Neurobeachin: A Protein Kinase A-Anchoring,beige/Chediak-Higashi Protein Homolog Implicated in Neuronal Membrane Traffic

Abstract

We describe the identification and initial characterization of neurobeachin, a neuron-specific multidomain protein of 327 kDa with a high-affinity binding site (K d , 10 nM) for the type II regulatory subunit of protein kinase A (PKA RII). Neurobeachin is peripherally associated with pleomorphic tubulovesicular endomembranes near the trans sides of Golgi stacks and throughout the cell body and cell processes. It is also found in a subpopulation of synapses, where it is concentrated at the postsynaptic plasma membrane. In live cells, perinuclear neurobeachin is dispersed by brefeldin A (BFA) within 1 min, and in permeabilized cells a recruitment of neurobeachin from cytosol to Golgi-near membranes is stimulated by GTP␥S and prevented by brefeldin A. Spots of neurobeachin recruitment are close to but distinct from recruitment sites of COP-I, AP-1, and AP-3 coat proteins involved in vesicle budding. These observations indicate that neurobeachin binding to membranes close to the trans-Golgi requires an ADP-ribosylation factor-like GTPase, possibly in association with a novel type of protein coat. A neurobeachin isoform that does not bind RII, beige-like protein (BGL), is expressed in many tissues. Neurobeachin, BGL, and ϳ10 other mammalian gene products share a characteristic C-terminal BEACH-WD40 sequence module, which is also present in gene products of invertebrates, plants, protozoans, and yeasts, thus defining a new protein family. The prototype member of this family of BEACH domain proteins, lysosomal trafficking regulator (LYST), is deficient in genetic defects of protein sorting in lysosome biogenesis (the beige mouse and Chediak-Higashi syndrome). Neurobeachin's subcellular localization, its coat proteinlike membrane recruitment, and its sequence similarity to LYST suggest an involvement in neuronal post-Golgi membrane traffic, one of its functions being to recruit protein kinase A to the membranes with which it associates. Key words: AKAP; ARF; BEACH domain; BGL; coat protein; Golgi complex; LYST; membrane traffic; neurobeachin; protein kinase A; scaffolding protein; synapse; TGNThe progression of membranes and proteins through the stages and compartments of the secretory and endocytic pathways is a highly organized and regulated process. The maintenance of the overall architecture of endomembranes and of the plasma membrane requires a balance of lipid flows into and out of the various compartments, and proteins destined for diverse organelles or plasma membrane domains must be appropriately sorted and targeted, whereas resident proteins of specific pathway stages must be retained or retrieved. These events require the interplay of lipids, membrane proteins, soluble cytosolic and lumenal proteins, and cytoskeletal and motor proteins. Their internal coordination and external regulation is known to involve protein phosphorylation and small and heterotrimeric G-proteins.In neurons, the mechanisms for the trafficking of membranes and membrane proteins must be particularly active and complex. Because of their m...

Mammalian class E Vps proteins, SBP1 and mVps2/CHMP2A, interact with and regulate the function of an AAA-ATPase SKD1/Vps4B

“…The antibody was raised with the carboxylterminal 89 kDa of the murine Beige protein, which contains the highly conserved BEACH domain and seven WD40 repeats (Perou et al, 1997). This region has 95% identity with human lyst in amino acid sequence (Wang et al, 2000). As expected, an approximately 400 kDa band, which corresponded to human lyst, was detected in the GM05655 lane (Fig.…”

Tandem mass spectrometry identifies many mouse brain O -GlcNAcylated proteins including EGF domain-specific O -GlcNAc transferase targets

“…We observed three O-GlcNAcylation sites (T1276, T1796, and T1797) on neurobeachin (Nbea), a protein implicated in membrane protein traffic and autism, and required for the formation and functioning of central synapses (28). This protein is a known phosphoprotein and binds to protein kinase A; however, its O-GlcNAcylation status was previously unknown (29). Literature provides examples of cross-talk between O-GlcNAcylation and phosphorylation (19,26,30), as well as examples of O-GlcNAc regulating ubiquitination through E1 ubiquitin-activating enzyme (26,31).…”

Subcellular Localization of Cyclic Nucleotide Phosphodiesterase Type 10A Variants, and Alteration of the Localization by cAMP-dependent Protein Kinase-dependent Phosphorylation

“…One role of PDE10A2 in the perinuclear region of cells is coordination of cyclic nucleotide signaling in this local space. The RII subunit of PKA binds to Neurobeachin, which anchors PKA to the Golgi/centrosome of PC12 cells and neuronal cells (41). The ␣ isoform catalytic subunit of PKA translocates from the Golgi area to the nucleus in neuronal cells via intracellular cAMP elevation induced by 24-h stimulation by ethanol, and phosphorylates the cAMP response element binding factor in the nucleus (42,43).…”