貝淵 弘三

Structural plasticity of dendritic spines in the nucleus accumbens (NAc) is crucial for learning from aversive experiences. Activation of NMDA receptors (NMDARs) stimulates Ca2+-dependent signaling that leads to changes in the actin cytoskeleton, mediated by the Rho family of GTPases, resulting in postsynaptic remodeling essential for learning. We investigated how phosphorylation events downstream of NMDAR activation drive the changes in synaptic morphology that underlie aversive learning. Large-scale phosphoproteomic analyses of protein kinase targets in mouse striatal/accumbal slices revealed that NMDAR activation resulted in the phosphorylation of 194 proteins, including RhoA regulators such as ARHGEF2 and ARHGAP21. Phosphorylation of ARHGEF2 by the Ca2+-dependent protein kinase CaMKII enhanced its RhoGEF activity, thereby activating RhoA and its downstream effector Rho-associated kinase (ROCK/Rho-kinase). Further phosphoproteomic analysis identified 221 ROCK targets, including the postsynaptic scaffolding protein SHANK3, which is crucial for its interaction with NMDARs and other postsynaptic scaffolding proteins. ROCK-mediated phosphorylation of SHANK3 in the NAc was essential for spine growth and aversive learning. These findings demonstrate that NMDAR activation initiates a phosphorylation cascade crucial for learning and memory.

Protein phosphorylation, a key regulator of cellular processes, plays a central role in brain function and is implicated in neurological disorders. Information on protein phosphorylation is expected to be a clue for understanding various neuropsychiatric disorders and developing therapeutic strategies. Nonetheless, existing databases lack a specific focus on phosphorylation events in the brain, which are crucial for investigating the downstream pathway regulated by neurotransmitters. To overcome the gap, we have developed a web-based database named “Kinase-Associated Neural PHOspho-Signaling (KANPHOS).” This paper presents the design concept, detailed features, and a series of improvements for KANPHOS. KANPHOS is designed to support data-driven research by fulfilling three key objectives: (1) enabling the search for protein kinases and their substrates related to extracellular signals or diseases; (2) facilitating a consolidated search for information encompassing phosphorylated substrate genes, proteins, mutant mice, diseases, and more; and (3) offering integrated functionalities to support pathway and network analysis. KANPHOS is also equipped with API functionality to interact with external databases and analysis tools, enhancing its utility in data-driven investigations. Those key features represent a critical step toward unraveling the complex landscape of protein phosphorylation in the brain, with implications for elucidating the molecular mechanisms underlying neurological disorders. KANPHOS is freely accessible to all researchers at https://kanphos.jp.

The unraveling of the regulatory mechanisms that govern neuronal excitability is a major challenge for neuroscientists worldwide. Neurotransmitters play a critical role in maintaining the balance between excitatory and inhibitory activity in the brain. The balance controls cognitive functions and emotional responses. Glutamate and γ-aminobutyric acid (GABA) are the primary excitatory and inhibitory neurotransmitters of the brain, respectively. Disruptions in the balance between excitatory and inhibitory transmission are implicated in several psychiatric disorders, including anxiety disorders, depression, and schizophrenia. Neuromodulators such as dopamine and acetylcholine control cognition and emotion by regulating the excitatory/inhibitory balance initiated by glutamate and GABA. Dopamine is closely associated with reward-related behaviors, while acetylcholine plays a role in aversive and attentional behaviors. Although the physiological roles of neuromodulators have been extensively studied neuroanatomically and electrophysiologically, few researchers have explored the interplay between neuronal excitability and cell signaling and the resulting impact on emotion regulation. This review provides an in-depth understanding of “cell signaling crosstalk” in the context of neuronal excitability and emotion regulation. It also anticipates that the next generation of neurochemical analyses, facilitated by integrated phosphorylation studies, will shed more light on this topic.

Abstract In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.

Clathrin-dependent endocytosis is a key process for secretory cells, in which molecules on the plasma membrane are both degraded and recycled in a stimulus-dependent manner. There are many reports showing that disruption of endocytosis is involved in the onset of various diseases. Recently, it has been reported that such disruption in pancreatic β-cells causes impaired insulin secretion and might be associated with the pathology of diabetes mellitus. Compared with exocytosis, there are few reports on the molecular mechanism of endocytosis in pancreatic β-cells. We previously reported that GDP-bound Rab27a regulates endocytosis through its GDP-dependent effectors after insulin secretion. In this study, we identified heat shock protein family A member 8 (HSPA8) as a novel interacting protein for GDP-bound Rab27a. HSPA8 directly bound GDP-bound Rab27a via the β2 region of its substrate binding domain (SBD). The β2 fragment was capable of inhibiting the interaction between HSPA8 and GDP-bound Rab27a, and suppressed glucose-induced clathrin-dependent endocytosis in pancreatic β-cells. The region also affected clathrin dynamics on purified clathrin-coated vesicles (CCVs). These results suggest that the interaction between GDP-bound Rab27a and HSPA8 regulates clathrin disassembly from CCVs and subsequent vesicle transport. The regulatory stages in endocytosis by HSPA8 differ from those for other GDP-bound Rab27a effectors. This study shows that GDP-bound Rab27a dominantly regulates each stage in glucose-induced endocytosis through its specific effectors in pancreatic β-cells.

INTRODUCTION: Since the emergence of the cholinergic hypothesis of Alzheimer's disease (AD), acetylcholine has been viewed as a mediator of learning and memory. Donepezil improves AD-associated learning deficits and memory loss by recovering brain acetylcholine levels. However, it is associated with side effects due to global activation of acetylcholine receptors. Muscarinic acetylcholine receptor M1 (M1R), a key mediator of learning and memory, has been an alternative target. The importance of targeting a specific pathway downstream of M1R has recently been recognized. Elucidating signaling pathways beyond M1R that lead to learning and memory holds important clues for AD therapeutic strategies. AREAS COVERED: This review first summarizes the role of acetylcholine in aversive learning, one of the outputs used for preliminary AD drug screening. It then describes the phosphoproteomic approach focused on identifying acetylcholine intracellular signaling pathways leading to aversive learning. Finally, the intracellular mechanism of donepezil and its effect on learning and memory is discussed. EXPERT OPINION: The elucidation of signaling pathways beyond M1R by phosphoproteomic approach offers a platform for understanding the intracellular mechanism of AD drugs and for developing AD therapeutic strategies. Clarifying the molecular mechanism that links the identified acetylcholine signaling to AD pathophysiology will advance the development of AD therapeutic strategies.

