Bacterial co-expression of human Tau protein with protein kinase A and 14-3-3 for studies of 14-3-3/phospho-Tau interaction

• Published in: PloS one Vol. 12; no. 6; p. e0178933
• Main Authors: Tugaeva, Kristina V, Tsvetkov, Philipp O, Sluchanko, Nikolai N
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 02-06-2017; Public Library of Science (PLoS)
• Subjects: 14-3-3 protein; 14-3-3 Proteins - analysis; 14-3-3 Proteins - genetics; 14-3-3 Proteins - metabolism; Alzheimer Disease - metabolism; Alzheimer's disease; Antifungal agents; Apoptosis; Bacteria; Binding; Binding sites; Biochemistry; Biology; Biology and Life Sciences; Biomarkers, Tumor - analysis; Biomarkers, Tumor - genetics; Biomarkers, Tumor - metabolism; Cloning; Cloning, Molecular; Cyclic AMP-Dependent Protein Kinases - analysis; Cyclic AMP-Dependent Protein Kinases - genetics; Cyclic AMP-Dependent Protein Kinases - metabolism; Disorders; Displays; E coli; Escherichia coli - genetics; Escherichia coli - metabolism; Exoribonucleases - analysis; Exoribonucleases - genetics; Exoribonucleases - metabolism; Fetuses; Functional anatomy; Gene Expression; Humans; Identification methods; In vitro methods and tests; Isoforms; Kinases; Neurodegenerative diseases; Neurological diseases; Parkinson Disease - metabolism; Phosphorylation; Physical Sciences; Physiology; Plasmids; Protein expression; Protein Interaction Maps; Protein Isoforms - analysis; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein kinase; Protein kinase A; Protein kinases; Protein-protein interactions; Proteins; Regulation; Research and Analysis Methods; Residues; Rodents; Signal transduction; Stoichiometry; Studies; Tau protein; tau Proteins - analysis; tau Proteins - genetics; tau Proteins - metabolism
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0178933

Abundant regulatory 14-3-3 proteins have an extremely wide interactome and coordinate multiple cellular events via interaction with specifically phosphorylated partner proteins. Notwithstanding the key role of 14-3-3/phosphotarget interactions in many physiological and pathological processes, they are dramatically underexplored. Here, we focused on the 14-3-3 interaction with human Tau protein associated with the development of several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Among many known phosphorylation sites within Tau, protein kinase A (PKA) phosphorylates several key residues of Tau and induces its tight interaction with 14-3-3 proteins. However, the stoichiometry and mechanism of 14-3-3 interaction with phosphorylated Tau (pTau) are not clearly elucidated. In this work, we describe a simple bacterial co-expression system aimed to facilitate biochemical and structural studies on the 14-3-3/pTau interaction. We show that dual co-expression of human fetal Tau with PKA in Escherichia coli results in multisite Tau phosphorylation including also naturally occurring sites which were not previously considered in the context of 14-3-3 binding. Tau protein co-expressed with PKA displays tight functional interaction with 14-3-3 isoforms of a different type. Upon triple co-expression with 14-3-3 and PKA, Tau protein could be co-purified with 14-3-3 and demonstrates complex which is similar to that formed in vitro between individual 14-3-3 and pTau obtained from dual co-expression. Although used in this study for the specific case of the previously known 14-3-3/pTau interaction, our co-expression system may be useful to study of other selected 14-3-3/phosphotarget interactions and for validations of 14-3-3 complexes identified by other methods.