Inhibition of ErbB-2 mitogenic and transforming activity by RALT, a mitogen-induced signal transducer which binds to the ErbB-2 kinase domain

The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen employing the ErbB-2 juxtamembrane and kinase domain s as bait. This interaction was reproduced in vitro with a glutathione S-tr ansferase fusion protein spanning positions 282 to 395 of the 459-residue g ene 33 protein. Activation of ErbB-2 catalytic function was required for Er bB-2-gene 33 physical interaction in living cells, whereas ErbB-2 autophosp horylation was dispensable. Expression of gene 33 protein was absent in gro wth-arrested NIH 3T3 fibroblasts but was induced within 60 to 90 min of ser um stimulation or activation of the ErbB-2 kinase and decreased sharply upo n entry into S phase. New differentiation factor stimulation of mitogen-dep rived mammary epithelial cells also caused accumulation of gene 33 protein, which could be found in a complex with ErbB-2. Overexpression of gene 33 p rotein in mouse fibroblasts inhibited (i) cell proliferation driven by ErbB -2 but not by serum, (ii) cell transformation induced by ErbB-2 but not by Ras or Src, and (iii) sustained activation of ERK 1 and 2 by ErbB-2 but not by serum. The gene 33 protein may convey inhibitory signals downstream to ErbB-2 by virtue of its association with SH3-containing proteins, including GRB-2, which was found to associate with gene 33 protein in living cells. These data indicate that the gene 33 protein is a feedback inhibitor of Erb B-2 mitogenic function and a suppressor of ErbB-2 oncogenic activity. We pr opose that the gene 33 protein be renamed with the acronym RALT (receptor-a ssociated late transducer).