Expression of the insulin receptor with a recombinant vaccinia virus. Biochemical evidence that the insulin receptor has intrinsic serine kinase activity.
We have previously reported the tight association of a serine kinase activity with the human insulin receptor (Lewis, R. E., Wu, G. P., MacDonald, R. G., and Czech, M. P. (1990) J. Biol. Chem. 265, 947-954). We tested the possibility that the associated serine kinase activity was intrinsic to the receptor catalytic domain. The ratio of phosphoserine to phosphotyrosine on insulin receptors phosphorylated in vitro was used as an index of the associated serine kinase activity. Phosphorylation and phosphoamino acid analysis of insulin proreceptors revealed associated serine kinase activity early in receptor synthesis. Insulin receptors were expressed in HeLa cells using a recombinant vaccinia virus. The ratio of phosphoserine to phosphotyrosine on insulin receptors expressed by the recombinant vaccinia virus was determined relative to endogenous insulin receptors in cells treated with alpha-amanitin to block host cell mRNA synthesis. alpha-Amanitin treatment had no effect on the ratio of phosphoserine to phosphotyrosine on insulin receptors expressed from the recombinant virus even though they were present in a 4000-fold excess above endogenous receptors. We conclude that the serine kinase activity associated with the insulin receptor is intrinsic to the receptor catalytic domain. Receptor-catalyzed autophosphorylation of serine may play an important role in modulating insulin receptor signaling.
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805, USA.
T J
TJ
Tauer
D J
DJ
Volle
S L
SL
Rhode
R E
RE
Lewis
D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D006801:Humans; D010449:Peptide Mapping; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D014616:Vaccinia virus / Q000235:genetics*
D000546:Amanitins; D012333:RNA, Messenger; D011972:Receptor, Insulin; D017346:Protein Serine-Threonine Kinases