GROWTH FACTOR-INDUCED TYROSINE PHOSPHORYLATION OF HRS, A NOVEL 115-KILODALTON PROTEIN WITH A STRUCTURALLY CONSERVED PUTATIVE ZINC-FINGER DOMAIN

The activation of growth factor receptor tyrosine kinases leads to tyr osine phosphorylation of many intracellular proteins which are thought to play crucial roles in growth factor signaling pathways. We previou sly showed that tyrosine phosphorylation of a 115-kDa protein is rapid ly induced in cells treated with hepatocyte growth factor. To clarify the structure and possible function of the 115-kDa protein (designated Hrs for hepatocyte growth factor-regulated tyrosine kinase substrate) , we purified this protein from B16-F1 mouse melanoma cells by anti-ph osphotyrosine immunoaffinity chromatography and determined its partial amino acid sequences. On the basis of the amino acid sequences, we mo lecularly cloned the cDNA for mouse Hrs. The nucleotide sequence of th e cDNA revealed that Hrs is a novel 775-amino-acid protein with a puta tive zinc finger domain that is structurally conserved in several othe r proteins. This protein also contained a proline-rich region and a pr oline- and glutamine-rich region. The expression of Hrs mRNA was detec ted in all adult mouse tissues tested and also in embryos. To analyze the Hrs cDNA product, we prepared a polyclonal antibody against bacter ially expressed Hrs. Using this antibody, we showed by subcellular fra ctionation that Hrs is localized to the cytoplasm; we also showed that that tyrosine phosphorylation of Hrs is induced in cells treated with epidermal growth factor or platelet-derived growth factor. These resu lts suggest that Brs plays a unique and important role in the signalin g pathway of growth factors.