Fig. 1. Insulin induces rapid and sustained tyrosine phosphorylation of annexin A2 in BHK cells stably expressing the human insulin receptor. (A) Time course of insulin-induced tyrosine phosphorylation of BHK-IR cell proteins. Serum-starved BHK-IR cells were treated with 100 nM insulin for the indicated times and total cellular lysates were probed by immunoblotting with anti-phosphotyrosine antibodies. Arrows indicate the positions of the insulin receptor β-subunit (IRβ) and the heavily phosphorylated 36 kDa protein (pp36). (B) The tyrosine-phosphorylated polypeptide of 36 kDa is annexin A2. Lysates of cells stimulated for the indicated periods of time with 100 nM insulin were subjected to immunoprecipitation (IP) with the monoclonal anti-annexin A2 antibody HH7. The precipitated protein was immunoblotted (WB) for phosphotyrosine (-pY) and the blot subsequently reprobed with anti-annexin A2 antibodies (-Anxa2). c, control lane (lysates were incubated with beads alone). (C) Annexin A2 phosphorylation increases during the first 3 hours of insulin treatment. Serum-starved cells were stimulated for the indicated times with 100 nM insulin. The intensity of the pp36 band was measured by densitometric scanning and is given as arbitrary units (mean values ± s.e.m.).