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Y. Kanan, H. Song, M. Sokolov, R. E. Anderson, R. V. S. Rajala; Characterization of Serine/Threonine Phosphatases, PHLPP and PHLPPL in the Rat Retina and its Regulation by Akt. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5190.
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Our purpose was to characterize the serine/threonine phosphatases PHLPP and PHLPPL, study their regulation, and determine their function in the rodent retina.
To determine the location of PHLPP and PHLPPL, 10 µm rat retina serial tangential cryosections were probed with PHLPP and PHLPPL antibody along with markers for photoreceptor inner and outer segments. To study the phosphatase activity of the proteins, the phosphatase domains of PHLPP and PHLPPL were cloned and expressed as GST tagged proteins and incubated with phospho-Akt substrate or the phosphorylated chemical substrate pNPP (para-nitro phenyl phosphate). The dephosphorylation of Akt was studied by Western blotting with phospho-Akt antibody and the dephosphorylation of pNPP was studied spectrophotometrically by measuring absorbance at 405 nm. To study the regulation of PHLPP and PHLPPL activity by insulin, rat retinal explants were incubated in DMEM media and 10 nM insulin was added in the presence or absence of phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and the activity assessed after immunoprecipitation of PHLPP and PHLPPL with their respective antibodies and incubation with the chemical substrate pNPP.
Both PHLPP and PHLPPL are expressed in the photoreceptor inner segments. PHLPP is expressed as 2 isoforms, whereas PHLPPL is expressed as a single isoform. In vitro expressed phosphatase domains of PHLPP and PHLPPL were catalytically active with the activity of PHLPP being higher than PHLPPL, and both phosphatases could dephosphorylate all three isoforms of Akt. We also show that insulin can modulate the activity of PHLPP through AKT. Insulin increased Akt phosphorylation which negatively regulated PHLPP and PHLPPL activity. Suppressing Akt phosphorylation by the addition of PI3K inhibitor in the presence of insulin increased the activity of PHLPP and PHLPPL.
PHLPP and PHLPPL are expressed in the photoreceptor inner segments where they dephosphorylate Akt. However, they can also be regulated by phosphorylated Akt. Our studies also suggest that the regulation of PHLPP and PHLPPL are insulin receptor-PI3K-dependent. Since Akt is an important neuron survival factor in the retina, these phosphatases may play an important role in modulating the neuroprotective potential of the retina.
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