Expression, purification and characterization of a 41 kDa insulin receptor tyrosine kinase domain.

An active 41 kDa cytoplasmic domain of the insulin receptor tyrosine kinase (CIRK-41) encompassing amino acid residues 946 and 1303 of the native protein with an additional three amino acids (HAI) at the N-terminus has been overexpressed using the baculovirus pAC 373 expression system. The recombinant protein termed CIRK-41 has been purified to homogeneity. CIRK-41 was capable of autophosphorylation and up to 1.9 moles of phosphate were incorporated per mole of enzyme when it was incubated in the presence of 10 mM manganese chloride and 0.5 mM ATP. Autophosphorylation resulted in stimulation of CIRK-41 activity towards its exogenous substrate, indicating that CIRK-41 may be used as a model molecule to study the role of phosphorylation and dephosphorylation in the control of the insulin receptor tyrosine kinase activity.