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Introduction: Alzheimer's disease (AD) is characterized by accumulation of amyloid beta (Aβ) and hyperphosphorylated tau (Tau-P) in the brain. Aβ enhances the activity of kinases involved in the formation of Tau-P. Phosphorylation at Thr 181 determines the propagation of multiple tau phosphorylations. Aβ is derived from the amyloid precursor protein (APP). Cleavage of APP by β-secretase also initiates release of heparan sulfate (HS) from the proteoglycan glypican-1 (GPC1)., Objectives: In this study, we have explored possible connections between GPC1 expression, HS release, APP processing and Tau-P formation in human neural stem cells., Methods: GPC1 formation was suppressed by using CRISPR/Cas9 and increased by using a vector encoding GPC1. HS release from GPC1 was increased by growing cells in medium containing Arg and ascorbate. Effects were monitored by immunofluorescence microscopy and slot immunoblotting using antibodies/antisera recognizing Aβ, GPC1, HS released from GPC1, total Tau, and Tau phosphorylated at Thr-181, 217 or 231. The latter have been used as blood biomarkers for AD., Results: Suppression of GPC1 expression resulted in increased phosphorylation at Thr 181 and Thr 217. When GPC1 was overexpressed, phosphorylation at Thr 217 decreased. Stimulation of HS release from GPC1 diminished tau phosphorylation at all of the three Thr positions, while expression of GPC1 was unaffected. Simultaneous stimulation of HS release and APP processing by the cytokine TNF-α also suppressed tau phosphorylation., Conclusion: The increased release of GPC1-derived HS may interfere with Aβ formation and/or Aβ interaction with tau., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The zebrafish, a widely used model in neurobiology, relies on hearing in aquatic environments. Unfortunately, its auditory pathways have mainly been studied in larvae. In this study, we examined the involvement of the anterior tuberal nucleus (AT) in auditory processing in adult zebrafish. Our tract-tracing experiments revealed that the dorsal subdivision of AT is strongly bidirectionally connected to the central nucleus of the torus semicircularis (TSc), a major auditory nucleus in fishes. Immunohistochemical visualization of the ribosomal protein S6 (pS6) phosphorylation to map neural activity in response to auditory stimulation substantiated this finding: the dorsal but not the ventral part of AT responded strongly to auditory stimulation. A similar response to auditory stimulation was present in the TSc but not in the nucleus isthmi, a visual region, which we used as a control for testing if the pS6 activation was specific to the auditory stimulation. We also measured the time course of pS6 phosphorylation, which was previously unreported in teleost fish. After auditory stimulation, we found that pS6 phosphorylation peaked between 100 and 130 min and returned to baseline levels after 190 min. This information will be valuable for the design of future pS6 experiments. Our results suggest an anatomical and functional subdivision of AT, where only the dorsal part connects to the auditory network and processes auditory information., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Corrales Parada et al.)

The Pcdhg gene cluster encodes 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules that critically regulate multiple aspects of neural development, including neuronal survival, dendritic and axonal arborization, and synapse formation and maturation. Each γ-Pcdh isoform has unique protein domains-a homophilically interacting extracellular domain and a juxtamembrane cytoplasmic domain-as well as a C-terminal cytoplasmic domain shared by all isoforms. The extent to which isoform-specific versus shared domains regulate distinct γ-Pcdh functions remains incompletely understood. Our previous in vitro studies identified protein kinase C (PKC) phosphorylation of a serine residue within a shared C-terminal motif as a mechanism through which γ-Pcdh promotion of dendrite arborization via myristoylated alanine-rich C-kinase substrate (MARCKS) is abrogated. Here, we used CRISPR/Cas9 genome editing to generate two new mouse lines expressing only non-phosphorylatable γ-Pcdhs, due either to a serine-to-alanine mutation (Pcdhg S/A ) or to a 15-amino acid C-terminal deletion resulting from insertion of an early stop codon (Pcdhg CTD ). Both lines are viable and fertile, and the density and maturation of dendritic spines remain unchanged in both Pcdhg S/A and Pcdhg CTD cortex. Dendrite arborization of cortical pyramidal neurons, however, is significantly increased in both lines, as are levels of active MARCKS. Intriguingly, despite having significantly reduced levels of γ-Pcdh proteins, the Pcdhg CTD mutation yields the strongest phenotype, with even heterozygous mutants exhibiting increased arborization. The present study confirms that phosphorylation of a shared C-terminal motif is a key γ-Pcdh negative regulation point and contributes to a converging understanding of γ-Pcdh family function in which distinct roles are played by both individual isoforms and discrete protein domains., (© 2024 The Author(s). Developmental Neurobiology published by Wiley Periodicals LLC.)

α-Synuclein (αS) is an abundant presynaptic protein that regulates neurotransmission. It is also a key protein implicated in a broad class of neurodegenerative disorders termed synucleinopathies, including Parkinson's disease (PD) and Lewy body dementia (LBD). Pathological αS deposits in these diseases, Lewy bodies (LBs)/neurites (LNs), contain about 90% of αS in its phospho-serine129 (pS129) form. Therefore, pS129 is widely used as a surrogate marker of pathology. However, recent findings demonstrate that pS129 is also physiologically triggered by neuronal activity to positively regulate synaptic transmission. In this opinion article, we contrast the literature on pathological and physiological pS129, with a special focus on the latter. We emphasize that pS129 is ambiguous and knowledge about the context is necessary to correctly interpret changes in pS129., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Huntington's disease is a neurodegenerative disorder caused by an expanded polyglutamine stretch near the N-terminus of the huntingtin (HTT) protein, rendering the protein more prone to aggregate. The first 17 residues in HTT (Nt17) interact with lipid membranes and harbor multiple post-translational modifications (PTMs) that can modulate HTT conformation and aggregation. In this study, we used a combination of biophysical studies and molecular simulations to investigate the effect of PTMs on the helicity of Nt17 in the presence of various lipid membranes. We demonstrate that anionic lipids such as PI4P, PI(4,5)P2, and GM1 significantly enhance the helical structure of unmodified Nt17. This effect is attenuated by single acetylation events at K6, K9, or K15, whereas tri-acetylation at these sites abolishes Nt17-membrane interaction. Similarly, single phosphorylation at S13 and S16 decreased but did not abolish the POPG and PIP2-induced helicity, while dual phosphorylation at these sites markedly diminished Nt17 helicity, regardless of lipid composition. The helicity of Nt17 with phosphorylation at T3 is insensitive to the membrane environment. Oxidation at M8 variably affects membrane-induced helicity, highlighting a lipid-dependent modulation of the Nt17 structure. Altogether, our findings reveal differential effects of PTMs and crosstalks between PTMs on membrane interaction and conformation of HTT. Intriguingly, the effects of phosphorylation at T3 or single acetylation at K6, K9, and K15 on Nt17 conformation in the presence of certain membranes do not mirror that observed in the absence of membranes. Our studies provide novel insights into the complex relationship between Nt17 structure, PTMs, and membrane binding.

Necroptosis, considered as a form of programmed cell death, contributes to neural loss. The 5-hydroxytryptamine 4 receptor (5-HT 4 R) is involved in neurogenesis in the enteric nervous system. However, whether the activation of 5-HT 4 R can alleviate diabetic enteric neuropathy by inhibiting receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is unclear. This study aimed to explore the beneficial effects of 5-HT 4 R agonist on enteric neuropathy in a mouse model of diabetes and the mechanisms underlying these effects. Diabetes developed neural loss in the colon of mice. 5-HT 4 Rs localized in submucosal and myenteric plexuses were confirmed. Administration of 5-HT 4 R agonist attenuated diabetes-induced colonic hypomotility and neural loss of the colon in mice. Remarkably, RIPK3, phosphorylated RIPK3, and its downstream target mixed lineage kinase domain-like protein (MLKL), two key proteins regulating necroptosis, were significantly up-regulated in the colon of diabetic mice. Treatment with 5-HT 4 R agonist appeared to inhibit diabetes-induced elevation of RIPK3, phosphorylated RIPK3, and MLKL in the colon of mice. Diabetes-induced up-regulation of MLKL in both the mucosa and the muscularis of the colon was prevented by Ripk3 deletion. Moreover, diabetes-evoked neural loss and delayed colonic transit were significantly inhibited by Ripk3 removal. These findings suggest that activation of 5-HT 4 Rs could potentially provide a protective effect against diabetic enteric neuropathy by suppressing RIPK3-mediated necroptosis., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is a progressive neurological disorder that impairs memory and cognitive abilities, primarily in the elderly. The burden of AD extends beyond patients, impacting families and caregivers due to the patients' reliance on assistance for daily tasks. The main features of the pathogenesis of AD are beta-amyloid plaques and neurofibrillary tangles (NFTs), that strongly correlate with oxidative stress and inflammation. NFTs result from misfolded and hyperphosphorylated tau proteins. Various studies have focused on tau phosphorylation, indicating protein phosphatase 2A (PP2A) as the primary tau phosphatase and glycogen synthase kinase-3 beta (GSK-3β) as the leading tau kinase. Experimental evidence suggests that inhibition of PP2A and increased GSK-3β activity contribute to neuroinflammation, oxidative stress, and cognitive impairment. Hence, targeting PP2A and GSK-3β with pharmacological approaches shows promise in treating AD. The use of natural compounds in the drug development for AD have been extensively studied for their antioxidant, anti-inflammatory, anti-cholinesterase, and neuroprotective properties, demonstrating therapeutic advantages in neurological diseases. Alongside the development of PP2A activator and GSK-3β inhibitor drugs, natural compounds are likely to have neuroprotective effects by increasing PP2A activity and decreasing GSK-3β levels. Therefore, based on the preclinical and clinical studies, the potential of PP2A and GSK-3β as therapeutic targets of natural compounds are highlighted in this review., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Exercise-induced activation of adenosine monophosphate-activated protein kinase (AMPK) and substrate phosphorylation modulate the metabolic capacity of mitochondria in skeletal muscle. However, the key effector(s) of AMPK and the regulatory mechanisms remain unclear. Here, we showed that AMPK phosphorylation of the folliculin interacting protein 1 (FNIP1) serine-220 (S220) controls mitochondrial function and muscle fuel utilization during exercise. Loss of FNIP1 in skeletal muscle resulted in increased mitochondrial content and augmented metabolic capacity, leading to enhanced exercise endurance in mice. Using skeletal muscle-specific nonphosphorylatable FNIP1 (S220A) and phosphomimic (S220D) transgenic mouse models as well as biochemical analysis in primary skeletal muscle cells, we demonstrated that exercise-induced FNIP1 (S220) phosphorylation by AMPK in muscle regulates mitochondrial electron transfer chain complex assembly, fuel utilization, and exercise performance without affecting mechanistic target of rapamycin complex 1-transcription factor EB signaling. Therefore, FNIP1 is a multifunctional AMPK effector for mitochondrial adaptation to exercise, implicating a mechanism for exercise tolerance in health and disease.

