MODULATION OF ERBB KINASE-ACTIVITY AND ONCOGENIC POTENTIAL BY SINGLE-POINT MUTATIONS IN THE GLYCINE LOOP OF THE CATALYTIC DOMAIN

Avian c-erbB is activated to a leukemia oncogene following truncation of its amino-terminal ligand-binding domain by retroviral insertion. T he insertionally activated transcripts encode protein products which h ave constitutive tyrosine kinase activity and can induce erythroleukem ia but not sarcomas. We have previously found that a valine-to-isoleuc ine point mutation at position 157 (V157I mutant) within the tyrosine kinase domain of this truncated erbB can dramatically activate the sar comagenic potential of the oncogene and increase the kinase activity o f this oncoprotein. This mutation lies at position 157 of the insertio nally activated c-erbB product, affecting a highly conserved valine re sidue of the glycine loop involved in ATP binding and phosphate transf er. To investigate the functional importance of this residue in the ca talytic activity of kinases, we have introduced at this position, by s ite-directed mutagenesis, codons representing the remaining 18 amino a cid residues. Most of the mutants have diminished activity, with six o f them completely devoid of kinase activity, indicating the sensitivit y of this region to conformational changes. Some of these mutants disp layed increased kinase activity and greater transforming potential in comparison with IA c-erbB, but none had levels as high as those of the V157I mutant. In general, the sarcomagenic potential of the various e rbB mutants correlated with their autophosphorylation state and their ability to cause phosphorylation of MAP kinase. However, there are imp ortant exceptions such as the V157G mutant, which lacks enhanced autop hosphorylation but is highly sarcomagenic. Studies of this and other a utophosphorylation site mutants point to the existence of an autophosp horylation-independent pathway in sarcomagenesis. The requirement for leukemo genic potential is much less stringent and correlates with pos itivity of kinase activity. When the valine-to-isoleucine substitution was put in context of the full-length erbB protein, the mutation rela xed the ligand dependence and had a positive effect on the transformin g potential of the full-length c-erbB