose activation can be controlled by a chemical dimerizer. This chimeric receptor will be expressed in mammary epithelial cells to map the regions that regulate glucose transport and Akt activation. Transgenic mice will be generated that express the chimeric prolactin receptor in the mammary gland to test the function of specific receptor mutants. These studies will be done under conditions where the production of prolactin by the pituitary is blocked thereby preventing activation of the endogenous prolactin receptor. Mammary gland development is altered in Src-/- mice but not in Fyn-/- mice. Primary mammary epithelial cells derived from Src-/- and Fyn-/- mice will be used to determine the role of these kinases in regulating Akt activation, and glucose transport. We will also test the role of specific signaling molecules (c-Cbl, Cbl-B, IRS-1, Gab2, Gab3, SHP 1, and SHP2) in regulating the PI3-kinase/Akt pathway and glucose transport. Finally, we will determine the mechanisms by which prolactin increases glucose transport in mammary epithelial cells. Until now, the mechanisms by which glucose transport in mammary epithelial cells is regulated have remained a mystery. These studies should provide important information about other roles for prolactin in the mammary gland and identify the mechanisms by which prolactin regulates these diverse processes.