The Small GTPase Rac1 is critical for various fundamental cellular processes, including cognitive functions. The cyclical activation and inactivation of Rac1, mediated by Rac guanine nucleotide exchange factors (RacGEFs) and Rac GTPase-activating proteins (RacGAPs), respectively, are essential for activating intracellular signaling pathways and controlling cellular processes. We have recently shown that the Alzheimer's disease (AD) therapeutic drug donepezil activates the Rac1-PAK pathway in the nucleus accumbens (NAc) for enhanced aversive learning. Also, PAK activation itself in the NAc enhances aversive learning. As aversive learning allows short-term preliminary AD drug screening, here we tested whether sustained Rac1 activation by RacGAP inhibition can be used as an AD therapeutic strategy for improving AD-learning deficits based on aversive learning. We found that the RacGAP domain of breakpoint cluster region protein (Bcr) (Bcr-GAP) efficiently inhibited Rac1 activity in a membrane ruffling assay. We also found that, in striatal/accumbal primary neurons, Bcr knockdown by microRNA mimic-expressing adeno-associated virus (AAV-miRNA mimic) activated Rac1-PAK signaling, while Bcr-GAP-expressing AAV inactivated it. Furthermore, conditional knockdown of Bcr in the NAc of wild-type adult mice enhanced aversive learning, while Bcr-GAP expression in the NAc inhibited it. The findings indicate that Rac1 activation by RacGAP inhibition enhances aversive learning, implying the AD therapeutic potential of Rac1 signaling.

Schizophrenia (SCZ) is a severe psychiatric disorder characterized by positive symptoms, negative symptoms, and cognitive deficits. Current antipsychotic treatment in SCZ improves positive symptoms but has major side effects and little impact on negative symptoms and cognitive impairment. The pathoetiology of SCZ remains unclear, but is known to involve small GTPase signaling. Rho kinase, an effector of small GTPase Rho, is highly expressed in the brain and plays a major role in neurite elongation and neuronal architecture. This study used a touchscreen-based visual discrimination (VD) task to investigate the effects of Rho kinase inhibitors on cognitive impairment in a methamphetamine (METH)-treated male mouse model of SCZ. Systemic injection of the Rho kinase inhibitor fasudil dose-dependently ameliorated METH-induced VD impairment. Fasudil also significantly suppressed the increase in the number of c-Fos-positive cells in the infralimbic medial prefrontal cortex (infralimbic mPFC) and dorsomedial striatum (DMS) following METH treatment. Bilateral microinjections of Y-27632, another Rho kinase inhibitor, into the infralimbic mPFC or DMS significantly ameliorated METH-induced VD impairment. Two proteins downstream of Rho kinase, myosin phosphatase-targeting subunit 1 (MYPT1; Thr696) and myosin light chain kinase 2 (MLC2; Thr18/Ser19), exhibited increased phosphorylation in the infralimbic mPFC and DMS, respectively, after METH treatment, and fasudil inhibited these increases. Oral administration of haloperidol and fasudil ameliorated METH-induced VD impairment, while clozapine had little effect. Oral administration of haloperidol and clozapine suppressed METH-induced hyperactivity, but fasudil had no effect. These results suggest that METH activates Rho kinase in the infralimbic mPFC and DMS, which leads to cognitive impairment in male mice. Rho kinase inhibitors ameliorate METH-induced cognitive impairment, perhaps via the cortico-striatal circuit.

Copy number variants in the ARHGAP10 gene are associated with schizophrenia (SCZ). We have previously demonstrated that Rho-kinase (ROCK) inhibitor, fasudil, ameliorates the decreased spine density in the medial prefrontal cortex (mPFC) of Arhgap10 S490P/NHEJ mice carrying the variants that mimic the ARHGAP10 variants found in a Japanese SCZ patient. Accordingly, we have proposed that ROCK is a potentially novel therapeutic target in SCZ. It is well known that there are two subtypes of ROCK, ROCK1 and ROCK2, and that fasudil inhibits both subtypes. Since ROCK2 is highly expressed in the brain, here we evaluated the effect of a selective ROCK2 inhibitor, belumosudil (KD025), on spine density in Arhgap10 S490P/NHEJ mice. We measured the spine density of pyramidal neurons in layer 2/3 of the mPFC in Arhgap10 S490P/NHEJ mice following daily oral administration of KD025 for one week. Moreover, we evaluated the general behaviors in an open field and systolic blood pressure after KD025 treatment. KD025 ameliorated decreased spine density of cortical neurons in the mPFC of Arhgap10 S490P/NHEJ mice, but had little effects on general behaviors and systolic blood pressure induced by fasudil. These observations suggest that ROCK2 is a more appropriate therapeutic target in SCZ, with little inducibility of hypotension.

Dopamine regulates psychomotor function by D1 receptor/PKA-dependent phosphorylation of DARPP-32. DARPP-32, phosphorylated at Thr34 by PKA, inhibits protein phosphatase 1 (PP1), and amplifies the phosphorylation of other PKA/PP1 substrates following D1 receptor activation. In addition to the D1 receptor/PKA/DARPP-32 signaling pathway, D1 receptor stimulation is known to activate Rap1/ERK signaling. Rap1 activation is mediated through the phosphorylation of Rasgrp2 (guanine nucleotide exchange factor; activation) and Rap1gap (GTPase-activating protein; inhibition) by PKA. In this study, we investigated the role of PP1 inhibition by phospho-Thr34 DARPP-32 in the D1 receptor-induced phosphorylation of Rasgrp2 and Rap1gap at PKA sites. The analyses in striatal and NAc slices from wild-type and DARPP-32 knockout mice revealed that the phosphorylation of Rasgrp2 at Ser116/Ser117 and Ser586, but not of Rasgrp2 at Ser554 or Rap1gap at Ser441 or Ser499 induced by a D1 receptor agonist, is under the control of the DARPP-32/PP1. The results were supported by pharmacological analyses using a selective PP1 inhibitor, tautomycetin. In addition, analyses using a PP1 and PP2A inhibitor, okadaic acid, revealed that all sites of Rasgrp2 and Rap1gap were regulated by PP2A. Thus, the interactive machinery of DARPP-32/PP1 may contribute to efficient D1 receptor signaling via Rasgrp2/Rap1 in the striatum.

