Diabetes Reduces Autophosphorylation of Retinal Insulin Receptor and Increases Protein-Tyrosine Phosphatase-1B Activity

Abstract

PURPOSE Protein-tyrosine phosphatase-1B (PTP1B) has been implicated in the negative regulation of insulin signaling. The expression, activity, and functional role of PTP1B in the retina are unknown. In this study, the authors examined the relationship between the retinal insulin receptor (IR) and PTP1B in normal and diabetic mouse retinas. METHODS IR and PTP1B localization was examined by immunohistochemistry. The activation of IR was analyzed using specific antibodies against phosphotyrosine. PTP1B activity was determined in anti–PTP1B immunoprecipitates. Glutathione-S-transferase fusion proteins containing wild-type and catalytically inactive mutant PTP1B was used to study the interaction between IR and PTP1B. Anti–IR immunoprecipitates and the cytoplasmic domain of purified IR were incubated in the presence of ATP, and the autophosphorylation of IR with antiphosphotyrosine antibody was analyzed. RESULTS Immunohistochemical analysis of PTP1B shows that it is predominantly expressed in nonphotoreceptor layers of the retina, though it is clearly expressed in the inner segments of the rod photoreceptors. The IR is predominately expressed in rod inner segments. Biochemical analysis of rod outer segments indicates the presence of IR and PTP1B. Retinal IR exhibits a high level of basal autophosphorylation, and this autophosphorylation is reduced in diabetic mouse retinas. In vitro, PTP1B is able to dephosphorylate the autophosphorylated IR. Substrate mutant-trap results indicate a stable interaction between IR and PTP1B. Further, PTP1B activity was increased in diabetic mouse retinas. CONCLUSIONS These studies indicate that diabetes reduces the autophosphorylation of retinal IR and increased PTP1B activity. Further, PTP1B regulates the state of IR phosphorylation in the retina.

Phosphorylated Grb14 Is an Endogenous Inhibitor of Retinal Protein Tyrosine Phosphatase 1B, and Light-Dependent Activation of Src Phosphorylates Grb14

“…We expressed GST and various domains of Grb14 in bacteria and purified these fusion proteins. The purified fusion proteins were incubated with recombinant PTP1B and examined for the dephosphorylation of a synthetic phosphopeptide substrate (R RLIEDAE P YAARG) as a function of the PTP1B activity in vitro (35). A significant Grb14-mediated inhibition of PTP1B activity in vitro was found (Fig.…”

Phosphoinositide 3-kinase signaling in the vertebrate retina

“…The IR, PI3K, and Akt pathway was found to be downregulated in diabetic retinopathy ( 132 ). Recently, we reported that diabetes downregulates IR activation and increases the activity of PTP1B ( 133 ).…”

Internal Pudental Artery Dysfunction in Diabetes Mellitus Is Mediated by NOX1-Derived ROS-, Nrf2-, and Rho Kinase–Dependent Mechanisms

“…A few studies corroborate our results. VSMC isolated from mesenteric artery from WKY (Wistar Kyoto) and SHRSP (spontaneously hypertensive stroke prone rat) rats 48 and retinal from rats and mice 49 exposed to hyperglycemia present increased expression of the PTP, PTP1B, Protein carbonylation is a post-translational modification where carbonyl groups are introduced into proteins. It is considered a biomarker for oxidative stress-induced irreversible damage and leads to loss of protein function.…”