FIG. 1.
Mutations in the M5 loop that dissociate Hsp90 promote ErbB2 hyperphosphorylation. (a) Mutations in the M5 loop disrupt ErbB2 association with Hsp90 complex. Wild-type (wt) and M5 mutant ErbB2 proteins transiently expressed in COS7 cells were immunoprecipitated (IP) with mouse antibodies. The association of Hsp90 and p50cdc37 was detected by Western blotting with rat and goat antibodies, respectively. ErbB2 protein was detected with rabbit antibody. (b) ErbB2-5M has higher activity in transactivating ErbB3. COS7 cells were transfected with ErbB3 plasmid together with either wild-type or mutant ErbB2 or empty vector pcDNA3, switched to serum-free medium 24 h after transfection, and continued in culture overnight. Half of the cells were treated with 1 nM HRG for 15 min at 37°C. ErbB3 proteins were precipitated with mouse anti-ErbB3 antibodies, and phosphorylation was detected with biotinylated mouse antiphosphotyrosine antibody and horseradish peroxidase-conjugated streptavidin. ErbB3 protein levels were detected with rabbit antibody. ErbB2 protein levels in total cell lysates were detected with mouse antibody. (c) ErbB2-5M is hyperphosphorylated. The phosphorylation of wild-type ErbB2 and ErbB2-5M in total cell lysates of transiently transfected COS7 cells was detected by Western blotting with specific antibodies. ErbB2 protein levels were confirmed by Western blotting. (d) Phase-contrast images of wild-type ErbB2- and ErbB2-5M-expressing NIH 3T3 cells demonstrate anchorage-independent growth. Stably transfected NIH 3T3 cells were plated in soft agar-containing six-well plates and incubated for 3 weeks. Acini were stained with p-iodonitrotetrazolium violet and photographed by bright-field microscopy. Note the larger size of ErbB2-5M acini and the absence of peripheral projections in the smaller ErbB2 acini.