Interaction of Tyrosine Hydroxylase and 14–3–3 Proteins in PC12 Cells

Abstract

The 14–3–3 protein is a family of acidic, dimeric proteins distributed widely among eukaryotic cells. This protein family binds to a variety of proteins in a phosphorylation-dependent manner and participates in the regulation of cell proliferation, differentiation and function. To date, more than 70 proteins, localized in the cell membrane to nuclei, are reported to bind with the 14–3–3 family. These proteins include at least 19 membrane components (receptors, ion channels and effectors), 22 signal transduction kinases and phosphatases, 5 cytoskelton proteins, 18 nuclear proteins including transcription factors, 10 metabolic enzymes, and others. We previously showed, using PC12nnr5 cells transfected with myc-tagged 14–3–3β and η) isoforms, that 14–3–3s form a complex with tyrosine hydroxylase (TH) particularly when the hydroxylase is phosphorylated at Ser-19 by endogeneous Ca²⁺, calmodulin-dependent protein kinase II¹. In this study we have employed proteomic techniques for systematic characterization of PC12 proteins and identified the TH protein as a major constituent of the proteins that are co-immunoprecipitated with 14–3–3. We have also shown that the TH protein is predominantly associated with 14–3-3ε and η isoforms, but not with ζ and θ isoforms. These results indicate that the activity of TH is regulated through phosphorylation-dependent interaction with limited members of the 14–3–3 family.