Copy-number variations in the ARHGAP10 gene encoding Rho GTPase-activating protein 10 are associated with schizophrenia. Model mice (Arhgap10 S490P/NHEJ mice) that carry "double-hit" mutations in the Arhgap10 gene mimic the schizophrenia in a Japanese patient, exhibiting altered spine density, methamphetamine-induced cognitive dysfunction, and activation of RhoA/Rho-kinase signaling. However, it remains unclear whether the activation of RhoA/Rho-kinase signaling due to schizophrenia-associated Arhgap10 mutations causes the phe-notypes of these model mice. Here, we investigated the effects of fasudil, a brain permeable Rho-kinase inhibitor, on altered spine density in the medial prefrontal cortex (mPFC) and on methamphetamine-induced cognitive impairment in a touchscreen-based visual discrimination task in Arhgap10 S490P/NHEJ mice. Fasudil (20 mg/ kg, intraperitoneal) suppressed the increased phosphorylation of myosin phosphatase-targeting subunit 1, a substrate of Rho-kinase, in the striatum and mPFC of Arhgap10 S490P/NHEJ mice. In addition, daily oral administration of fasudil (20 mg/kg/day) for 7 days ameliorated the reduced spine density of layer 2/3 pyra-midal neurons in the mPFC. Moreover, fasudil (3-20 mg/kg, intraperitoneal) rescued the methamphetamine (0.3 mg/kg)-induced cognitive impairment of visual discrimination in Arhgap10 S490P/NHEJ mice. Our results suggest that Rho-kinase plays significant roles in the neuropathological changes in spine morphology and in the vulnerability of cognition to methamphetamine in mice with schizophrenia-associated Arhgap10 mutations.

The N-methyl-D-aspartate receptor (NMDAR)-mediated structural plasticity of dendritic spines plays an important role in synaptic transmission in the brain during learning and memory formation. The Rho family of small GTPase RhoA and its downstream effector Rho-kinase/ROCK are considered as one of the major regulators of synaptic plasticity and dendritic spine formation, including long-term potentiation (LTP). However, the mechanism by which Rho-kinase regulates synaptic plasticity is not yet fully understood. Here, we found that Rho-kinase directly phosphorylated discs large MAGUK scaffold protein 2 (DLG2/PSD-93), a major postsynaptic scaffold protein that connects postsynaptic proteins with NMDARs; an ionotropic glutamate receptor, which plays a critical role in synaptic plasticity. Stimulation of striatal slices with an NMDAR agonist induced Rho-kinase-mediated phosphorylation of PSD-93 at Thr612. We also identified PSD-93-interacting proteins, including DLG4 (PSD-95), NMDARs, synaptic Ras GTPase-activating protein 1 (SynGAP1), ADAM metallopeptidase domain 22 (ADAM22), and leucine-rich glioma-inactivated 1 (LGI1), by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among them, Rho-kinase increased the binding of PSD-93 to PSD-95 and NMDARs. Furthermore, we found that chemical-LTP induced by glycine, which activates NMDARs, increased PSD-93 phosphorylation at Thr612, spine size, and PSD-93 colocalization with PSD-95, while these events were blocked by pretreatment with a Rho-kinase inhibitor. These results indicate that Rho-kinase phosphorylates PSD-93 downstream of NMDARs, and suggest that Rho-kinase mediated phosphorylation of PSD-93 increases the association with PSD-95 and NMDARs to regulate structural synaptic plasticity.

Primary cilia are antenna-like organelles that regulate growth and development via extracellular signals. However, the molecular mechanisms underlying cilia dynamics, particularly those regulating their disassembly, are not well understood. Here, we show that leucine-rich repeat kinase 1 (LRRK1) plays a role in regulating cilia disassembly. The depletion of LRRK1 impairs primary cilia resorption following serum stimulation in cultured cells. Polo-like kinase 1 (PLK1) plays an important role in this process. During ciliary resorption, PLK1 phosphorylates LRRK1 at the primary cilia base, resulting in its activation. We identified nuclear distribution protein nudE-like 1 (NDEL1), which is known to positively regulate cilia disassembly, as a target of LRRK1 phosphorylation. Whereas LRRK1 phosphorylation of NDEL1 on Ser-155 promotes NDEL1 interaction with the intermediate chains of cytoplasmic dynein-2, it is also crucial for triggering ciliary resorption through dynein-2-driven retrograde intraflagellar transport. These findings provide evidence that a novel PLK1-LRRK1-NDEL1 pathway regulates cilia disassembly.

Dopamine regulates emotional behaviors, including rewarding and aversive behaviors, through the mesolimbic dopaminergic pathway, which projects dopamine neurons from the ventral tegmental area to the nucleus accumbens (NAc). Protein phosphorylation is critical for intracellular signaling pathways and physiological functions, which are regulated by neurotransmitters in the brain. Previous studies have demonstrated that dopamine stimulated the phosphorylation of intracellular substrates, such as receptors, ion channels, and transcription factors, to regulate neuronal excitability and synaptic plasticity through dopamine receptors. We also established a novel database called KANPHOS that provides information on phosphorylation signals downstream of monoamines identified by our kinase substrate screening methods, including dopamine, in addition to those reported in the literature. Recent advances in proteomics techniques have enabled us to clarify the mechanisms through which dopamine controls rewarding and aversive behaviors through signal pathways in the NAc. In this review, we discuss the intracellular phosphorylation signals regulated by dopamine in these two emotional behaviors.

Dysfunctional dopamine signaling is implicated in various neuropsychological disorders. Previously, we reported that dopamine increases D1 receptor (D1R)-expressing medium spiny neuron (MSN) excitability and firing rates in the nucleus accumbens (NAc) via the PKA/Rap1/ERK pathway to promote reward behavior. Here, the results show that the D1R agonist, SKF81297, inhibits KCNQ-mediated currents and increases D1R-MSN firing rates in murine NAc slices, which is abolished by ERK inhibition. In vitro ERK phosphorylates KCNQ2 at Ser414 and Ser476; in vivo, KCNQ2 is phosphorylated downstream of dopamine signaling in NAc slices. Conditional deletion of Kcnq2 in D1R-MSNs reduces the inhibitory effect of SKF81297 on KCNQ channel activity, while enhancing neuronal excitability and cocaine-induced reward behavior. These effects are restored by wild-type, but not phospho-deficient KCNQ2. Hence, D1R-ERK signaling controls MSN excitability via KCNQ2 phosphorylation to regulate reward behavior, making KCNQ2 a potential therapeutical target for psychiatric diseases with a dysfunctional reward circuit.

Abstract Granule cell progenitors (GCPs) and granule cells (GCs) in the cerebellum are excellent models for studying the differentiation of neural progenitors into neurons. Although gradual degradation of ATOH1 protein in GCPs leads to their differentiation into GCs, the underlying regulatory mechanism is unclear. We show that a homeodomain-less isoform of MEIS1 (MEIS1-HdL) regulates ATOH1 degradation and GCP differentiation in a transcriptional regulation-independent manner. BMP signaling phosphorylates Ser328 of ATOH1 via ERK. CUL3 was identified as an E3-ligase that polyubiquitinates Ser328 phosphorylated ATOH1, leading to ATOH1 degradation. MEIS1-HdL and full-length MEIS1 form a trimeric complex with CUL3 and COP9 signalosome that inhibits ATOH1 ubiquitination and degradation. MEIS1-HdL is exclusively expressed in GCPs and suppresses ATOH1 degradation and GCP differentiation into GCs, despite high BMP signaling activities in the cells. Our study provides insight into the precise regulatory machinery of the degradation of the pivotal protein ATOH1 and differentiation of neural progenitors.

