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V.L. Bonilha, E. Rodriguez-Boulan; Pka Activation Promotes Microvilli Assembly Mediated by Ezrin in Retinal Pigment Epithelium . Invest. Ophthalmol. Vis. Sci. 2003;44(13):372.
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Purpose: Previous work has shown that Ezrin, an adaptor protein that links plasma membrane and the actin cytoskeleton promotes the formation of microvilli and basal infoldings in cultured RPE cells (Bonilha et al., J. Cell Biology, 1999). Independent work has shown that ezrin phosphorylation promotes its association with both plasma membrane and the actin cytoskeleton. Here, we tested the hypothesis that PKA promotes morphogenesis of RPE microvilli by phosphorylating ezrin. Methods:In vivo studies quantified whole cells lysates from rat RPE collected during the 3 weeks after birth. To further investigate the role of PKA in elongation of RPE microvilli, we treated rat RPE primary cultures (which preserve microvilli and and express high levels of ezrin) with myristoylated-PKI (14-22) amide a specific inhibitor of PKA. On the other hand, D407 cells (human RPE cell line with scattered short microvilli at their apical surface, and low levels of ezrin) were treated with the PKA activators Sp-cAMPS and 8-bromo-cAMP. In addition, these cells were infected with a replication-deficient adenovirus carring the ezrin cDNA. Results were analyzed by immunofluorescence, scanning electron microscopy and western blot. Results:Quantification of western blots of post-natal rat RPE (P1 to adult) demonstrated that PKA expression increased proportionally to the elongation of the microvilli during postnatal maturation, as previously shown for ezrin. PKA inhibition decreased the length and number of apical microvilli and the triton X-100 resistant immunofluorescent staining of PKAc (PKA catalytic subunit) and ezrin in primary cultures of RPE. In D407 cells, PKAc localized to the Golgi complex but shifted to a punctated plasma membrane pattern (consistent with the appearance of microvilli) after overexpression of ezrin cDNA and PKA activation with Sp-cAMP and 8-bromo cAMP. We are currently investigating whether PKA activation results in direct ezrin phosphorylation. Conclusions: PKA activation promotes plasma membrane localization of PKAc and ezrin and increased number of microvilli in RPE cells.
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