Human insulin receptor beta-subunit transmembrane/cytoplasmic domain expressed in a baculovirus expression system: purification, characterization, and polylysine effects on the protein tyrosine kinase activity.
We have expressed, purified, and characterized the insulin receptor protein tyrosine kinase (PTK) retaining the transmembrane and downstream domains. The proteins expressed in insect cells using a baculovirus expression system were identified as membrane-bound by immunofluorescence staining and biochemical characterization. One-step purification by immunoaffinity chromatography from Triton X-100 cell extracts resulted in a approximately 360-fold increase in the specific kinase activity with a yield of approximately 50%. An appMr = approximately 60,000 protein was the major component identified by both silver staining of the purified enzyme and immunostaining of the crude extracts after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Using nondenaturing conditions, the molecular weight was estimated to be approximately 250,000 and approximately 500,000 by glycerol gradient centrifugation and gel permeation chromatography, respectively, suggesting that oligomers of the beta-subunit domains such as tetramers and octamers are formed. The basal PTK activity of this enzyme was much higher than those of previously reported soluble-form insulin receptor PTKs expressed in insect cells or the native receptor. Km and Vmax for two substrates, src-related peptide and poly(Glu, Tyr) (4:1), were 2.4 mM and 2.5 mumol min-1 mg-1 and 0.26 mM and 1.2 mumol min-1 mg-1, respectively. Specific activities measured under two previously reported conditions using histone H2B as a substrate were 100 or 135 nmol min-1 mg-1, in contrast to those of soluble PTKs which were reported to be 20 or 70 nmol min-1 mg-1, respectively. The purified enzyme was autophosphorylated at Tyr residues. Autophosphorylation activated the enzyme approximately 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)
Li
S L
SL
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Yan
P F
PF
Paz
I B
IB
Fujita-Yamaguchi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007915:Lepidoptera; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D011107:Polylysine / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D011107:Polylysine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin