title: Identification of Phosphorylation Consensus Sequences and Endogenous Neuronal Substrates of the Psychiatric Risk Kinase TNIK.
abstract: Traf2- and Nck-interacting kinase (TNIK) is a serine/threonine kinase highly expressed in the brain and enriched in the postsynaptic density of glutamatergic synapses in the mammalian brain. Accumulating genetic evidence and functional data have implicated TNIK as a risk factor for psychiatric disorders. However, the endogenous substrates of TNIK in neurons are unknown. Here, we describe a novel selective small molecule inhibitor of the TNIK kinase family. Using this inhibitor, we report the identification of endogenous neuronal TNIK substrates by immunoprecipitation with a phosphomotif antibody followed by mass spectrometry. Phosphorylation consensus sequences were defined by phosphopeptide sequence analysis. Among the identified substrates were members of the delta-catenin family including p120-catenin, δ-catenin, and armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), each of which is linked to psychiatric or neurologic disorders. Using p120-catenin as a representative substrate, we show TNIK-induced p120-catenin phosphorylation in cells requires intact kinase activity and phosphorylation of TNIK at T181 and T187 in the activation loop. Addition of the small molecule TNIK inhibitor or knocking down TNIK by two shRNAs reduced endogenous p120-catenin phosphorylation in cells. Together, using a TNIK inhibitor and phosphomotif antibody, we identify endogenous substrates of TNIK in neurons, define consensus sequences for TNIK, and suggest signaling pathways by which TNIK influences synaptic development and function linked to psychiatric and neurologic disorders.
authors.affiliation: Neuroscience & Pain Research Unit, BioTherapeutics Research and Development, Pfizer Inc. Cambridge, Massachusetts (Q.W., S.P.A., D.M.R., N.J.B., M.D.E.); Center of Chemistry Innovation and Excellence, Pfizer Inc., Groton, Connecticut (M.M.H.); Neuroscience Medicinal Chemistry, Pfizer Inc., Cambridge, Massachusetts (B.L.K., P.R.V.);and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts (L.X.) Qi.Wang@azneuro.com Michael.Ehlers@pfizer.com.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Neuroscience & Pain Research Unit, BioTherapeutics Research and Development, Pfizer Inc. Cambridge, Massachusetts (Q.W., S.P.A., D.M.R., N.J.B., M.D.E.); Center of Chemistry Innovation and Excellence, Pfizer Inc., Groton, Connecticut (M.M.H.); Neuroscience Medicinal Chemistry, Pfizer Inc., Cambridge, Massachusetts (B.L.K., P.R.V.);and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts (L.X.).
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Amato
authors.affiliation: Neuroscience & Pain Research Unit, BioTherapeutics Research and Development, Pfizer Inc. Cambridge, Massachusetts (Q.W., S.P.A., D.M.R., N.J.B., M.D.E.); Center of Chemistry Innovation and Excellence, Pfizer Inc., Groton, Connecticut (M.M.H.); Neuroscience Medicinal Chemistry, Pfizer Inc., Cambridge, Massachusetts (B.L.K., P.R.V.);and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts (L.X.).
authors.forename: David M
authors.initials: DM
authors.lastname: Rubitski
authors.affiliation: Neuroscience & Pain Research Unit, BioTherapeutics Research and Development, Pfizer Inc. Cambridge, Massachusetts (Q.W., S.P.A., D.M.R., N.J.B., M.D.E.); Center of Chemistry Innovation and Excellence, Pfizer Inc., Groton, Connecticut (M.M.H.); Neuroscience Medicinal Chemistry, Pfizer Inc., Cambridge, Massachusetts (B.L.K., P.R.V.);and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts (L.X.).
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Hayward
authors.affiliation: Neuroscience & Pain Research Unit, BioTherapeutics Research and Development, Pfizer Inc. Cambridge, Massachusetts (Q.W., S.P.A., D.M.R., N.J.B., M.D.E.); Center of Chemistry Innovation and Excellence, Pfizer Inc., Groton, Connecticut (M.M.H.); Neuroscience Medicinal Chemistry, Pfizer Inc., Cambridge, Massachusetts (B.L.K., P.R.V.);and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts (L.X.).
authors.forename: Bethany L
authors.initials: BL
authors.lastname: Kormos
authors.affiliation: Neuroscience & Pain Research Unit, BioTherapeutics Research and Development, Pfizer Inc. Cambridge, Massachusetts (Q.W., S.P.A., D.M.R., N.J.B., M.D.E.); Center of Chemistry Innovation and Excellence, Pfizer Inc., Groton, Connecticut (M.M.H.); Neuroscience Medicinal Chemistry, Pfizer Inc., Cambridge, Massachusetts (B.L.K., P.R.V.);and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts (L.X.).
authors.forename: Patrick R
authors.initials: PR
authors.lastname: Verhoest
authors.affiliation: Neuroscience & Pain Research Unit, BioTherapeutics Research and Development, Pfizer Inc. Cambridge, Massachusetts (Q.W., S.P.A., D.M.R., N.J.B., M.D.E.); Center of Chemistry Innovation and Excellence, Pfizer Inc., Groton, Connecticut (M.M.H.); Neuroscience Medicinal Chemistry, Pfizer Inc., Cambridge, Massachusetts (B.L.K., P.R.V.);and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts (L.X.).
authors.forename: Lan
authors.initials: L
authors.lastname: Xu
authors.affiliation: Neuroscience & Pain Research Unit, BioTherapeutics Research and Development, Pfizer Inc. Cambridge, Massachusetts (Q.W., S.P.A., D.M.R., N.J.B., M.D.E.); Center of Chemistry Innovation and Excellence, Pfizer Inc., Groton, Connecticut (M.M.H.); Neuroscience Medicinal Chemistry, Pfizer Inc., Cambridge, Massachusetts (B.L.K., P.R.V.);and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts (L.X.).
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Brandon
authors.affiliation: Neuroscience & Pain Research Unit, BioTherapeutics Research and Development, Pfizer Inc. Cambridge, Massachusetts (Q.W., S.P.A., D.M.R., N.J.B., M.D.E.); Center of Chemistry Innovation and Excellence, Pfizer Inc., Groton, Connecticut (M.M.H.); Neuroscience Medicinal Chemistry, Pfizer Inc., Cambridge, Massachusetts (B.L.K., P.R.V.);and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts (L.X.) Qi.Wang@azneuro.com Michael.Ehlers@pfizer.com.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Ehlers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D016384:Consensus Sequence / Q000502:physiology*; D005260:Female; D000081410:Germinal Center Kinases; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D000081410:Germinal Center Kinases; D010446:Peptide Fragments; D017346:Protein Serine-Threonine Kinases; C000631454:TNIK protein, human