Studying the structure of the intracellular moiety of the insulin receptor with a kinase-splitting membranal proteinase.
A kinase-splitting membranal proteinase specifically clips the cytoplasmic moiety of the insulin receptor beta-subunit (95 kd) to yield an 84-kd fragment. Using antibodies against different domains in the receptor, cleavage is shown to remove an 11-kd 'tail' (rooted at the C-terminal end of the kinase domain) which includes tyrosines 1316 and 1322. This cleavage impairs the ability of the clustered tyrosines 1146, 1150 and 1151 to undergo autophosphorylation. Nevertheless, the clipped beta-subunit is as active as the intact subunit if its kinase activity is measured at high exogenous substrate concentrations (greater than or equal to 2 mg/ml) indicating that autophosphorylation is not obligatory for insulin-dependent phosphotransferase activity. With low substrate concentrations (e.g. 0.2 mg/ml) a severe damage to the kinase activity is detected, which may reflect an important structural contribution of the 'tail' and/or the clustered phosphotyrosines in creating the preferential affinity of the kinase for its in vivo substrate(s). The membranal proteinase strictly recognizes the native conformation of the kinase domain, and fails to cleave it after denaturation. Since such a conformation-dependent cleavage occurs also in the case of the cytoplasmic moiety of the EGF receptor and the catalytic subunit of cAMP-dependent protein kinase, it is suggested that the similarity between these three kinase domains extends beyond their reported sequence homology to reflect a similarity in conformation.
Seger
R
R
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Zick
Y
Y
Shaltiel
S
S
D000818:Animals; D001665:Binding Sites; D003593:Cytoplasm / Q000378:metabolism; D010450:Endopeptidases; D066298:In Vitro Techniques; D007700:Kinetics; D015394:Molecular Structure; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*
D011972:Receptor, Insulin; D010450:Endopeptidases; C059784:kinase-splitting membranal proteinase