In Vivo Regulation of Phosphoinositide 3-Kinase in Retina through Light-induced Tyrosine Phosphorylation of the Insulin Receptor β-Subunit

Abstract

Recently, we have shown that phosphoinositide 3-kinase (PI3K) in bovine rod outer segment (ROS) is activated in vitro by tyrosine phosphorylation of the C-terminal tail of the insulin receptor (Rajala, R. V. S., and Anderson, R. E. (2001) Invest. Ophthal. Vis. Sci. 42, 3110 -3117). In this study, we have investigated the in vivo mechanism of PI3K activation in the rodent retina and report the novel finding that light stimulates tyrosine phosphorylation of the ␤-subunit of the insulin receptor (IR␤) in ROS membranes, which leads to the association of PI3K enzyme activity with IR␤. Retinas from light-or dark-adapted mice and rats were homogenized and immunoprecipitated with antibodies against phosphotyrosine, IR␤, or the p85 regulatory subunit of PI3K, and PI3K activity was measured using PI-4,5-P 2 as substrate. We observed a light-dependent increase in tyrosine phosphorylation of IR␤ and an increase in PI3K enzyme activity in isolated ROS and in anti-phosphotyrosine and anti-IR␤ immunoprecipitates of retinal homogenates. The light effect was localized to photoreceptor neurons and is independent of insulin secretion. Our results suggest that light induces tyrosine phosphorylation of IR␤ in outer segment membranes, which leads to the binding of p85 through its N-terminal Src homology 2 domain and the generation of PI-3,4,5-P 3 . We suggest that the physiological role of this process may be to provide neuroprotection of the retina against light damage by activating proteins that protect against stressinduced apoptosis.Many cell proliferation and cell survival pathways are initiated upon activation of tyrosine kinase receptors, which transduce their signals by recruiting a variety of cytoplasmic signaling proteins (1, 2). Many of the signaling proteins contain phosphotyrosine binding domains, Src homology 2 (SH2) 1 domains, and Src homology 3 (SH3) domains, which are involved in mediating protein-protein interactions (4, 5).The phosphotyrosine-dependent interaction between different phosphotyrosine binding and SH2 domain-containing proteins with activated receptors initiates cellular changes that take place in response to a wide range of extracellular signals (2).One of the SH2 domain-containing proteins, phosphoinositide 3-kinase (PI3K), consists of an ϳ85-kDa regulatory subunit (p85) and a ϳ110-kDa catalytic subunit (p110), the latter being responsible for the phosphorylation of phosphoinositides at the D3 position and of serine residues in proteins (7, 8). The p85 subunit contains an SH3 domain capable of binding to proline-rich sequences, a p110 binding domain, and two SH2 domains. PI3K was initially found to be associated with middle-T/pp60c-Src (9), pp60v-Src, and platelet-derived growth factor receptors in both normal and transformed NIH3T3 fibroblast cells (10, 11). PI3K activity increases in response to platelet-derived growth factor binding to its receptor, in large part because the p85-p110 complex is translocated from the cytosol to the plasma membrane, by the direct binding of the p85 SH2 domain to tyro...

Loss of Neuroprotective Survival Signal in Mice Lacking Insulin Receptor Gene in Rod Photoreceptor Cells

“…We showed previously that the important anti-apoptotic enzyme PI3K is regulated through light-activated IR in rod photoreceptor cells (2). In this study, we observed that light stress induced tyrosine phosphorylation of the IR in ROS membranes.…”

“…IR activation has been shown to rescue retinal neurons from apoptosis through a phosphoinositide 3-kinase (PI3K) cascade (1). We previously reported that light induces tyrosine phosphorylation of the retinal IR and that this activation leads to the binding of PI3K to rod outer segment (ROS) membranes (2). More recently, we demonstrated that IR activation is mediated through the G-protein-coupled receptor rhodopsin (3).…”

“…Preparation of Mouse Rod Outer Segments-ROS were prepared from mouse retinas by using a discontinuous sucrose gradient centrifugation as described previously (2). Eight retinas from four mice were homogenized in 1.25 ml of ice-cold 47% sucrose solution containing 100 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 mM Tris-HCl (pH 7.4) (buffer A).…”

“…Generation of the Conditional IR Knock-out Mice-We previously reported that the light-induced tyrosine phosphorylation of retinal IR leads to the activation of PI3K in rod photoreceptor cells (2). During light stress the IR in the rat retina was tyrosine-phosphorylated, which led to increased binding of PI3K (Fig.…”

“…Specificity of Cell-specific Deletion of IR-To further assess the specificity of IR deletion or reduction in rod photoreceptor cells from the knock-out mice, we examined the expression of proteins involved in the IR signaling pathway in the non-ROS fraction (band II), which represents the retina minus the ROS membranes (2). The non-ROS fraction from the wild-type and the conditional IR knock-out retinas from both the 0.2-kb (supplemental Fig.…”