Abstract Granule cell progenitors (GCPs) and granule cells (GCs) in the cerebellum are excellent models for studying the differentiation of neural progenitors into neurons. Although gradual degradation of ATOH1 protein in GCPs leads to their differentiation into GCs, the underlying regulatory mechanism is unclear. We show that a homeodomain-less isoform of MEIS1 (MEIS1-HdL) regulates ATOH1 degradation and GCP differentiation in a transcriptional regulation-independent manner. BMP signaling phosphorylates Ser328 of ATOH1 via ERK. CUL3 was identified as an E3-ligase that polyubiquitinates Ser328 phosphorylated ATOH1, leading to ATOH1 degradation. MEIS1-HdL and full-length MEIS1 form a trimeric complex with CUL3 and COP9 signalosome that inhibits ATOH1 ubiquitination and degradation. MEIS1-HdL is exclusively expressed in GCPs and suppresses ATOH1 degradation and GCP differentiation into GCs, despite high BMP signaling activities in the cells. Our study provides insight into the precise regulatory machinery of the degradation of the pivotal protein ATOH1 and differentiation of neural progenitors.

Current antipsychotics used to treat schizophrenia have associated problems, including serious side effects and treatment resistance. We recently identified a significant association of schizophrenia with exonic copy number variations in the Rho GTPase activating protein 10 (ARHGAP10) gene using genome-wide analysis. ARHGAP10 encodes a RhoGAP superfamily member that is involved in small GTPase signaling. In mice, Arhgap10 gene variations result in RhoA/Rho-kinase pathway activation. We evaluated the pharmacokinetics of fasudil and hydroxyfasudil using liquid chromatography-tandem mass spectrometry in mice. The antipsychotic effects of fasudil on hyperlocomotion, social interaction deficits, prepulse inhibition deficits, and novel object recognition deficits were also investigated in a MK-801-treated pharmacological mouse schizophrenia model. Fasudil and its major metabolite, hydroxyfasudil, were detected in the brain at concentrations above their respective Ki values for Rho-kinase after intraperitoneal injection of 10 mg kg-1 fasudil. Fasudil improved the hyperlocomotion, social interaction deficits, prepulse inhibition deficits, and novel object recognition deficits in MK-801-treated mice in a dose-dependent manner. Following oral administration of fasudil, brain hydroxyfasudil was detected at concentration above the Ki value for Rho-kinase whilst fasudil was undetectable. MK-801-induced hyperlocomotion was also improved by oral fasudil administration. These results suggest that fasudil has antipsychotic-like effects on the MK-801-treated pharmacological mouse schizophrenia model. There are two isoforms in Rho-kinase, and further investigation is needed to clarify the isoforms involved in the antipsychotic-like effects of fasudil in the MK-801-treated mouse schizophrenia model.

Acetylcholine is a neuromodulator critical for learning and memory. The cholinesterase inhibitor donepezil increases brain acetylcholine levels and improves Alzheimer's disease (AD)-associated learning disabilities. Acetylcholine activates striatal/nucleus accumbens dopamine receptor D2-expressing medium spiny neurons (D2R-MSNs), which regulate aversive learning through muscarinic receptor M1 (M1R). However, how acetylcholine stimulates learning beyond M1Rs remains unresolved. Here, we found that acetylcholine stimulated protein kinase C (PKC) in mouse striatal/nucleus accumbens. Our original kinase-oriented phosphoproteomic analysis revealed 116 PKC substrate candidates, including Rac1 activator β-PIX. Acetylcholine induced β-PIX phosphorylation and activation, thereby stimulating Rac1 effector p21-activated kinase (PAK). Aversive stimulus activated the M1R-PKC-PAK pathway in mouse D2R-MSNs. D2R-MSN-specific expression of PAK mutants by the Cre-Flex system regulated dendritic spine structural plasticity and aversive learning. Donepezil induced PAK activation in both accumbal D2R-MSNs and in the CA1 region of the hippocampus and enhanced D2R-MSN-mediated aversive learning. These findings demonstrate that acetylcholine stimulates M1R-PKC-β-PIX-Rac1-PAK signaling in D2R-MSNs for aversive learning and imply the cascade's therapeutic potential for AD as aversive learning is used to preliminarily screen AD drugs.

The nucleus accumbens (NAc) plays critical roles in emotional behaviors, including aversive learning. Aversive stimuli such as an electric foot shock increase acetylcholine (ACh) in the NAc, and muscarinic signaling appears to increase neuronal excitability and aversive learning. Muscarinic signaling inhibits the voltage-dependent potassium KCNQ current which regulates neuronal excitability, but the regulatory mechanism has not been fully elucidated. Phosphorylation of KCNQ2 at threonine 217 (T217) and its inhibitory effect on channel activity were predicted. However, whether and how muscarinic signaling phosphorylates KCNQ2 in vivo remains unclear. Here, we found that PKC directly phosphorylated KCNQ2 at T217 in vitro. Carbachol and a muscarinic M1 receptor (M1R) agonist facilitated KCNQ2 phosphorylation at T217 in NAc/striatum slices in a PKC-dependent manner. Systemic administration of the cholinesterase inhibitor donepezil, which is commonly used to treat dementia, and electric foot shock to mice induced the phosphorylation of KCNQ2 at T217 in the NAc, whereas phosphorylation was suppressed by an M1R antagonist. Conditional deletion of Kcnq2 in the NAc enhanced electric foot shock induced aversive learning. Our findings indicate that muscarinic signaling induces the phosphorylation of KCNQ2 at T217 via PKC activation for aversive learning.

Protein kinases exert physiological functions through phosphorylating their specific substrates; however, the mode of kinase–substrate recognition is not fully understood. Rho-kinase is a Ser/Thr protein kinase that regulates cytoskeletal reorganization through phosphorylating myosin light chain (MLC) and the myosin phosphatase targeting subunit 1 (MYPT1) of MLC phosphatase (MLCP) and is involved in various diseases, due to its aberrant cellular contraction, morphology, and movement. Despite the importance of the prediction and identification of substrates and phosphorylation sites, understanding of the precise regularity in phosphorylation preference of Rho-kinase remains far from satisfactory. Here we analyzed the Rho-kinase–MYPT1 interaction, to understand the mode of Rho-kinase substrate recognition and found that the three short regions of MYPT1 close to phosphorylation sites (referred to as docking motifs (DMs); DM1 (DLQEAEKTIGRS), DM2 (KSQPKSIRERRRPR), and DM3 (RKARSRQAR)) are important for interactions with Rho-kinase. The phosphorylation levels of MYPT1 without DMs were reduced, and the effects were limited to the neighboring phosphorylation sites. We further demonstrated that the combination of pseudosubstrate (PS) and DM of MYPT1 (PS1 + DM3 and PS2 + DM2) serves as a potent inhibitor of Rho-kinase. The present information will be useful in identifying new substrates and developing selective Rho-kinase inhibitors.

