Receptor-mediated phosphorylation of the hepatic insulin receptor: Evidence that the M _r 95,000 receptor subunit is its own kinase

Abstract

Insulin stimulates the phosphorylation of its own receptor. In the work reported here, the kinase activity responsible for the insulin-stimulated phosphorylation of the insulin receptor was localized. In a first approach, partially purified insulin receptors derived from normal rat hepatocytes were immunoprecipitated with antibodies specific for the insulin receptor; thereafter, the immunoprecipitates were incubated with [gamma-(32)P]-ATP in the absence or presence of insulin (1 muM). NaDodSO(4)/polyacrylamide gel electrophoretic analysis of the immunoprecipitates under reducing conditions revealed autophosphorylation of the beta subunit (M(r) 95,000) of the insulin receptor; the alpha subunit (M(r) 130,000) was not phosphorylated. Further, insulin specifically increased 3- to 4-fold the labeling of its own receptor beta subunit, indicating that anti-receptor antibodies precipitate a functional and insulin-stimulable protein kinase that appears to be independent of cyclic AMP and calcium. To localize more precisely the insulin receptor-related kinase activity, we searched for an ATP-binding site on solubilized insulin receptors. By using covalent labeling with oxidized [alpha-(32)P]ATP, a labeled polypeptide with precisely the same electrophoretic mobility as that of the beta subunit of the insulin receptor (M(r) 95,000) was specifically immunoprecipitated with anti-receptor antibodies. Further, its appearance was prevented when the immunoprecipitation was preceded by incubation with unlabeled insulin. In conclusion, we have shown that an insulin-stimulated phosphorylation site and an ATP-binding site coexist on the beta subunit of the insulin receptor. The simultaneous presence of these two sites on the same receptor subunit indicates that the insulin receptor acts as its own protein kinase.

Protein kinase activity of the insulin receptor

“…Subsequently, insulin-stimulated phosphorylation of the f-subunit of insulin receptor was demonstrated in cell-free systems using [y-32P]ATP in solubilized and partially purified receptor preparations from rat liver ( Fig. 3) and human placenta, and analysis of phosphoaminoacids showed predominant labelling of phosphotyrosine and to a smaller degree of phosphoserine (Kasuga et al, 1982b,;Van Obberghen et al, 1983: Avruch et al, 1982Petruzzelli et al, 1982;Roth & Cassell, 1983;. In intact cells phosphoaminoacid analysis of the phosphorylated fisubunit of insulin receptors showed phosphoserine, phosphothreonine and phosphotyrosine under basal conditions.…”

“…The purification scheme was based on sequential affinity chromatography on wheat germ agglutinin-and insulin-agarose (FujitaYamaguchi et al, 1983;Roth et al, 1983b;Petruzzelli et al, 1984;Nemenoffet al, 1984;Kasuga et al, 1983a,b). Alternatively, the lectin-purified receptors were immunoprecipitated with antibodies to insulin receptors obtained from patients with severe insulin-resistance and acanthosis nigricans (Kasuga et al, 1982b;Van Obberghen et al, 1983), or monoclonal IgG directed against insulin receptors (Roth et al, 1982;Kull et al, 1983). These purified receptor preparations exhibited insulinstimulated protein kinase activity which catalysed phosphorylation of both the f-subunit and exogenous substrates like casein, histones and synthetic tyrosinecontaining peptides.…”

“…In the proteins, phosphoaminoacid analysis showed only phosphorylation on tyrosine residues. Among the synthetic peptides, even the dipeptide Tyr-Arg was a substrate, although with very high Km (Stadtmauer & Rosen, 1983 Kasuga et al (1982a-c), Van Obberghen et al (1983, Haring et al (1982Haring et al ( , 1984b, Velicelebi & Aiyer (1984), Burant et al (1984), Petruzzelli et al (1982, Roth & Cassell (1983), (1982b, 1983a,b), Van Obberghen et al (1983), Roth et al (1982, Petruzzelli et al (1984 Gazzano et al (1983, Kasuga et al (1983b), Pike et al (1984), Stadtmauer & Rosen (1983), Sadoul et al (1985), …”

Immunoprecipitation of insulin receptors by antibodies against Class 1 antigens of the murine H‐2 major histocompatibility complex

“…I~RO~UCTION The insulin receptor is a multimeric complex composed of two major subunits: the a-subunit (A.& 130~) containing the insulin binding site [1,2], and the &subunit (Mr 95000) containing a protein kinase activity leading to a selfphosphorylation of the receptor in the presence of insulin [3,4]. In addition to these 2 major components, a MI 45000 polypeptide has also been observed [5,6] and is thought to represent a proteolytic fragment of the &subunit [6].…”

Protein kinase activity of the insulin receptor from muscle

“…The kinase activity of receptor from human placenta was preserved after purification of the receptor protein to near homogeneity by insulin-affinity chromatography [ 131. Furthermore, the 95-kDa subunit of receptor from liver was labelled with ATP-affinity reagents suggesting that the catalytic domain is located in this subunit [14].…”