Determining the interactome and function of the neurabins across cell and tissue types.

About This Grant

Project Summary & Abstract: The major serine/threonine phosphatase, protein phosphatase 1 (PP1), is involved in many different biological processes throughout the body. Phosphatases like PP1 are known to “keep things in check” to limit adaptive changes from occurring under pathological conditions. Unlike their kinase brethren, serine/threonine phosphatases are more substrate promiscuous. This has historically led to the notion that they are “undruggable.” However, phosphatases like PP1 associate with myriad targeting and inhibitory proteins that permit appropriate regulation of spatial and temporal signaling. Specifically, PP1 associates with over 200 proteins that target and/or inhibit its activity at specific sets of substrates. However, knowledge of how specific proteins regulate PP1 targeting and function are poorly described. Spinophilin and neurabin, collectively termed the neurabins, are the two most abundant brain postsynaptic density (PSD)-enriched PP1 targeting proteins. While the neurabins are PSD and brain enriched, they are also located throughout the body. Moreover, we and others have found that the neurabins play roles in myriad different disease-induced adaptations, including neurological disorders, cancers, immune function, and obesity and diabetes. However, due to a lack of genetic tools, our understanding of how the neurabins impact pathological adaptive changes associated with disease is unclear, In this “Focused Technology Research and Development” R01 proposal, we will leverage our current cell type-specific spinophilin and neurabin knockout mice, as well as develop novel tools to identify and compare the spinophilin- and neurabin-specific interactomes across cell types. We have generated constructs that allow for Cre-dependent epitope tagging of spinophilin and neurabin with an ALFA epitope tag as well as an UltraID proximity ligase that we show leads to robust, specific, and selective pulldown of spinophilin and neurabin protein interaction networks in overexpression studies. To permit in vivo studies, we will generate transgenic animals to allow for tissue and cell type-specific isolation of endogenous spinophilin and neurabin interactors. These studies will validate that these approaches allow us to delineate unique and overlapping spinophilin- and neurabin- specific interacting proteins within specific cell types, setting the stage for future studies to determine how disruption of specific or overlapping interactions impacts pathological adaptations associated with a range of disorders. Successful completion of these studies will enhance our knowledge and ability to “drug” selective PP1 holoenzymes.