Glutamate induces Ca²⁺ influx in neurons through NMDA receptors (NMDARs) and activates Ca²⁺-dependent protein kinases, including CaMKII, which play critical roles in synaptic plasticity and learning. However, how these kinases regulate synaptic plasticity and learning remains largely unknown. Here, we performed phosphoproteomics and identified 160 proteins including ArhGEF2 whose phosphorylation were promoted by NMDA. CaMKII phosphorylated ArhGEF2 and stimulated its RhoGEF activity. Aversive stimuli induced CaMKII-mediated ArhGEF2 phosphorylation and Rho-kinase/ROCK activation in the nucleus accumbens (NAc). Inhibition of Rho-kinase in the NAc attenuated aversive learning. We also screened Rho-kinase substrates and identified 221 proteins including Shank3 which links actin filaments with NMDARs and AMPA receptors via Dlgap3. The Rho-kinase-mediated phosphorylation of Shank3 increased its interaction with Dlgap3. Manipulation of Shank3 in the NAc regulated dendritic spine formation and aversive learning in a phosphorylation-dependent manner. These results demonstrate that NMDA activates the CaMKII-ArhGEF2-Rho-kinase pathway to induce Shank3 phosphorylation for aversive learning.

Protein phosphorylation plays critical roles in a variety of intracellular signaling pathways and physiological functions that are controlled by neurotransmitters and neuromodulators in the brain. Dysregulation of these signaling pathways has been implicated in neurodevelopmental disorders, including autism spectrum disorder, attention deficit hyperactivity disorder and schizophrenia. While recent advances in mass spectrometry-based proteomics have allowed us to identify approximately 280,000 phosphorylation sites, it remains largely unknown which sites are phosphorylated by which kinases. To overcome this issue, previously, we developed methods for comprehensive screening of the target substrates of given kinases, such as PKA and Rho-kinase, upon stimulation by extracellular signals and identified many candidate substrates for specific kinases and their phosphorylation sites. Here, we developed a novel online database to provide information about the phosphorylation signals identified by our methods, as well as those previously reported in the literature. The “KANPHOS” (Kinase-Associated Neural Phospho-Signaling) database and its web portal were built based on a next-generation XooNIps neuroinformatics tool. To explore the functionality of the KANPHOS database, we obtained phosphoproteomics data for adenosine-A2A-receptor signaling and its downstream MAPK-mediated signaling in the striatum/nucleus accumbens, registered them in KANPHOS, and analyzed the related pathways.

Dopamine type 1 receptor (D1R) signaling activates protein kinase A (PKA), which then activates mitogen-activated protein kinase (MAPK) through Rap1, in striatal medium spiny neurons (MSNs). MAPK plays a pivotal role in reward-related behavior through the activation of certain transcription factors. How D1R signaling regulates behavior through transcription factors remains largely unknown. CREB-binding protein (CBP) promotes transcription through hundreds of different transcription factors and is also important for reward-related behavior. To identify transcription factors regulated by dopamine signaling in MSNs, we performed a phosphoproteomic analysis using affinity beads coated with CBP. We obtained approximately 40 novel candidate proteins in the striatum of the C57BL/6 mouse brain after cocaine administration. Among them, the megakaryoblastic leukemia-2 (MKL2) protein, a transcriptional coactivator of serum response factor (SRF), was our focus. We found that the interaction between CBP and MKL2 was increased by cocaine administration. Additionally, MKL2, CBP and SRF formed a ternary complex in vivo. The C-terminal domain of MKL2 interacted with CBP-KIX and was phosphorylated by MAPK in COS7 cells. The activation of PKA-MAPK signaling induced the nuclear localization of MKL2 and increased SRF-dependent transcriptional activity in neurons. These results demonstrate that dopamine signaling regulates the interaction of MKL2 with CBP in a phosphorylation-dependent manner and thereby controls SRF-dependent gene expression.

Dysregulation of epigenetic processes involving histone methylation induces neurodevelopmental impairments and has been implicated in schizophrenia (SCZ) and autism spectrum disorder (ASD). Variants in the gene encoding lysine demethylase 4C (KDM4C) have been suggested to confer a risk for such disorders. However, rare genetic variants in KDM4C have not been fully evaluated, and the functional impact of the variants has not been studied using patient-derived cells. In this study, we conducted copy number variant (CNV) analysis in a Japanese sample set (2605 SCZ and 1141 ASD cases, and 2310 controls). We found evidence for significant associations between CNVs in KDM4C and SCZ (p = 0.003) and ASD (p = 0.04). We also observed a significant association between deletions in KDM4C and SCZ (corrected p = 0.04). Next, to explore the contribution of single nucleotide variants in KDM4C, we sequenced the coding exons in a second sample set (370 SCZ and 192 ASD cases) and detected 18 rare missense variants, including p.D160N within the JmjC domain of KDM4C. We, then, performed association analysis for p.D160N in a third sample set (1751 SCZ and 377 ASD cases, and 2276 controls), but did not find a statistical association with these disorders. Immunoblotting analysis using lymphoblastoid cell lines from a case with KDM4C deletion revealed reduced KDM4C protein expression and altered histone methylation patterns. In conclusion, this study strengthens the evidence for associations between KDM4C CNVs and these two disorders and for their potential functional effect on histone methylation patterns.

© 2020, The Author(s). Schizophrenia (SCZ) is known to be a heritable disorder; however, its multifactorial nature has significantly hampered attempts to establish its pathogenesis. Therefore, in this study, we performed genome-wide copy-number variation (CNV) analysis of 2940 patients with SCZ and 2402 control subjects and identified a statistically significant association between SCZ and exonic CNVs in the ARHGAP10 gene. ARHGAP10 encodes a member of the RhoGAP superfamily of proteins that is involved in small GTPase signaling. This signaling pathway is one of the SCZ-associated pathways and may contribute to neural development and function. However, the ARHGAP10 gene is often confused with ARHGAP21, thus, the significance of ARHGAP10 in the molecular pathology of SCZ, including the expression profile of the ARHGAP10 protein, remains poorly understood. To address this issue, we focused on one patient identified to have both an exonic deletion and a missense variant (p.S490P) in ARHGAP10. The missense variant was found to be located in the RhoGAP domain and was determined to be relevant to the association between ARHGAP10 and the active form of RhoA. We evaluated ARHGAP10 protein expression in the brains of reporter mice and generated a mouse model to mimic the patient case. The model exhibited abnormal emotional behaviors, along with reduced spine density in the medial prefrontal cortex (mPFC). In addition, primary cultured neurons prepared from the mouse model brain exhibited immature neurites in vitro. Furthermore, we established induced pluripotent stem cells (iPSCs) from this patient, and differentiated them into tyrosine hydroxylase (TH)-positive neurons in order to analyze their morphological phenotypes. TH-positive neurons differentiated from the patient-derived iPSCs exhibited severe defects in both neurite length and branch number; these defects were restored by the addition of the Rho-kinase inhibitor, Y-27632. Collectively, our findings suggest that rare ARHGAP10 variants may be genetically and biologically associated with SCZ and indicate that Rho signaling represents a promising drug discovery target for SCZ treatment.

