Protein tyrosine kinase activity and its substrates in rat intestinal microvillus membranes

Abstract

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 °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.