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Z. Huang, R. Rajula, R.E. Anderson; Light Regulation of Type II Phosphatidylinositol Phosphate Kinase in Mouse Retina and Rat Rod Outer Segments . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5510.
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© ARVO (1962-2015); The Authors (2016-present)
In our previous work, we reported that the activity of type II phosphatidylinositol phosphate kinase (PIPKII) is mediated by tyrosine phosphorylation in bovine rod outer segments (ROS). This enzyme uses PI–5–P as substrate for biosynthesis of PI–4,5–P2 (PIP2), a molecule putatively involved in regulation of phototransduction and neuroprotection in the vertebrate retina. The purpose of this study was to identify the isoforms of PIPK in the retina and dissect the effect of light on the kinase activities and their mechanism of activation in retina and ROS.
Total RNA extracted from mouse retina was used for RT–PCR to clone the isoforms of PIPK and for real time PCR to measure the transcription levels of the kinases. PIPK isoform expression in photoreceptor cells was detected by single rod cell cDNA PCR. The PIPK activity assay was used to study the light regulation of PIPKactivity in rat ROS and mouse retina lysates.
Five isoforms of PIPK were cloned from mouse retina, which include type I alpha and beta, and type II alpha, beta, and gamma. Among the isoforms of PIPK expressed in mouse retina, the highest transcription level is type I beta and the lowest level is type II alpha. In single rod photoreceptor cell cDNA, four PIPK isoforms, including type I alpha and beta, and type II alpha and gamma, were positive in their PCR reaction. We found increased binding of PIPK activity to light–adapted ROS compared to dark–adapted ROS. Immunoprecipitates with an antibody against phosphotyrosine had higher PIP kinase activity in lysates from light–adapted retinas than from dark–adapted retinas.
Light–dependent regulation of PIPKII was observed in rat rod outer segments. The presence of several isoforms of type I and type II PIPK indicate a complex regulation of PIP2 biosynthesis in the retina.
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