Disabled 1 (DAB1) is an intracellular adaptor protein in the Reelin signaling pathway and plays an essential role in correct neuronal migration and layer formation in the developing brain. DAB1 has been repeatedly reported to be associated with neurodevelopmental disorders including schizophrenia (SCZ) and autism spectrum disorders (ASD) in genetic, animal, and postmortem studies. Recently, increasing attention has been given to rare single-nucleotide variants (SNVs) found by deep sequencing of candidate genes. In this study, we performed exon-targeted resequencing of DAB1 in 370 SCZ and 192 ASD patients using next-generation sequencing technology to identify rare SNVs with a minor allele frequency <1%. We detected two rare missense mutations (G382C, V129I) and then performed a genetic association study in a sample comprising 1763 SCZ, 380 ASD, and 2190 healthy control subjects. Although no statistically significant association with the detected mutations was observed for either SCZ or ASD, G382C was found only in the case group, and in silico analyses and in vitro functional assays suggested that G382C alters the function of the DAB1 protein. The rare variants of DAB1 found in the present study should be studied further to elucidate their potential functional relevance to the pathophysiology of SCZ and ASD.

For normal neurogenesis and circuit formation, delamination of differentiating neurons from the proliferative zone must be precisely controlled; however, the regulatory mechanisms underlying cell attachment are poorly understood. Here, we show that Down syndrome cell adhesion molecule (DSCAM) controls neuronal delamination by local suppression of the RapGEF2–Rap1–N-cadherin cascade at the apical endfeet in the dorsal midbrain. <italic>Dscam</italic> transcripts were expressed in differentiating neurons, and DSCAM protein accumulated at the distal part of the apical endfeet. Cre-<italic>loxP</italic>–based neuronal labeling revealed that <italic>Dscam</italic> knockdown impaired endfeet detachment from ventricles. DSCAM associated with RapGEF2 to inactivate Rap1, whose activity is required for membrane localization of N-cadherin. Correspondingly, <italic>Dscam</italic> knockdown increased N-cadherin localization and ventricular attachment area at the endfeet. Furthermore, excessive endfeet attachment by <italic>Dscam</italic> knockdown was restored by co-knockdown of <italic>RapGEF2</italic> or <italic>N-cadherin</italic>. Our findings shed light on the molecular mechanism that regulates a critical step in early neuronal development.

AIM: A Japanese individual with schizophrenia harboring a novel exonic deletion in RELN was recently identified by genome-wide copy-number variation analysis. Thus, the present study aimed to generate and analyze a model mouse to clarify whether Reln deficiency is associated with the pathogenesis of schizophrenia. METHODS: A mouse line with a novel RELN exonic deletion (Reln-del) was established using the CRISPR/Cas9 method to elucidate the underlying molecular mechanism. Subsequently, general behavioral tests and histopathological examinations of the model mice were conducted and phenotypic analysis of the cerebellar granule cell migration was performed. RESULTS: The phenotype of homozygous Reln-del mice was similar to that of reeler mice with cerebellar atrophy, dysplasia of the cerebral layers, and abrogated protein levels of cerebral reelin. The expression of reelin in heterozygous Reln-del mice was approximately half of that in wild-type mice. Conversely, behavioral analyses in heterozygous Reln-del mice without cerebellar atrophy or dysplasia showed abnormal social novelty in the three-chamber social interaction test. In vitro reaggregation formation and neuronal migration were severely altered in the cerebellar cultures of homozygous Reln-del mice. CONCLUSION: The present results in novel Reln-del mice modeled after our patient with a novel exonic deletion in RELN are expected to contribute to the development of reelin-based therapies for schizophrenia.

In Drosophila, dopamine signaling to the mushroom body intrinsic neurons, Kenyon cells (KCs), is critical to stabilize olfactory memory. Little is known about the downstream intracellular molecular signaling underlying memory stabilization. Here we address this question in the context of sugar-rewarded olfactory long-term memory (LTM). We show that associative training increases the phosphorylation of MAPK in KCs, via Dop1R2 signaling. Consistently, the attenuation of Dop1R2, Raf, or MAPK expression in KCs selectively impairs LTM, but not short-term memory. Moreover, we show that the LTM deficit caused by the knockdown of Dop1R2 can be rescued by expressing active Raf in KCs. Thus, the Dop1R2/Raf/MAPK pathway is a pivotal downstream effector of dopamine signaling for stabilizing appetitive olfactory memory.SIGNIFICANCE STATEMENT Dopaminergic input to the Kenyon cells (KCs) is pivotal to stabilize memory in Drosophila This process is mediated by dopamine receptors like Dop1R2. Nevertheless, little is known for its underlying molecular mechanism. Here we show that the Raf/MAPK pathway is specifically engaged in appetitive long-term memory in KCs. With combined biochemical and behavioral experiments, we reveal that activation of the Raf/MAPK pathway is regulated through Dop1R2, shedding light on how dopamine modulates intracellular signaling for memory stabilization.

<title>Abstract</title>Here we report that CyclinD1 (CCND1) directly regulates both the proliferative and immature states of cerebellar granule cell progenitors (GCPs). CCND1 not only accelerates cell cycle but also upregulates ATOH1 protein, an essential transcription factor that maintains GCPs in an immature state. In cooperation with CDK4, CCND1 directly phosphorylates Ser309 of ATOH1, which inhibits additional phosphorylation at S328, consequently preventing Ser328 phosphorylation-dependent ATOH1 degradation. PROX1 downregulates Ccnd1 expression by histone-deacetylation of Ccnd1 promoter in GCPs, leading to cell cycle exit and differentiation. WNT signaling upregulates PROX1 expression in GCPs. These findings suggest that WNT-PROX1-CCND1-ATOH1 signaling cascade cooperatively controls proliferation and immaturity of GCPs. We revealed that the expression and phosphorylation levels of these molecules dynamically change during cerebellar development, which was suggested to determine appropriate differentiation rates from GCPs to GCs at distinct developmental stages. This study contributes to understanding the regulatory mechanism of GCPs as well as neural progenitors.

