Expression, characterization, and crystallization of the catalytic core of the human insulin receptor protein-tyrosine kinase domain.
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.
Wei
L
L
W. M. Keck Center for Genome Informatics, Texas A & M University, Houston 77030, USA.
Hubbard
S R
SR
Hendrickson
W A
WA
Ellis
L
L
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D003460:Crystallization; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera; D014443:Tyrosine / Q000378:metabolism
D018076:DNA, Complementary; D014443:Tyrosine; D011972:Receptor, Insulin