2 SEQUENCES FLANKING THE MAJOR AUTOPHOSPHORYLATION SITE OF THE INSULIN-RECEPTOR ARE ESSENTIAL FOR TYROSINE KINASE ACTIVATION

The tyrosine kinase domain of the human insulin receptor (IR) contains several short amino acid motifs which are strictly conserved in all p rotein kinases and two sequence motifs which are specific to the tyros ine kinases (AAR or RAA and P-I/(K)(V)/W-R(T)/M). In the serine/threon ine kinases these motifs are replaced by the sequences KPE and G(T)/(S )XX(Y)/(P)X respectively. In the present work, the tyrosine kinase-spe cific sequences of the IR ((1134)AAR(1136) and (1172)PVRWM(1176)) were replaced using site-directed mutagenesis by sequences which confer a serine kinase specificity on the receptor. Five different IR mutants w ere expressed in Chinese hamster ovary (CHO) or COS cells and their st ructural and functional properties compared with those of the wild-typ e recombinant human IR. These mutants are processed normally and bind insulin with normal affinities. None of the mutants containing a putat ive serine kinase-specific sequence display detectable autophosphoryla tion or tyrosine kinase activity in response to insulin, either in vit ro or in vivo. These mutants were also unable to phosphorylate serine/ threonine kinase substrates after insulin stimulation. Unexpectedly, t hey showed impaired ATP binding, as studied by an original technique c onsisting of cross-linking adenosine 5'-{[S-35]thio}tri-phosphate to p artially purified receptors. Finally, none of the studied mutants tran smit the insulin signal necessary to stimulate either DNA or glycogen synthesis. These data provide evidence for the importance of these con served sequences in the kinase domain for both receptor activation and kinase activity. Furthermore, they demonstrate that the exchange of s equences specific to the catalytic domain of tyrosine kinases for thos e specific to the serine/threonine kinases is not sufficient to confer serine/threonine specificity on the insulin receptor.