Scapinin-induced Inhibition of Axon Elongation Is Attenuated by Phosphorylation and Translocation to the Cytoplasm

Abstract

Scapinin is an actin-and PP1-binding protein that is exclusively expressed in the brain; however, its function in neurons has not been investigated. Here we show that expression of scapinin in primary rat cortical neurons inhibits axon elongation without affecting axon branching, dendritic outgrowth, or polarity. This inhibitory effect was dependent on its ability to bind actin because a mutant form that does not bind actin had no effect on axon elongation. Immunofluorescence analysis showed that scapinin is predominantly located in the distal axon shaft, cell body, and nucleus of neurons and displays a reciprocal staining pattern to phalloidin, consistent with previous reports that it binds actin monomers to inhibit polymerization. We show that scapinin is phosphorylated at a highly conserved site in the central region of the protein (Ser-277) by Cdk5 in vitro. Expression of a scapinin phospho-mimetic mutant (S277D) restored normal axon elongation without affecting actin binding. Instead, phosphorylated scapinin was sequestered in the cytoplasm of neurons and away from the axon. Because its expression is highest in relatively plastic regions of the adult brain (cortex, hippocampus), scapinin is a new regulator of neurite outgrowth and neuroplasticity in the brain. The brain has a remarkable capacity to restructure its neuronal circuitry. This phenomenon is known as neuroplasticity and is critical for higher order functions, such as cognition, calculation, adaptation, and memory formation. It is especially active during development but persists in regions of the adult brain associated with higher order functions, especially the cortex and hippocampus. Neuroplasticity is achieved via two mechanisms: 1) altering the strength of existing synapses and circuits and 2) forming new synapses and circuits. The former mechanism primarily involves changes in the release of neurotransmitters at the pre-synapse or expression of neurotransmitter receptors at the post-synapse, whereas the latter mechanism involves neurite outgrowth and new synapse formation. Defects in neuroplasticity occur with aging and are dramatic in neurodegenerative diseases, such as Alzheimer disease. Conversely, enhancing neuroplasticity might be an effective therapy for improved mental function following brain insults, including stroke, dementia, and age-related cognitive decline. Therefore, it is important to understand the molecular mechanisms controlling neuroplasticity to discover new therapeutic targets for the maintenance of healthy brain function.Scapinin 2 (scaffold-associated PP1-inhibiting protein) is a brain-specific member of the Phactr (phosphatase and actin regulator) family of proteins (also called Phactr3 (1-3)). There are three splice variants of scapinin in mammals encoding proteins of 559 (62.5 kDa), 518 (58.5 kDa), and 448 (51.5 kDa) amino acids (1), with isoform 2 (518 amino acids) being predominant in human brain (4). It was originally identified as a component of the nuclear matrix intermediate filament scaffold (1). Since then,...

Actin-regulated feedback loop based on Phactr4, PP1 and cofilin maintains the actin monomer pool

“…2). Similarly, it has been reported that in rat cortical neurons the localization of Phactr1 does not respond to Latrunculin A or Cytochalasin D treatments (Farghaian et al, 2011). Our FRAP experiments suggested that there is continuous import of Phactr4 to the nucleus (Fig.…”

“…Thereby only extensive changes in the actin monomer pool, which cannot be corrected by simple modulation of cofilin activity, would evoke a transcriptional response. Alternatively, also other signals may contribute to the regulation, and for example both Phactr proteins (Farghaian et al, 2011) and MRTF-A (Muehlich et al, 2008) have been shown to be phosphorylated in cells. Structural studies on the Phactr4-actin complex, as well as development of methods to both measure and perturb the different stoichiometric complexes between the RPEL domain and actin, in the context of both MRTF and Phactr proteins, are required to resolve these questions.…”

“…The subcellular localization of Phactr proteins has not been studied systematically and variable localization patterns ranging from nuclear to cytoplasmic have been reported (Allen et al, 2004;Farghaian et al, 2011;Favot et al, 2005;Kim et al, 2007;Sagara et al, 2009;Sagara et al, 2003;Zhang et al, 2012). Phactr proteins have been implicated in many distinct biological processes, from angiogenesis (Allain et al, 2012;Jarray et al, 2011) to cell spreading, migration (Sagara et al, 2009) and axon elongation (Farghaian et al, 2011), which are also dependent on the actin cytoskeleton. Mice carrying a specific mutation of Phactr4 gene called the humdy exhibit a defect of neural tube and optic fissure closure resulting in anomalies such as exencephaly.…”

IPP5 inhibits neurite growth in primary sensory neurons by maintaining TGF- /Smad signaling

“…Neurabin-I and spinophilin are both regulatory subunits targeting PP1 to F-actin during neuronal development, which regulates spine development or axonal growth by affecting the cytoskeletal dynamics in hippocampal and cortical neurons (Bielas et al, 2007;Oliver et al, 2002). Scapinin is another PP1-inhibiting protein that inhibits axonal growth without affecting neurite branching in primary rat cortical neurons (Farghaian et al, 2011). As a regulatory subunit of PP1, IPP5 is selectively expressed in primary sensory neurons localized in the DRG.…”

G-actin regulates the shuttling and PP1 binding of the RPEL protein Phactr1 to control actomyosin assembly

“…In Phactr3, sequences required for high-affinity PP1 binding overlap its RPEL3 motif (Sagara et al, 2003), suggesting that actin and PP1 might compete for Phactr binding. Actin binding appears required for morphological changes induced by Phactr3 overexpression, and in some systems PP1 binding is also required (Sagara et al, 2009;Farghaian et al, 2011). Phactr3 inhibits PP1 activity against a model substrate (Sagara et al, 2003), but it remains unclear whether this reflects a bona fide general inhibitory function of Phactr3 or a positive function as a regulatory subunit for a different substrate (for discussion see Ceulemans and Bollen, 2004;Roy and Cyert, 2009).…”