Mechanistic effects of autophosphorylation on receptor tyrosine kinase catalysis: Enzymatic characterization of Tie2 and phospho-Tie2

Activation of receptor tyrosine kinases by autophosphorylation is one of th e most common and critical transformations in signal transduction, yet its role in catalysis remains controversial. Autophosphorylation of the angioge nic receptor tyrosine kinase Tie2 was studied in terms of the autophosphory lation sites, sequence of phosphorylation at these sites, kinetic effects, and mechanistic consequences. Isoelectric focusing electrophoresis and mass spectrometric analysis of a Tie2 autophosphorylation time course showed th at Tyr992 on the putative activation loop was phosphorylated first followed by Tyr1108 in the C-terminal tail (previously unidentified autophosphoryla tion site). Autophosphorylation of Tie2 to produce pTie2 resulted in a 100- fold increase in k(cat) and a 460-fold increase in k(cat)/K-m. Viscosity st udies showed that the unphosphorylated Tie2 was partially limited by produc t diffusion ((k(cat))(eta) = 0.67 +/- 0.06), while product release was more rate-limiting ((k(cat))(eta) = 0.94 +/- 0.08) for autophosphorylated Tie2 (pTie2). Furthermore, autophosphorylation did not significantly affect the phosphoacceptor dissociation constants. There was a significant (k(cat))(H) /(k(cat))(D) solvent isotope effect (SIE) for unphosphorylated Tie2 (2.42 /- 0.12) and modest SIE (1.28 +/- 0.04) for pTie2, which is consistent with the chemistry step being more rate-limiting for Tie2 as compared to pTie2. The pH-rate profiles of Tie2 and pTie2 revealed a >0.5 unit shift in the p K(a) values of catalytically relevant ionizable residues upon autophosphory lation. The shift in rate-limiting step will result in a different distribu tion of enzyme pools (e.g., E, E.S, E.P, etc.) which may modulate the susce ptibility to inhibition. Tie2 and pTie2 were profiled with a panel of known ATP-competitive kinase inhibitors. Tie2 activation perturbs catalytic resi due ionizations, shifts the rate-limiting step to almost exclusive diffusio n-control, and transforms the kinase into a more perfect catalyst.