( p using tyrosine-phosphoryl...)TKB1 Cells Identify Receptor Tyrosine Kinase Interacting Proteins

p> using tyrosine-phosphorylated GST-fusion proteins produced in bacteria. Because E. coli do not contain detectable tyrosine kinases, we used the recombinant bacterial strain TKB1, which harbors an inducible Elk⁶ RTK domain. Plasmids encoding GST-fusion proteins were transformed into TKB1 cells or the parental RTK-deficient BL21 cells, and the fusion proteins were induced with IPTG, which is also capable of inducing expression of the Elk RTK domain in TKB1 bacteria but not in BL21 bacteria (Figure 1). The in vitro association assays carried out with bacteria produced unphosphorylated and phosphorylated fusions that recapitulate associations observed in vivo.

Figure 2

Neu contains five sites of tyrosine phosphorylation (Y1028, Y1144, Y1201, Y1226/7, and Y1253), which we renamed (to simplify) sites A through E in a membrane proximal to distal fashion. Three GST fusion proteins were constructed to individually contain phosphorylation sites Y1201, Y1226/7, and Y1253 (gT-YC, YD, and YE, respectively), as well as a fusion mutated at each of the three sites (gT-F3) (Figure 2). Plasmids encoding each GST-fusion protein were transformed into competent BL21 cells or TKB1 cells (following manufacturers instructions), and the fusion proteins from 3 ml of bacterial cultures were purified on glutathione sepharose as described.⁷ While Coomassie Blue staining revealed roughly equivalent protein levels from each strain, immunoblot analyses showed that gT-YC, gT-YD, and gT-YE were tyrosine phosphorylated when purified from TKB1 but were not phosphorylated from the parental BL21 strain (Figure
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