Expression, Characterization, and Crystallization of the Catalytic Core of the Human Insulin Receptor Protein-tyrosine Kinase Domain

Abstract

The deduced primary sequence of the cytoplasmic protein-tyrosine kinase domain of the insulin receptor contains a conserved kinase homology region (receptor residues 1002-1257) flanked by a juxtamembrane region and a C-terminal tail. A soluble 48-kDa derivative (residues 959-1355) containing these regions but lacking the first six residues of the juxtamembrane region had earlier been synthesized in Sf9 cells using a baculovirus expression system. The catalytic core of the kinase domain was studied first by proteolytic analysis of the 48-kDa kinase and then by expressing a series of truncated kinase domains in transiently transfected COS cells. Based on these studies, two core kinases of 34 (residues 985-1283) and 35 (residues 978-1283) kDa, respectively, were overexpressed in Sf9 cells. Biochemical characterization of the 35-kDa kinase revealed that the core kinase conserved the major functional properties of the native receptor kinase domain. Activity of the 35-kDa kinase toward a synthetic peptide increased more than 200-fold upon autophosphorylation, which occurred exclusively at Tyr-1158, Tyr-1162, and Tyr-1163; the largest increase was observed between bis- and trisphosphorylation of the kinase. The activated 35- and 48-kDa kinases were similar with respect to specific activity and ATP and Mg2+ requirements for peptide phosphorylation. Moreover, autophosphorylation appeared to initiate predominantly at Tyr-1162, immediately followed by phosphorylation at Tyr-1158 and then at Tyr-1163. The rate of autophosphorylation was dependent on enzyme concentration, consistent with a trans-phosphorylation mechanism. Finally, the 35-kDa kinase was crystallized, making possible elucidation of its three-dimensional structure by x-ray crystallography.

Biochemical characterization of the protein tyrosine kinase homology domain of the ErbB3 (HER3) receptor protein

“…We have attempted to detect intrinsic protein tyrosine kinase activity in the ErbB3 protein by various approaches. As the protein tyrosine kinase domains of a variety of other receptors have been produced in catalytically active form with the baculovirus system [29,39,[40][41][42], we used this sytem in the generation of an ErbB3 cytosolic domain protein (hisTKD-B3). CD spectroscopic measurements indicated that the hisTKD-B3 protein was folded in a conformation similar to that of the corresponding EGF receptor cytosolic domain (hisTKD61), which displayed robust catalytic activity.…”

The Insulin Receptor: Both a Prototypical and Atypical Receptor Tyrosine Kinase

“…In the presence of cellular (millimolar) concentrations of Mg-ATP, the unphosphorylated activation loop probably adopts multiple conformations, including the pseudosubstrate conformation, as well as conformations in which Mg-ATP is bound and the active site is available for transphosphorylation of a tyrosine from the neighboring kinase domain. It was shown many years ago that Y1162 was the first of the three tyrosines in the IRK activation loop to undergo autophosphorylation, followed by Y1158 and Y1163 (Wei et al 1995). Several years ago, a crystal structure of the IGF1R kinase domain (IGF1RK) was determined in which the tyrosine equivalent to Y1162, Y1135, was bound in the active site of a symmetry-related kinase domain (in trans) (Wu et al 2008a).…”

Tyrosine 740 Phosphorylation of Discoidin Domain Receptor 2 by Src Stimulates Intramolecular Autophosphorylation and Shc Signaling Complex Formation

“…These studies have demonstrated that phosphorylation of three tyrosine residues (Tyr-1158, Tyr-1161, and Tyr-1162) within the activation loop of IRTK is critical for the activation of IRTK autophosphorylation as well as for its tyrosine kinase activity toward exogenous substrates (19,25,26).…”

“…Three Tyrosine Residues in the DDR2 Activation Loop Are the Targets of Src Phosphorylation-The activation mechanism of IRTK has been well characterized (18,19,22,25,26). These studies have demonstrated that phosphorylation of three tyrosine residues (Tyr-1158, Tyr-1161, and Tyr-1162) within the activation loop of IRTK is critical for the activation of IRTK autophosphorylation as well as for its tyrosine kinase activity toward exogenous substrates (19,25,26).…”