June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Light-Dependent Phosphorylation of BBS5 in Photoreceptors and Its Interaction with Arrestin1
Author Affiliations & Notes
  • Tyler Smith
    Ophthalmology, University of Florida, Gainesville, FL
  • Donald Dugger
    Ophthalmology, University of Florida, Gainesville, FL
  • Susan Bolch
    Ophthalmology, University of Florida, Gainesville, FL
  • J Hugh McDowell
    Ophthalmology, University of Florida, Gainesville, FL
  • W Clay Smith
    Ophthalmology, University of Florida, Gainesville, FL
  • Footnotes
    Commercial Relationships Tyler Smith, None; Donald Dugger, None; Susan Bolch, None; J Hugh McDowell, None; W Clay Smith, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1226. doi:
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      Tyler Smith, Donald Dugger, Susan Bolch, J Hugh McDowell, W Clay Smith; Light-Dependent Phosphorylation of BBS5 in Photoreceptors and Its Interaction with Arrestin1. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1226.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: We have previously demonstrated that Bardet-Biedl Syndrome 5 (BBS5) protein is one member of the phosphoproteome in photoreceptors. Here we provide an analysis of the kinase involved in BBS5 phosphorylation and investigate the functional effect of BBS5 phosphorylation on its interaction with other proteins.

Methods: BBS5 was cloned from murine retinal mRNA, and heterologously expressed in bacteria with either a His(6) tag or as a fusion protein with glutathione-S-transferase (GST). BBS5 was phosphorylated in situ by endogenous kinases; phosphorylation of BBS5 was tested in vitro using a panel of six kinases. Interaction of BBS5 with arrestin was assessed in vitro via immunoprecipitation. Tissue localization of BBS5 was examined in cryosections of Xenopus laevis retina using a monoclonal antibody prepared against heterologously expressed BBS5.

Results: BBS5 is phosphorylated in a light-dependent manner, showing a threshold of phosphorylation that correlates with the initiation of arrestin1 translocation. Phosphorylation of BBS5 can also be stimulated in vivo by phorbol-12,13-diacetate, a protein kinase C agonist. In vitro analysis demonstrates that BBS5 is phosphorylated by PKC, but not by protein kinase A, cGMP-dependent protein kinase, casein kinase I or II, or calcium/calmodulin-dependent protein kinase II. Arrestin1 co-immunoprecipitates with BBS5/GST; phosphorylation of BBS5 reduces the interaction between arrestin1 and BBS5. Under identical conditions, arrestin1 shows negligible co-immunoprecipitation with BBS8. In photoreceptors, BBS5 principally localizes to the axonemes of rods and cones, photoreceptor inner segments, and synaptic regions.

Conclusions: Phosphorylation of BBS5 in retina is initiated by light, apparently through PKC activation based on in vitro studies. Activation of PKC in situ using phorbol ester leads to BBS5 phosphorylation, supporting this conclusion. The three-fold observation that (1) BBS5 directly interacts with arrestin1, (2) that phosphorylation of BBS5 reduces the affinity of arrestin1 for BBS5, and (3) that BBS5 principally localizes to the axoneme suggests that phosphorylation of BBS5 may play a role in regulating light-dependent translocation of arrestin1.

Keywords: 648 photoreceptors • 646 phosphorylation • 657 protein modifications-post translational  
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