The accumulation of phosphorylated tau protein (pTau) in the entorhinal cortex (EC) is the earliest tau pathology in Alzheimer's disease (AD). Tau tubulin kinase-1 (TTBK1) is a neuron-specific tau kinase and expressed in the EC and hippocampal regions in both human and mouse brains. Here we report that collapsin response mediator protein-2 (CRMP2), a critical mediator of growth cone collapse, is a new downstream target of TTBK1 and is accumulated in the EC region of early stage AD brains. TTBK1 transgenic mice show severe axonal degeneration in the perforant path, which is exacerbated by crossing with Tg2576 mice expressing Swedish familial AD mutant of amyloid precursor protein (APP). TTBK1 mice show accumulation of phosphorylated CRMP2 (pCRMP2), in the EC at 10 months of age, whereas age-matched APP/TTBK1 bigenic mice show pCRMP2 accumulation in both the EC and hippocampal regions. Amyloid-β peptide (Aβ) and TTBK1 suppress the kinetics of microtubule polymerization and TTBK1 reduces the neurite length of primary cultured neurons in Rho kinase-dependent manner in vitro. Silencing of TTBK1 or expression of dominant-negative Rho kinase demonstrates that Aβ induces CRMP2 phosphorylation at threonine 514 in a TTBK1-dependent manner, and TTBK1 enhances Aβ-induced CRMP2 phosphorylation in Rho kinase-dependent manner in vitro. Furthermore, TTBK1 expression induces pCRMP2 complex formation with pTau in vitro, which is enhanced upon Aβ stimulation in vitro. Finally, pCRMP2 forms a complex with pTau in the EC tissue of TTBK1 mice in vivo, which is exacerbated in both the EC and hippocampal tissues in APP/TTBK1 mice. These results suggest that TTBK1 and Aβ induce phosphorylation of CRMP2, which may be causative for the neurite degeneration and somal accumulation of pTau in the EC neurons, indicating critical involvement of TTBK1 and pCRMP2 in the early AD pathology.

Neurons are highly polarized cells that have structurally and functionally distinct processes called axons and dendrites. How neurons establish polarity is one of the fundamental questions of neuroscience. In the last decade, significant progress has been made in identifying and understanding the molecular mechanisms responsible for neuronal polarization, primarily through researches conducted on cultured neurons. Advances in phosphoproteomics technologies and molecular tools have enabled comprehensive signal analysis and visualization and manipulation of signaling molecules for analyzing neuronal polarity. Furthermore, advances in gene transfer techniques have revealed the role of extracellular and intracellular signaling molecules in neuronal polarization in vivo. This review discusses the latest insights and techniques for the elucidation of the molecular mechanisms that control neuronal polarity.

Prickle2 has been identified in genetic studies of subjects with autism spectrum disorder (ASD) and epilepsy, but the pathological mechanism of Prickle2 remains to be fully understood. Proteomic analysis of Prickle2 with mass spectrometry revealed twenty-eight Prickle2 interactors, including immunoglobulin superfamily member 9b (Igsf9b), in the brain. Here, because Igsf9 family proteins are associated with psychiatric diseases and seizures, we studied the physiological interaction between Prickle2 and Igsf9b. Prickle2 colocalized with Igsf9b in cultured hippocampal neurons. Knockdown of Prickle2 affected the subcellular localization of Igsf9b. Interestingly, Igsf9b localized along axonal processes in a pattern opposite to the ASD-related molecule ANK3/AnkG. AnkG is a major component of the axon initial segment (AIS), where a variety of ASD and epilepsy susceptibility proteins accumulate. Igsf9b-knockdown neurons displayed altered AnkG localization. Prickle2 depletion caused defects in AnkG and voltage-gated Na+ channel localization, resulting in altered network activity. These results support the idea that Prickle2 regulates AnkG distribution by controlling the proper localization of Igsf9b. The novel function of Prickle2 in AIS cytoarchitecture provides new insights into the shared pathology of ASD and epilepsy.

Dopamine (DA) activates mitogen-activated protein kinase (MAPK) via protein kinase A (PKA)/Rap1 in medium spiny neurons (MSNs) expressing the dopamine D1 receptor (D1R) in the nucleus accumbens (NAc), thereby regulating reward-related behavior. However, how MAPK regulates reward-related learning and memory through gene expression is poorly understood. Here, to identify the relevant transcriptional factors, we perform proteomic analysis using affinity beads coated with cyclic AMP response element binding protein (CREB)-binding protein (CBP), a transcriptional coactivator involved in reward-related behavior. We identify more than 400 CBP-interacting proteins, including Neuronal Per Arnt Sim domain protein 4 (Npas4). We find that MAPK phosphorylates Npas4 downstream of PKA, increasing the Npas4-CBP interaction and the transcriptional activity of Npas4 at the brain-derived neurotrophic factor (BDNF) promoter. The deletion of Npas4 in D1R-expressing MSNs impairs cocaine-induced place preference, which is rescued by Npas4-wild-type (WT), but not by a phospho-deficient Npas4 mutant. These observations suggest that MAPK phosphorylates Npas4 in D1R-MSNs and increases transcriptional activity to enhance reward-related learning and memory.

The process by which future behavioral responses are shaped by past experiences is one of the central questions in neuroscience. To gain insight into this process at the molecular and cellular levels, we have applied zebrafish larvae to explore behavioral desensitization to sound. A sudden loud noise often evokes a defensive response known as the acoustic startle response (ASR), which is triggered by firing Mauthner cells in teleosts and amphibians. The probability of evoking ASR by suprathreshold sound is reduced after exposure to repetitive auditory stimuli insufficient in amplitude to evoke the ASR (subthreshold). Although it has been suggested that the potentiation of inhibitory glycinergic inputs into Mauthner cell is involved in this desensitization of the ASR, the molecular basis for the potentiation of glycinergic transmission has been unclear. Through the in vivo monitoring of fluorescently-tagged glycine receptors (GlyRs), we here showed that behavioral desensitization to sound in zebrafish is governed by GlyR clustering in Mauthner cells. We further revealed that CaMKII-dependent phosphorylation of the scaffolding protein gephyrin at serine 325 promoted the synaptic accumulation of GlyR on Mauthner neurons through the enhancement of the gephyrin-GlyR binding, which was indispensable for and could induce desensitization of the ASR. Our study demonstrates an essential molecular and cellular basis of sound-induced receptor dynamics and thus of behavioral desensitization to sound.SIGNIFICANCE STATEMENT Behavioral desensitization in the acoustic startle response of fish is known to involve the potentiation of inhibitory glycinergic input to the Mauthner cell, which is a command neuron for the acoustic startle response. However, the molecular and cellular basis for this potentiation has been unknown. Here we show that an increase in glycine receptor (GlyR) clustering at synaptic sites on zebrafish Mauthner cells is indispensable for and could induce desensitization. Furthermore, we demonstrate that CaMKII-mediated phosphorylation of the scaffolding protein gephyrin promotes GlyR clustering by increasing the binding between the β-loop of GlyRs and gephyrin. Thus, the phosphorylation of gephyrin is a key event which accounts for the potentiation of inhibitory glycinergic inputs observed during sound-evoked behavioral desensitization.

