The overall objectives of this grant are to determine the role of Bcr sequences in the leukemogenic effects of Bcr-Abl oncoproteins, and to determine the role of the normal Bcr protein in Philadelphia chromosome (PhI)-positive leukemias. The hypothesis is that normal Bcr can: 1) supplement the oncogenic response as a result of tyrosine phosphorylation of Bcr; 2) play a negative role, which would be carried out by non-tyrosine phosphorylated Bcr. They have established that P160 BCR forms stable complexes with P210 BCR-ABL and P185 BCR-ABL. Importantly, cell lines from CML and ALL patients expressing Bcr-Abl contain tyrosine phosphorylated P160 BCR, but not cells lacking Bcr-Abl. Moreover, two cell lines lacking functional Bcr grow well in vitro and have an active Bcr-Abl tyrosine kinase, thus indicating that normal Bcr is not required for Bcr-Abl function. The 3' BCR anti-sense studies raise the possibility that normal Bcr opposes the effects of Bcr-Abl. In addition, the results indicate that the Bcr-Abl tyrosine kinase inhibits the Bcr autophosphorylation activity. In other studies, Bcr sequences within the first exon of Bcr-Abl and the same sequences within normal Bcr can by tyrosine phosphorylated by Bcr-Abl in vitro at residues located within the first Bcr exon. They have identified one of these residues as phosphotyrosine 177, which is involved in binding Grb2/Sos proteins known to activate the Ras signalling pathway. They have identified two other phosphotyrosine residues (Tyr 283 and 360) within the first exon of Bcr. These findings indicate that Bcr sequences play a positive role in the transformation events as a result of tyrosine phosphorylation.
The specific aims are: 1) identify phosphotyrosine residues encoded by the first exon of BCR; 2) determine the biological activity of BCR-ABL and BCR first exon mutants either alone or together in transformation assays; 3) identify proteins that interact with tyrosine phosphorylated Bcr sequences, determine their binding site within Bcr, and probe the role of these proteins in leukemic effects of Bcr-Abl; 4) determine whether Bcr plays a stimulatory and/or a negative role in Bcr-Abl oncogenic effects; and what role Bcr plays in cells not expressing Bcr- Abl; 5) determine the effects of tyrosine phosphorylation of P160 BCR on its kinase and GAP biochemical activities; 6) identify proteins that interact with domains of P160 Bcr using the yeast 2 hybrid system. The investigators are confident that these studies will provide important new information on the possible mechanisms employed by the Bcr-Abl oncoprotein and those of normal Bcr in these leukemias.
