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P Ramulu, Y Tao, J Nathans; The Rod Photoreceptor-specific Phosphatase PPEF-2 Interacts with 14-3-3 zeta in a Phosphorylation-dependent Manner . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1406.
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Purpose: The PPEFs represent a family of neuron-specific protein phosphatases characterized by calcium-binding EF-hands at their C-termini. The original member of the PPEF family, Drosophila rdgC, is known to dephosphorylate rhodopsin and to cause a light-dependent retinal degeneration. Two vertebrate PPEFs have also been identified, including PPEF-2, which is specifically found in the rod photoreceptor inner segment and the pineal body. Mice lacking both PPEF-1 and PPEF-2 have been generated, and show normal rhodopsin dephosphorylation and no evidence of retinal degeneration. Here, we investigated the function of PPEF-2 by searching for proteins interacting with PPEF-2. Methods: To identify proteins that interact with PPEF-2, a yeast two-hybrid screen was performed using the entire coding region of the PPEF-2 cDNA as bait. Veracity of putative interactors was confirmed by in vitro analysis, in which GST fusions of the putative interacting proteins bound to gluatathione-sepharose beads were mixed with bovine retina lysates and then analyzed by SDS-PAGE and Western blotting using PPEF-2 specific antibodies. To determine if interactions were phosphorylation dependent, bovine retina lysates were pre-treated with lambda phosphatase before binding to the GST fusion. Results: The yeast two-hybrid analysis with the coding region of human PPEF-2 cDNA as bait suggested interaction of PPEF-2 with 14-3-3 zeta. In vitro analysis demonstrated that a GST-14-3-3 zeta fusion was able to bind PPEF-2 from bovine retina lysates, but that this binding was eliminated with short treatments with lambda phosphatase. Conclusion: These results demonstrate that PPEF-2 interacts with members of the 14-3-3 family of proteins. Members of the 14-3-3 family of proteins are known to bind the consensus sequence RSXpSXP, where X represents any amino acid and pS represents phosphoserine. Indeed, PPEF-2 contains this consensus sequence in a surface loop interrupting the phosphatase domain. That the PPEF-2 interaction with 14-3-3 zeta could be eliminated by treatment with lambda phsophatase suggusts that the serine in the consensus sequence is phosphorylated, and that dephosphorylation of this serine eliminates 14-3-3 zeta binding. Phosphorylation of PPEF-2 and binding to 14-3-3 protiens may serve to regulate PPEF-2 enzymatic activity, subcellular localization or interaction with substrates.
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