Mutations in the microtubule-associated protein tau (MAPT) gene are known to cause familial frontotemporal dementia (FTD). The R406W tau mutation is a unique missense mutation whose patients have been reported to exhibit Alzheimer's disease (AD)-like phenotypes rather than the more typical FTD phenotypes. In this study, we established patient-derived induced pluripotent stem cell (iPSC) models to investigate the disease pathology induced by the R406W mutation. We generated iPSCs from patients and established isogenic lines using CRISPR/Cas9. The iPSCs were induced into cerebral organoids, which were dissociated into cortical neurons with high purity. In this neuronal culture, the mutant tau protein exhibited reduced phosphorylation levels and was increasingly fragmented by calpain. Furthermore, the mutant tau protein was mislocalized and the axons of the patient-derived neurons displayed morphological and functional abnormalities, which were rescued by microtubule stabilization. The findings of our study provide mechanistic insight into tau pathology and a potential for therapeutic intervention.

Endocytosis after insulin secretion plays a pivotal role in the regulation of insulin secretion in pancreatic β-cells. Our recent study suggested that EPI64, a GTPase activating protein for Rab27a, contributes to the regulation of glucose-induced endocytosis, which is mediated by the GDP-bound form of Rab27a. Here, we identified insulin receptor-related receptor (IRR) as an EPI64-interacting protein. Knockdown of IRR inhibited glucose-induced uptake of transferrin, a marker of endocytosis, translocation of the guanine-nucleotide-exchange factor ARNO to the plasma membrane, and generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3). These results suggest that IRR functions upstream of PIP3 generation and controls endocytosis after insulin secretion.

Ligand-induced activation of epidermal growth factor receptor (EGFR) initiates trafficking events that re-localize the receptor from the cell surface to intracellular endocytic compartments. EGFR-containing endosomes are transported to lysosomes for degradation by the dynein-dynactin motor protein complex. However, this cargo-dependent endosomal trafficking mechanism remains largely uncharacterized. Here, we show that GTP-bound Rab7 is phosphorylated on S72 by leucine-rich repeat kinase 1 (LRRK1) at the endosomal membrane. This phosphorylation promotes the interaction of Rab7 (herein referring to Rab7a) with its effector RILP, resulting in recruitment of the dynein-dynactin complex to Rab7-positive vesicles. This, in turn, facilitates the dynein-driven transport of EGFR-containing endosomes toward the perinuclear region. These findings reveal a mechanism regulating the cargo-specific trafficking of endosomes.

Accumulating information on eukaryotic protein phosphorylation implies a large and complicated phospho-signalling network in various cellular processes. Although a large number of protein phosphorylation sites have been detected, their physiological consequences and the linkage between each phosphorylation site and the responsible protein kinase remain largely unexplored. To understand kinase-oriented phospho-signalling pathways, we have developed novel substrate screening technologies. In this review, we described the in vitro and in vivo screening methods named kinase-interacting substrate screening analysis and kinase-oriented substrate screening analysis, respectively.

Protein phosphorylation plays a critical role in the regulation of cellular function. Information on protein phosphorylation and the responsible kinases is important for understanding intracellular signaling. A method for in vivo screening of kinase substrates named KIOSS (kinase-oriented substrate screening) has been developed. This protocol provides a method that utilizes phosphoprotein-binding modules such as 14-3-3 protein, the pin1-WW domain, and the chek2-FHA domain as biological filters to successfully enrich phosphorylated proteins related to intracellular signaling rather than housekeeping and/or structural proteins. More than 1000 substrate candidates for PKA, PKC, MAPK, and Rho-kinase in HeLa cells, as well as phosphorylation downstream of D1R, NMDAR, adenosine A2a receptor, PKA, PKC, MAPK, and Rho-kinase in mouse brain slice cultures have been identified by this method. An online database named KANPHOS (Kinase-Associated Neural Phospho-Signaling) provides the phosphorylation signals identified by these studies, as well as those previously reported in the literature. © 2019 by John Wiley & Sons, Inc.

Medium spiny neurons (MSNs) expressing dopamine D1 receptor (D1R) or D2 receptor (D2R) are major components of the striatum. Stimulation of D1R activates protein kinase A (PKA) through Golf to increase neuronal activity, while D2R stimulation inhibits PKA through Gi. Adenosine A2A receptor (A2AR) coupled to Golf is highly expressed in D2R-MSNs within the striatum. However, how dopamine and adenosine co-operatively regulate PKA activity remains largely unknown. Here, we measured Rap1gap serine 563 phosphorylation to monitor PKA activity and examined dopamine and adenosine signals in MSNs. We found that a D1R agonist increased Rap1gap phosphorylation in striatal slices and in D1R-MSNs in vivo. A2AR agonist CGS21680 increased Rap1gap phosphorylation, and pretreatment with the D2R agonist quinpirole blocked this effect in striatal slices. D2R antagonist eticlopride increased Rap1gap phosphorylation in D2R-MSNs in vivo, and the effect of eticlopride was blocked by the pretreatment with the A2AR antagonist SCH58261. These results suggest that adenosine positively regulates PKA in D2R-MSNs through A2AR, while this effect is blocked by basal dopamine in vivo. Incorporating computational model analysis, we propose that the shift from D1R-MSNs to D2R-MSNs or vice versa appears to depend predominantly on a change in dopamine concentration.

<p>Protein phosphorylation is a major and essential post-translational modification in eukaryotic cells that plays a critical role in various cellular processes. While recent advances in mass spectrometry based proteomics allowed us to identify approximately 200,000 phosphorylation sites, it is not fully understood which sites are phosphorylated by a specific kinase and which extracellular stimuli regulate the protein phosphorylation via intracellular signaling cascades. Recently, we have developed an in vitro approach termed the kinase-interacting substrate screening (KISS) method and an in vivo approach termed kinase-oriented substrate screening (KIOSS) method. Using KIOSS method, we analyzed the phosphorylation signals downstream of dopamine in mouse striatal slices, and found that about 100 proteins including ion channels and transcription factors were phosphorylated probably by PKA or MAPK. Here, we present an on-line database system which provides the phosphorylation signals identified by our KISS and KIOSS methods as well as those previously reported in the literature. The database system and its web portal, named KANPHOS (Kinase-Associated PHOspho-Signaling), were built based on the Next Generation XooNIps. We also demonstrate how to retrieve proteins and pathways in striatal medium-sized spiny neurons modulated by extracellular dopaminergic stimulation.</p>