PT  - JOURNAL ARTICLE
AU  - Alicyia Walczyk Mooradally
AU  - Jennifer Holborn
AU  - Karamjeet Singh
AU  - Marshall Tyler
AU  - Debasis Patnaik
AU  - Hendrik Wesseling
AU  - Nicholas J Brandon
AU  - Judith Steen
AU  - Steffen P Graether
AU  - Stephen J Haggarty
AU  - Jasmin Lalonde
TI  - Phosphorylation-dependent control of Arc protein by synaptic plasticity regulator TNIK
AID  - 10.1101/2021.04.04.438383
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.04.04.438383
4099  - http://biorxiv.org/content/early/2021/04/04/2021.04.04.438383.short
4100  - http://biorxiv.org/content/early/2021/04/04/2021.04.04.438383.full
AB  - Activity-regulated cytoskeleton-associated protein (Arc) is an immediate-early gene product that support neuroplastic changes important for cognitive function and memory formation. As a protein with homology to the retroviral Gag protein, a particular characteristic of Arc is its capacity to self-assemble into virus-like capsids that can package mRNAs and transfer those transcripts to other cells. Although a lot has been uncovered about the contributions of Arc to neuron biology and behavior, very little is known about how different functions of Arc are coordinately regulated both temporally and spatially in neurons. The answer to this question we hypothesized must involve the occurrence of different protein post-translational modifications acting to confer specificity. In this study, we used mass spectrometry and sequence prediction strategies to map novel Arc phosphorylation sites. Our approach led us to recognize serine 67 (S67) and threonine 278 (T278) as residues that can be modified by TNIK, which is a kinase abundantly expressed in neurons that shares many functional overlaps with Arc and has, along with its interacting proteins such as the NMDA receptor, been implicated as a risk factor for psychiatric disorders. Furthermore, characterization of each residue using site-directed mutagenesis to create S67 and T278 mutant variants revealed that TNIK action at those amino acids can strongly influence Arc’s subcellular distribution and self-assembly as capsids. Together, our findings reveal an unsuspected connection between Arc and TNIK. Better understanding of the interplay between these two proteins in neuronal cells could lead to new insights about apparition and progression of psychiatric disorders.Competing Interest StatementThe authors have declared no competing interest.AMPARα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptorArcactivity-regulated cytoskeleton-associated proteinBSAbovine serum albuminCaMKIIcalcium/calmodulin-dependent protein kinase IIDAPI4′,6-diamidino-2-phenylindoleDISC1disrupted in schizophrenia 1DLSdynamic light scatteringDMEMDulbecco’s modified eagle’s mediumDTTdithiothreitolECLenhanced chemiluminescenceEDTAethylenediaminetetraacetic acidFPLCfast protein liquid chromatographyGAPDHglyceraldehyde 3-phosphate dehydrogenaseHIVhuman immunodeficiency virusHPLChigh performance liquid chromatographyHRPhorseradish peroxidaseICCimmunocytochemistryIPimmunoprecipitationIPTGisopropyl β-D-1-thiogalactopyranosideKOknockoutLBLuria–Bertani mediumLC/MSliquid chromatography–mass spectrometryLTDlong-term depressionLTPlong-term potentiationmGLURmetabotropic glutamate receptorNRDnuclear retention domainPCRpolymerase chain reactionPMSFphenylmethylsulfonyl fluoridePSD-95postsynaptic density protein 95PTMspost-translational modificationsRIPAradioimmunoprecipitation assaySDS-PAGEsodium dodecyl sulphate-polyacrylamide gel electrophoresisTARPγ2transmembrane AMPAR regulatory protein γ2TNIKthe tumor necrosis factor receptor (Traf2) and noncatalytic region of tyrosine kinase (Nck) interacting kinaseWBwestern blotWTwildtype