Increased Protein Kinase Activity Associated With Photoreceptor Degeneration Onset

Abstract

Purpose: : The precise mechanisms that activate cell death and survival pathways in photoreceptors, in response to environmental and genetic factors, have yet to be defined. In Pde6b^rd1 and Pde6b^H620Q mutant mice, elevated levels of cGMP and Ca²⁺ second messengers have been postulated to initiate degeneration. Indeed, in both mutants, peak cGMP levels correlate with degeneration onset. Kinases stimulated by cGMP and/or Ca²⁺, may exhibit abnormally high activity in Pde6b mutants resulting in increased phosphorylation of kinase effectors. Thus, measuring kinase activity may provide a means to identify kinases, substrates, and signalling pathways that mediate the pathogenic response.

Methods: : To test if Pde6b^H620Q mutants exhibit abnormal kinase activities, prior to degeneration, we used biochemical and immunohistological approaches. All comparisons were made between age-matched Pde6b^H620Q mutant and DBA/2J control mice. The activity of protein kinase A, G, C and B (AGC kinases) was measured, using in vitro kinase assays, on P14 and P18 mutant and control retinal lysates. To extend our analysis to the entire kinome, we performed peptide microarray analysis on mutant and control lysates. To confirm the presence of abnormal activity in vivo, we analyzed the expression of PKB substrates using phospho-specific antibodies and compared staining patterns between mutant and control retinal sections.

Results: : Our in vitro assays demonstrate elevated AGC kinase activity in Pde6b^H620Q retinal lysates. PKG exhibits the greatest difference in activity (10 fold higher than control, p<0.001), while PKC (2.6 fold, p<0.001), PKA (1.6 fold, p<0.001), and PKB (1.6 fold, p1.5, p<0.05). Finally, our immunohistochemical data show increased staining of mutant photoreceptors using phospho-PDK1, Phospho-Tuberin, and phospho-ribosomal protein S6 antibodies.

Conclusions: : These studies suggest that cGMP- and/or Ca²⁺-regulated kinases may mediate the early pathogenic response to Pde6b loss of function. While all members of the AGC kinase family exhibited elevated activity, the fold difference in cGMP-regulated kinase (PKG) activity was greatest, suggesting that part of the degeneration response is mediated by PKG substrates. Our in vitro evidence of increased PKB activity and PKB pathway substrate phosphorylation suggests that the PKB cell growth and survival pathway is activated in Pde6b mutants prior to photoreceptor degeneration onset. Our data suggest that elevated second messengers generate a number of signalling changes, some pro-survival, others pro-apoptotic, with the balance resulting in cell death in Pde6b^H620Q mutants.

Keywords: retinal degenerations: cell biology • second messengers • phosphorylation