Phospho-Site-Specific Antibody Microarray to Study the State of Protein Phosphorylation in the Retina

Abstract

Neurodegeneration is an important component of diabetic retinopathy as demonstrated by increased neural apoptosis in the retina during experimental and human diabetes. Accumulation of sorbitol and fructose and the generation or enhancement of oxidative stress has been reported in the whole retina of diabetic animals. Aldose reductase (AR), the first and the rate limiting enzyme in the pathway reduces glucose to sorbitol and the diabetic complications are prevented by drugs that inhibit AR. In this study we examined the phosphorylation state of various retinal proteins in response to sorbitol-treatment by phosphor-site-specific antibody microarray. Our results suggest that various retinal protein kinases and cytoskeletal proteins either activated or down regulated in response to sorbitol treatment. Further, our study also indicates the activation of retinal insulin- and insulin growth factor 1 receptor and their downstream signaling proteins such as phosphoinositide 3-kinanse and protein kinase B (Akt). Understanding the regulation of retinal proteins involved in polyol (sorbitol) pathway would help to design therapeutic agents for the treatment of diabetic retinopathy.

Figure 1

Sorbitol-induced tyrosine phosphorylation of several retinal proteins. Retinas were cultured in DMEM in the presence or the absence various concentrations of sorbitol for 30 min at 37 °C. Thirty micrograms of retinal proteins were subjected to Western blot analysis with anti-PY 99 antibody (A). The blot was reprobed with the anti-actin antibody to ensure equal amount of protein in each lane (B). All experiments were carried out in duplicate.

Figure 2

Sorbitol-induced activation of IR and IGF-1R. Retinal proteins from control and 1.0 M sorbitol-treated organotypic cultures were immunoprecipitated with anti-IRβ (B) or anti-IGF-1R (D) antibody followed by Western blot analysis with anti-PY99 antibody (A and C). The blots were stripped and reprobed with anti-IRβ antibody or anti-IGF-1R antibody to ensure equal amounts of IR and IGF-1R in each immunoprecipitate. Sorbitol-induced activation IR associated PI3K activity. Retinas were cultured in DMEM and treated with various concentrations of sorbitol for 30 min at 37 °C. TLC autoradiogram of PI3K activity measured in anti-IRb immunoprecipitates of retinas using PI-4,5-P₂ and [g³²P]ATP as substrates. The radioactive spots of PI-3,4,5-P₃ were scraped from the TLC plate and counted (E). Sorbitol-induced activation of Akt. Sorbitol treated and untreated retinal proteins were subjected to Western blot analysis with anti-pAkt (S473) (F) and anti-Akt (G) antibodies. All experiments were carried out in duplicate.

Figure 3

Antibody microarray analysis. The technique utilize antibody microarrays to track the differential binding of dye-labeled proteins in lysates prepared from retinal tissues.