The Neuronal Actin-binding Proteins, Neurabin I and Neurabin II, Recruit Specific Isoforms of Protein Phosphatase-1 Catalytic Subunits

Abstract

Neurabins are protein phosphatase-1 (PP1) targeting subunits that are highly concentrated in dendritic spines and post-synaptic densities. Immunoprecipitation of neurabin I and neurabin II/spinophilin from rat brain extracts sedimented PP1␥1 and PP1␣ but not PP1␤. In vitro studies showed that recombinant peptides representing central regions of neurabins also preferentially bound PP1␥1 and PP1␣ from brain extracts and associated poorly with PP1␤. Analysis of PP1 binding to chimeric neurabins suggested that sequences flanking a conserved PP1-binding motif altered their selectivity for PP1␤ and their activity as regulators of PP1 in vitro. Assays using recombinant PP1 catalytic subunits and a chimera of PP1 and protein phosphatase-2A indicated that the C-terminal sequences unique to the PP1 isoforms contributed to their recognition by neurabins. Collectively, the results from several different in vitro assays established the rank order of PP1 isoform selection by neurabins to be PP1␥1 > PP1␣ > PP1␤. This PP1 isoform selectivity was confirmed by immunoprecipitation of neurabin I and II from brain extracts from wild type and mutant PP1␥ null mice. In the absence of PP1␥1, both neurabins showed enhanced association with PP1␣ but not PP1␤. These studies identified some of the structural determinants in PP1 and neurabins that together contribute to preferential targeting of PP1␥1 and PP1␣ to the mammalian synapse.Protein phosphatase-1 (PP1), 1 a major eukaryotic protein serine/threonine phosphatase, is encoded by multiple genes in both plants and animals. Disruption of PP1 genes in fungi (1), fruit flies (2), and mice (3) suggested that PP1 isoforms encoded by individual genes control distinct but overlapping physiological functions. Three mammalian isoforms, PP1␣, PP1␤, and PP1␥1, are expressed in all tissues (4) with PP1␥2, an alternately spliced product of the PP1␥ gene, present predominantly in testes (5, 6). Immunocytochemistry using isoform-specific antibodies suggested that expression of PP1 isoforms varied in different brain regions where they are also localized to different subcellular compartments (6, 7). For example, PP1␤ was the predominant isoform associated with microtubules in the neuronal cell body, whereas PP1␥1 and PP1␣ were preferentially concentrated in dendritic spines (6,8). Furthermore, by analyzing endogenous PP1 movement during the cell cycle, Andreassen et al. (9) showed that the distribution of PP1 isoforms in cells was highly dynamic. This placed new emphasis on understanding the mechanisms that target individual PP1 isoforms to cellular organelles.Isolation of PP1 bound to skeletal muscle glycogen (10) and myosin (11) established the paradigm that regulatory or targeting subunits bound to PP1 catalytic subunits dictate the subcellular localization, substrate recognition, and hormonal control of PP1. The search for PP1 regulators that control functions as diverse as protein synthesis, gene expression, cell division, and motility has thus far yielded more than 50 PP1-binding proteins (12). Wh...

Proteomics Analysis of Rat Brain Postsynaptic Density

“…As actin microfilaments are associated with structural plasticity, the identification of a number of actin-regulatory proteins in the present study is entirely consistent with the idea of rapid structural changes driven by reorganization of the actin-microfilament in the spine. These proteins include cofilin, which depolymerizes actin filaments from its pointed end thereby increasing actin dynamics (54); F-actin capping protein Z and capping protein ␣2, which bind to the barbed end of actin filaments (54); neurabin 2, which binds to actin and recruits protein phosphatase to actin that controls actin rearrangement (55); the LIM and SH3 domain-containing protein, which is proposed to play important roles in the regulation of dynamic actin-based, cytoskeletal activities (56). Another LIM domain containing protein present in the PSD fraction, the cysteine rich protein, has been shown to interact with the actin cytoskeleton and may be involved in muscle differentiation (57).…”

Binding of the Concave Surface of the Sds22 Superhelix to the α4/α5/α6-Triangle of Protein Phosphatase-1

“…The fourth isoform, PP1␤/␦, makes up the ␤-subtype. Interestingly, some regulatory subunits are selectively associated with a particular PP1 isoform or subtype, such as the PP1␤/␦-specific myosin-targeting subunits (22) or the ␣-subtype-specific neurabins (23). Thus far, the only mammalian PP1 isoform that has been shown to interact with Sds22 is PP1␥ 2 (24).…”

Synaptic NMDA receptor stimulation activates PP1 by inhibiting its phosphorylation by Cdk5

“…In neurons, PP1 is mainly concentrated in the cell body (Strack et al, 1999), whereas PP1 and PP11 are concentrated in dendritic spines (Ouimet et al, 1995;Strack et al, 1999;Terry-Lorenzo et al, 2002a;Carmody et al, 2008). All PP1 isoforms can be regulated by phosphorylation of a conserved threonine residue near the C terminus (Dohadwala et al, 1994).…”