Cis-Autophosphorylation of Juxtamembrane Tyrosines in the Insulin Receptor Kinase Domain

Receptor tyrosine kinases undergo ligand-induced dimerization that promotes kinase domain trans-autophosphorylation. However, the kinase domains of the insulin receptor are effectively dimerized because of the covalent α2β2 holomeric structure. This fact has made it difficult to determine the molecular mechanism of intraholomeric autophosphorylation, but there is evidence for both cis- and trans-autophosphorylation in the absence and presence of insulin. Here, using the cytoplasmic kinase domain (CKD) of the human insulin receptor, we demonstrate that autophosphorylation in the juxtamembrane (JM) subdomain follows a cis-reaction pathway. JM autophosphorylation was independent of CKD concentration over the range 6 nM−3 μM and was characterized kinetically: Half-saturation (K ATP) was observed at 75 μM ATP [5 mM Mn(CH3CO2)2] with a maximal rate of 0.24 mol of PO4 (mol of CKD)-1 min-1. Pairwise substitutions of Phe for Tyr in the other two autophosphorylation subdomains, generated by site-directed mutagenesis, altered the kinetics of JM autophosphorylation but did not change the pathway from a cis-reaction. Tyr1328,1334 to Phe (in the carboxy-terminal subdomain) yielded