RESUMO
Ezrin-Radixin-Moesin (ERM) binding phosphoprotein 50 (EBP50, a.k.a. NHERF-1) is a scaffold protein essential for the localization and coordinated activity of apical transporters, enzymes and receptors in epithelial cells. EBP50 acts via multiple protein binding interactions, including oligomerization through interactions of its PSD95-Dlg-ZO1 (PDZ) domains. EBP50 can be phosphorylated on multiple sites and phosphorylation of specific sites modulates the extent of oligomerization. The aim of the present study was to test the capacity of protein kinase C (PKC) to phosphorylate EBP50 and to regulate its oligomerization. In vitro experiments showed that the catalytic subunit of PKC directly phosphorylates EBP50. In HEK-293 cells transfected with rat EBP50 cDNA, a treatment with 12 myristate 13-acetate (PMA) induced a translocation of PKCalpha and beta isoforms to the membrane and increased 32P incorporation into EBP50. In co-transfection/co-precipitation studies, PMA treatment stimulated EBP50 oligomerization. Mass spectrometry analysis of full-length EBP50 and phosphorylation analyses of specific domains, and of mutated or truncated forms of EBP50, indicated that PKC-induced phosphorylation of EBP50 occurred on the Ser337/Ser338 residue within the carboxyl-tail domain of the protein. Truncation of Ser337/Ser338 also diminished PKC-induced oligomerization of EBP50. These results suggest the PKC signaling pathway can impact EBP50-dependent cellular functions by regulating EBP50 oligomerization.
Assuntos
Proteínas de Transporte/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Proteínas de Transporte/genética , Linhagem Celular , Meios de Cultura Livres de Soro/farmacologia , Humanos , Dados de Sequência Molecular , Mutação , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Trocadores de Sódio-Hidrogênio , Acetato de Tetradecanoilforbol/farmacologia , Treonina/metabolismo , TransfecçãoRESUMO
Shank proteins are a family of multidomain scaffolding proteins best known for their role in organizing the postsynaptic density region in neurons. Unlike Shank1 and Shank3, Shank2 [also known as Pro-SAP1 (proline-rich synapse-associated protein 1), CortBP1 (cortactin binding protein 1) or Spank-3] has been described as a truncated family member without an N-terminal ankyrin repeat domain. The present study utilized bioinformatics to demonstrate the presence of exons encoding ankyrin repeats in the region preceding the previously described Shank2 gene. cDNA sequencing of mRNA from epithelial cells revealed a novel spliceoform of Shank2, termed Shank2E, that encodes a predicted 200 kDa protein with six N-terminal ankyrin repeats. Shank2 mRNA from epithelial tissues was larger than transcripts in brain. Likewise, the apparent mass of Shank2 protein was larger in epithelial tissues (230 kDa) when compared with brain (165/180 kDa). Immunofluorescence and membrane fractionation found Shank2E concentrated at the apical membrane of liver epithelial cells. In cultured cholangiocytes, co-immunoprecipitation and detergent solubility studies revealed Shank2E complexed with actin and co-distributed with actin in detergent-insoluble lipid rafts. These findings indicate epithelial cells express an ankyrin repeat-containing Shank2 isoform, termed Shank2E, that is poised to co-ordinate actin-dependent events at the apical membrane.
