+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

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



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



Biochemistry 31(49): 12455-12462



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 apprx 360-fold increase in the specific kinase activity with a yield of apprx 50%. An appM-r = apprx 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 apprx 250,000 and apprx 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. K-m and V-max for two substrates, src-related peptide and poly(Glu, Tyr) (4:1), were 2.4 mM and 2.5 mu-mol min-1 mg-1 and 0.26 mM and 1.2 mu-mol 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 apprx 3-fold. In the presence of 1 mu-M polylysine, the kinase activity was stimulated on average apprx 4-fold. Polylysine treatment concurrently resulted in both aggregation and activation of the enzyme. An oligomeric form of the enzyme with extremely high basal kinase activity and an additional apprx 4-fold activation and aggregation of the enzyme by polylysine is consistent with the notion that aggregation and concomitant activation of the receptor kinase can occur through the beta-subunit domain without participation of the alpha subunit.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 008788719

Download citation: RISBibTeXText

PMID: 1334428

DOI: 10.1021/bi00164a023


Related references

Human insulin receptor b-subunit transmembrane/cytoplasmic domain expressed in a baculovirus expression system: purification, characterization, and polylysine effects on the protein tyrosine kinase activity. Biochemistry (American Chemical Society) 31: 455-62, 1992

Synthesis, purification, and characterization of the cytoplasmic domain of the human insulin receptor using a baculovirus expression system. Journal of Biological Chemistry 263(12): 5560-5568, 1988

Expression, purification, and characterization of the cytoplasmic domain of the human IGF-1 receptor using a baculovirus expression system. Biochemical and Biophysical Research Communications 260(3): 724-728, 1999

The full-length, cytoplasmic C-terminus of the beta 2-adrenergic receptor expressed in E. coli acts as a substrate for phosphorylation by protein kinase A, insulin receptor tyrosine kinase, GRK2, but not protein kinase C and suppresses desensitization when expressed in vivo. Protein Expression and Purification 20(3): 451-461, 2000

Purification and characterization of human ZAP-70 protein-tyrosine kinase from a baculovirus expression system. Journal of Biological Chemistry 271(26): 15753-15761, 1996

Interaction of the human insulin receptor tyrosine kinase from the baculovirus expression system with protein kinase C in a cell-free system. Journal of Biological Chemistry 268(10): 7571-7576, 1993

Production, purification and characterization of non-myristylated human T-cell protein tyrosine kinase in a baculovirus expression system. Gene 169(2): 275-279, 1996

Efficient expression in insect cells of a soluble, active human insulin receptor protein-tyrosine kinase domain by use of a baculovirus vector. Journal of Virology 62(5): 1634-1639, 1988

The role of COOH-terminal and acidic domains in the activity and stability of human insulin receptor protein tyrosine kinase studied by purified deletion mutant of the beta subunit domain. Journal of Biological Chemistry 268(30): 22444-22449, 1993

Protein tyrosine kinase activity of native and carboxyl terminal domain- and acidic domain-deleted mutants of insulin receptor kinase expressed in insect cells. FASEB Journal 7(7): A1159, 1993

The purified COOH-terminal domain of the insulin receptor carries activity to stimulate protein kinase activity or autophosphorylation of the beta subunit domain of insulin receptor. Biochemical and Biophysical Research Communications 200(2): 777-783, 1994

Expression, purification and characterization of a 41 kDa insulin receptor tyrosine kinase domain. Biochemical and Biophysical Research Communications 168(2): 616-624, 1990

Relationship of site-specific beta subunit tyrosine autophosphorylation to insulin activation of the insulin receptor (tyrosine) protein kinase activity. Journal of Biological Chemistry 263(10): 4593-4601, 1988

Expression, characterization, and crystallization of the catalytic core of the human insulin receptor protein-tyrosine kinase domain. Journal of Biological Chemistry 270(14): 8122-8130, 1995

An extracellular domain of the insulin receptor beta-subunit with regulatory function on protein-tyrosine kinase. Journal of Biological Chemistry 264(15): 8627-8635, 1989