Neuron-specific protein interactions of Drosophila CASK-β are revealed by mass spectrometry

Abstract

Modular scaffolding proteins are designed to have multiple interactors. CASK, a member of the membrane-associated guanylate kinase (MAGUK) superfamily, has been shown to have roles in many tissues, including neurons and epithelia. It is likely that the set of proteins it interacts with is different in each of these diverse tissues. In this study we asked if within the Drosophila central nervous system, there were neuron-specific sets of CASK-interacting proteins. A YFP-tagged CASK-β transgene was expressed in genetically defined subsets of neurons in the Drosophila brain known to be important for CASK function, and proteins present in an anti-GFP immunoprecipitation were identified by mass spectrometry. Each subset of neurons had a distinct set of interacting proteins, suggesting that CASK participates in multiple protein networks and that these networks may be different in different neuronal circuits. One common set of proteins was associated with mitochondria, and we show here that endogenous CASK-β co-purifies with mitochondria. We also determined CASK-β posttranslational modifications for one cell type, supporting the idea that this technique can be used to assess cell- and circuit-specific protein modifications as well as protein interaction networks.

CASK stabilizes neurexin and links it to liprin-α in a neuronal activity-dependent manner

“…Immunoprecipitation of CASK using a CASK antibody resulted in co-precipitation of stoichiometric amounts of Mint1 and a few peptides of Caskin, suggesting that Mint1 is the predominant binding partner of CASK’s CaMK domain in the brain [13,14]. This endogenous Mint1-CASK interaction has also been demonstrated in C. elegans , and we have recently confirmed it in Drosophila, indicating that the CASK-Mint1 interaction is evolutionarily conserved [29]. Similarly, affinity chromatography using the CaMK domain of CASK identified Mint1, Caskin and Nedd4 as potential CASK binding partners.…”

Regulation of dopamine release by CASK-β modulates locomotor initiation in Drosophila melanogaster

“…Because CASK has so many potential binding partners, it is important to have a methodology to identify putative cell-specific interactors. Recently, we developed a method for mass spectrometry identification of co-immunoprecipitated proteins bound to full length CASK- β -YFP expressed in defined neuronal populations using the GAL4/UAS system (Mukherjee et al, 2014 ). This published data set allowed us to obtain a list of cell-specific binding partners for CASK- β .…”

“…Using directed expression via the GAL4/UAS system (Fischer et al, 1988 ; Brand and Perrimon, 1993 ; Phelps and Brand, 1998 ), we show here that a number of cells within this subset are dopaminergic neurons, and that reconstitution of CASK- β specifically in these cells selectively rescues the motor initiation component of the mutant’s complex locomotor phenotype. The motor initiation disruption in CASK-β mutants likely stems from aberrations in vesicle release at dopaminergic synapses, which may be driven by a recently identified interaction between CASK- β and the molecular chaperone Hsc70-4 (Mukherjee et al, 2014 ). Interestingly, while this interaction appears to be present in multiple cell types throughout the invertebrate nervous system, our results indicate that the association between these two proteins is only behaviorally relevant to locomotion in dopaminergic cells.…”

A genomic approach to understanding metabolic insecticide resistance to diamides in diamondback moth (Plutella xylostella)

“…Following the liquid chromatography-mass spectrometry performed on P. One study to have made use of the GAL4/UAS system linked with biochemical studies incorporating LC-MS, is that by Mukherjee et al where the transgenic GAL4/UAS system has been used incorporating the reporter gene, yellow fluorescent protein (YFP) to aid in organ tissue dissection. LC-MS was then subsequently used to identify proteins in these tissues [342]. As discussed in chapter 3, the majority of pesticide-based studies published focus on the use of LC-MS in the detection of pesticide residues in crops, and those that do focus on insecticide metabolism in insects tend to use in-vitro platforms on which to perform LC-MS.…”