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

Biochemistry. 2001 Aug 28;40(34):10243-53. doi: 10.1021/bi010959e.

Abstract

Activation of receptor tyrosine kinases by autophosphorylation is one of the most common and critical transformations in signal transduction, yet its role in catalysis remains controversial. Autophosphorylation of the angiogenic receptor tyrosine kinase Tie2 was studied in terms of the autophosphorylation 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 that Tyr992 on the putative activation loop was phosphorylated first followed by Tyr1108 in the C-terminal tail (previously unidentified autophosphorylation 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 studies showed that the unphosphorylated Tie2 was partially limited by product 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 pK(a) values of catalytically relevant ionizable residues upon autophosphorylation. The shift in rate-limiting step will result in a different distribution of enzyme pools (e.g., E, E*S, E*P, etc.) which may modulate the susceptibility to inhibition. Tie2 and pTie2 were profiled with a panel of known ATP-competitive kinase inhibitors. Tie2 activation perturbs catalytic residue ionizations, shifts the rate-limiting step to almost exclusive diffusion-control, and transforms the kinase into a more perfect catalyst.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Catalysis
  • Cell Line
  • Chromatography, High Pressure Liquid
  • Histones / metabolism
  • Humans
  • Isoelectric Focusing
  • Kinetics
  • Mass Spectrometry
  • Phosphopeptides / chemistry
  • Phosphoproteins / chemistry
  • Phosphoproteins / isolation & purification
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Receptor Protein-Tyrosine Kinases / chemistry*
  • Receptor Protein-Tyrosine Kinases / isolation & purification
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Receptor, TIE-2
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Spodoptera
  • Transfection
  • Viscosity

Substances

  • Histones
  • Phosphopeptides
  • Phosphoproteins
  • Recombinant Proteins
  • Adenosine Triphosphate
  • Receptor Protein-Tyrosine Kinases
  • Receptor, TIE-2