Increased in vivo phosphorylation of insulin receptor at serine 994 in the liver of obese insulin-resistant Zucker rats

Abstract

Serine phosphorylation of the insulin receptor (IR) has been proposed to exert an inhibitory influence on its tyrosine kinase activity. Previous works using site-directed mutagenesis suggested that serine 994 of the IR (IR Ser 994) might be part of an inhibitory domain of the receptor. In this study we examined whether this residue is subjected to phosphorylation in vivo. We used a sitephosphospecific antibody to determine the extent of phosphorylation of IR Ser 994 in insulin target tissues from two animal models of insulin resistance with different IR kinase (IRK) activity: obese (fa/fa) Zucker rats and transgenic mice overexpressing bovine growth hormone (PEPCK-bGH mice).Phosphorylation at IR Ser 994 was markedly increased in liver of obese rats. This alteration appeared to be tissue-selective since no phosphorylation on Ser 994 was detected in IRs isolated from skeletal muscle of these animals. On the other hand, the phosphorylation level of IR Ser 994 was very low in liver of PEPCK-bGH mice and did not differ from that of the control group. We have also demonstrated that protein kinase (PK) C isoforms , I and are able to promote the in vitro phosphorylation of the IR on Ser 994. Differential findings in these two models of insulin resistance might thus reflect increased PKC activity resulting from increased lipid availability in obese Zucker rats. Our results suggest that Ser 994 is a novel in vivo IR phosphorylation site that might be involved in the regulation of the IRK in some states of insulin resistance.

Regulation of insulin receptor function

“…Similarly, there is evidence that serine phosphorylation of the IR impairs the autophosphorylation response to insulin binding, and likely contributes to impaired insulin signaling related to obesity as well as that resulting from glucose toxicity. In studies of cells or with isolated proteins, protein kinase C (PKC) -mediated phosphorylation of the IR has been demonstrated on serine residues 967 and 968 of the juxtamembrane region [93], serines 1006, 1035 and 1037 in the catalytic domain [94,95], and serines 1288, 1305, 1306, 1321, 1327 and threonine 1348 in the C-terminus [93, 96 -100] (Fig. 4b).…”

“…Pharmacological inhibition of PKC activity can reverse the impaired insulin stimulation of glucose transport in muscle strips obtained from obese subjects [45]. Serine phosphorylation of the IR likely contributes to this effect as increased phosphorylation of the IR on serine 944 has been demonstrated in muscle and liver from obese insulin-resistant rodents [94,106]. In humans, serine phosphorylation of muscle IR has been observed in muscle from PCOS patients [44].…”

TANK-binding kinase 1 mediates phosphorylation of insulin receptor at serine residue 994: a potential link between inflammation and insulin resistance

“…4), 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM phenylmethylsulphonyl fluoride, and 0 . 1 mg/ml aprotinin) at 4 8C as described previously (Coba et al 2004). Liver extracts were centrifuged at 100 000 g for 1 h at 4 8C to eliminate insoluble material, and protein concentration in the supernatants was measured using the Bradford method as described previously (Coba et al 2004).…”

“…Immune complexes were collected by incubation with protein A-Sepharose 6 MB as described previously (Coba et al 2004). SDS-PAGE and western transfer of proteins to PVDF membranes (Hybond-P, GE Healthcare Bio-Sciences, Piscataway, NJ, USA) were performed as previously described (Coba et al 2004).…”

Regulation of hepatic insulin receptor activity following injury

“…As the initial molecule of the signaling cascade, moderate changes in IR content or function may not impact insulin action, and studies focusing on the regulation of the IR have produced inconsistent results (5,19,23). However, defects in the ability of insulin to induce IR tyrosine phosphorylation have been found in various chronic models of insulin resistance, including models of Type 2 diabetes and obesity (5,19). Studies with heterozygous IR knockout mice indicated that a reduction of the number of IRs can have a significant physiological impact, decreasing insulin signal transduction and insulin action (2).…”