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P.S. Zelenka, D. Ledee, R. Seth, C. Gao, R. Fariss; The Cdk5 Activating Protein, p39, Complexes With Muskelin to Regulate Cell Adhesion on Thrombospondin . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3262.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previous studies have implicated cyclin dependent kinase 5 (Cdk5) in cell adhesion and cytoskeletal regulation in the lens. This study seeks to determine molecular mechanisms underlying these functions, by identifying proteins that interact with Cdk5 and its regulatory proteins, p35 and p39. Methods: An embryonic rat lens yeast two hybrid library was constructed and screened using Cdk5, p35 and p39 as baits. GST-pulldown experiments were performed using purified recombinant GST-tagged prey protein and in vitro translated baits. Intracellular interaction was determined by co-immunoprecipitation of epitope-tagged constructs from co-transfected Cos1 cells. Subcellular localization and function were assessed in Cos1 using fluorescence-tagged constructs. In vitro kinase assays used purified recombinant protein and immunoprecipitated Cdk5/p39. mRNA expression was determined by RT-PCR and in situ hybridization. Results: Library screening identified muskelin, an intracellular mediator of cell adhesion to thrombospondin-1, as a specific interactor of p39. GST-pulldown experiments and co-immunoprecipitations from transfected cells confirmed that muskelin interacted specifically with p39, and not p35 or Cdk5. GST-pulldowns with deletion constructs of muskelin indicated that its fifth kelch domain contains the p39 binding site. Similar analysis of p39 showed that a short, unique sequence in the C-terminus of p39 (amino acids 329-369) contains the muskelin binding site. Fluorescence-tagged muskelin and p39 co-localized at the leading edge of lamellopodia of transfected Cos1 cells. Co-expression of p39 and muskelin inhibited muskelin-dependent attachment to thrombospondin. RT-PCR demonstrated that both muskelin and p39 mRNAs are expressed in lens and in situ hybridization localized muskelin expression to equatorial epithelial cells and peripheral lens fibers. In vitro kinase assays showed that muskelin can be phosphorylated by Cdk5/p39. Conclusions: The interaction between p39 and muskelin provides a possible mechanism for regulation of cell adhesion by Cdk5 and provides the first evidence that p39 may have unique functions not shared by p35.
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