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Fig. 4. Reggie-1 is phosphorylated at several tyrosine residues. (A) Eight tyrosine residues of reggie-1 predicted to become phosphorylated were mutated to phenylalanine and the phosphorylation of these mutants was analyzed in pervanadate-stimulated cells after immunoprecipitation with anti-GFP. All single or double mutants exhibited a similar degree of phosphorylation as the wild-type protein. Note the increase in the amount of the lower band in the Y27F mutant, probably owing to increased processing. (B) Seven Tyr residues in R1-EGFP (excluding Tyr27) were mutated into Phe within one construct (7xYF) and six residues (excluding Tyr27 and Tyr163) in another one (6xYF), and the phosphorylation was analyzed upon EGF stimulation (5 minutes) in the absence of pervanadate. The 7xYF mutant was found to be unphosphorylated, indicating that multiple phosphorylation of reggie-1 can take place. The 6xYF mutant was phosphorylated, although somewhat less than the wt protein, indicating that Y163 is indeed phosphorylated. (C) Y158F and Y163F mutants were phosphorylated in equal amounts as the wild-type protein when treated with pervanadate. A-C, HeLa cells.
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