Thompson J F, Buikhuisen W A
Department of Pediatrics, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts.
Gastroenterology. 1990 Aug;99(2):370-9. doi: 10.1016/0016-5085(90)91018-2.
Tyrosine phosphorylation has recently been recognized as a unique system involved in the regulation of cellular growth and differentiation. The role of tyrosine kinase activity in regulating intestinal proliferation has received little attention. The aim of this study was to document the presence of tyrosine kinase activity in intestinal microvillus membranes and to characterize the major endogenous tyrosine kinase substrates in microvillus membranes. Microvillus membranes, prepared from 21-day gestation fetal and adult CD rats by the calcium precipitation method, were solubilized in 0.1% Triton X-100 and incubated with [32P]adenosine triphosphate and (Glu80Tyr20)n, which is a heterogeneous population of synthetic peptides containing glutamate and tyrosine. Fetal, and to a lesser extent adult, microvillus membranes were shown to phosphorylate (Glu80Tyr20)n assayed by trichloroacetic acid-precipitable 32P incorporation as well as autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Preliminary identification of the endogenous substrates of microvillus membrane tyrosine kinase activity was determined by three techniques. First, phosphorylated microvillus membrane proteins were solubilized in sodium dodecyl sulfate and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resultant gel was incubated in 1 mol/L KOH at 55 degrees C to selectively retain phosphotyrosine proteins. The patterns of tyrosine phosphorylation were dissimiliar in fetal and adult microvillus membranes; specifically, two major bands, 80 and 190 kilodaltons, were present in fetal microvillus membranes and were not prominent in adult microvillus membranes. These proteins were both phosphorylated on tyrosine residues as determined by phosphoamino acid analysis. Second, a specific antiphosphotyrosine monoclonal antibody was used to immunoprecipitate phosphotyrosine proteins from solubilized phosphorylated microvillus membranes. This antibody specifically immunoprecipitated proteins of molecular weights 36 and 68 from fetal and 33, 54, and 68 from adult microvillus membranes. Third, using a polyclonal antiphosphotyrosine antibody, Western blot analysis showed that the 68-kilodalton protein is the most abundant phosphotyrosine protein in fetal and adult microvillus membranes. These data will focus new investigations into the cellular mechanisms of the regulation of intestinal growth, particularly the role of luminal factors that may modulate microvillus membrane tyrosine kinase, and thus modulate enterocyte proliferation, differentiation, and function.
酪氨酸磷酸化最近被认为是参与细胞生长和分化调节的独特系统。酪氨酸激酶活性在调节肠道增殖中的作用很少受到关注。本研究的目的是证明肠道微绒毛膜中存在酪氨酸激酶活性，并鉴定微绒毛膜中的主要内源性酪氨酸激酶底物。通过钙沉淀法从妊娠21天的胎儿和成年CD大鼠制备微绒毛膜，将其溶解在0.1% Triton X-100中，并与[32P]三磷酸腺苷和(Glu80Tyr20)n一起孵育，(Glu80Tyr20)n是一种含有谷氨酸和酪氨酸的合成肽异质群体。通过三氯乙酸可沉淀的32P掺入以及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳的放射自显影显示，胎儿微绒毛膜以及程度较轻的成年微绒毛膜可使(Glu80Tyr20)n磷酸化。通过三种技术对微绒毛膜酪氨酸激酶活性的内源性底物进行了初步鉴定。首先，将磷酸化的微绒毛膜蛋白溶解在十二烷基硫酸钠中，并在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上分离，然后将所得凝胶在55℃下于1mol/L氢氧化钾中孵育，以选择性保留磷酸酪氨酸蛋白。胎儿和成年微绒毛膜中的酪氨酸磷酸化模式不同；具体而言，胎儿微绒毛膜中有两条主要条带，分子量分别为80和190千道尔顿，而在成年微绒毛膜中不明显。通过磷酸氨基酸分析确定，这些蛋白的酪氨酸残基均被磷酸化。其次，使用特异性抗磷酸酪氨酸单克隆抗体从溶解的磷酸化微绒毛膜中免疫沉淀磷酸酪氨酸蛋白。该抗体从胎儿微绒毛膜中特异性免疫沉淀分子量为36和68的蛋白，从成年微绒毛膜中特异性免疫沉淀分子量为33、54和68的蛋白。第三，使用多克隆抗磷酸酪氨酸抗体，蛋白质印迹分析表明，68千道尔顿的蛋白是胎儿和成年微绒毛膜中最丰富的磷酸酪氨酸蛋白。这些数据将为肠道生长调节的细胞机制的新研究提供重点，特别是腔内因子的作用，这些因子可能调节微绒毛膜酪氨酸激酶，从而调节肠上皮细胞的增殖、分化和功能。
