MassIVE MSV000096575

Partial Public PXD058487

Proximity labeling and orthogonal nanobody pulldown (ID-oPD) approaches to map the spinophilin interactome uncover a role for spinophilin in protein regulation.

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Description

The major synaptic protein phosphatase 1 (PP1) targeting protein, spinophilin can inhibit or target PP1 activity towards diverse substrates. To detail the full panoply of substrates at which spinophilin modulates PP1 activity, we have validated a novel approach that permits proximity labeling and orthogonal nanobody pulldown (ID-oPD) of spinophilin protein complexes. We identified 614 specific and 217 selective spinophilin interacting proteins in a HEK293 overexpression system, many of which are involved in mRNA translation or metabolism. We validated a subset of these interactors using neutravidin and ALFA-tag nanobody pulldowns followed by immunoblotting. Additionally, using wildtype or an F451A mutant spinophilin that limits PP1 binding, we found that the PP1 binding-deficient spinophilin mutant enhanced its own expression, but limited the expression of proteins involved in mRNA translation and metabolism Moreover, in the striatum, spinophilin mRNA is highly neuropil localized and endogenous spinophilin limits its own expression. Overall, our studies demonstrate a novel role for spinophilin in mediating synaptic protein expression and that care must be taken to not over-control when using proximity labeling to detect PP1 targeting protein interactomes. [doi:10.25345/C59C6SC6Q] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: spinophilin ; protein phosphatase 1 ; proximity labeling ; orthogonal nanobody pulldown ; proximity labeling and orthogonal nanobody pulldown (ID-oPD) ; ALFA-tag ; TurboID ; DatasetType:Proteomics

Contact

Principal Investigators:
(in alphabetical order) 		A.J. Baucum II, Indiana University School of Medicine, United States
Amber L. Mosley, Indiana University School of Medicine, USA
Submitting User: edoud

Publications

Claeboe ET, Blake KL, Shah NR, Morris CW, Hens B, Atwood BK, Absalon S, Mosley AL, Doud EH, Baucum AJ.
Proximity labeling and orthogonal nanobody pulldown (ID-oPD) approaches to map the spinophilin interactome uncover a putative role for spinophilin in protein homeostasis.
bioRxiv. Epub 2025 Jan 23.

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