Traumatic brain injury (TBI) is a major cause of death and disability in adults. The pathological process of TBI involves a multifactorial cascade in which kinases have been proven contribute to interactions between relevant factors and amplification of signaling cascades. Cyclin-dependent kinase 5 (Cdk5) is a promising kinase that has been implicated in various brain disorders, including TBI. However, the mechanism by which Cdk5 induces neuronal damage remains unclear. Here, we show for the first time that Drosha, a key enzyme in microRNA biogenesis, is a pivotal substrate of abnormally activated Cdk5. Cdk5-mediated phosphorylation decreases Drosha expression and exacerbates nerve injury in TBI. We proved that maintaining Drosha expression via the administration of repurposed Cdk5 inhibitors that were previously studied in clinical trials is a promising approach for the early treatment of TBI. Together, our work identifies Drosha as a novel target for neuroprotective strategies after TBI and suggests Cdk5-mediated regulation of Drosha expression as a potential therapeutic strategy for early TBI intervention., (© 2024. The Author(s).)

Phosphosignaling in bacteria is mediated by two distinct systems, the two-component systems (TCSs) and the protein Ser/Thr/Tyr, or O-phosphorylation systems. These two arms of phosphosignaling are currently thought to be largely independent from one another. We mined a deep Mycobacterium tuberculosis (Mtb) phosphoproteome and identified over 170 O-phosphorylation sites on histidine kinases and response regulators of TCSs, suggesting that the two signaling pathways extensively intersect. Several TCSs were phosphorylated on multiple sites, and many by multiple Ser/Thr protein kinases, suggesting convergent and cooperative regulatory interactions. To test in which way these O-phosphorylation sites affect TCS activity, we reconstituted the NarSL phosphorelay in vitro. The Ser/Thr protein kinase PknL phosphorylated the histidine kinase NarS and activated its autophosphorylating activity. A phosphoablative mutation at the PknL phosphorylation site Thr380 resulted in low autophosphorylating activity, whereas a phosphomimetic mutation strongly activated autophosphorylation. The phosphomimetic mutation also resulted in more efficient phosphotransfer from NarS to the response regulator NarL and suppression of gene expression. These data show control of NarSL signaling by STPKs through a phosphoswitch and point to extensive, functional crosstalk between TCSs and O-phosphorylation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The potassium chloride cotransporter KCC2 is crucial for Cl - extrusion from mature neurons and thus key to hyperpolarizing inhibition. Auditory brainstem circuits contain well-understood inhibitory projections and provide a potent model to study the regulation of synaptic inhibition. Two peculiarities of the auditory brainstem are (i) posttranslational activation of KCC2 during development and (ii) extremely negative reversal potentials in specific circuits. To investigate the role of the potent phospho-site serine 937 therein, we generated a KCC2 Thr 934Ala /Ser 937Asp double mutation, in which Ser 937 is replaced by aspartate mimicking the phosphorylated state, and the neighbouring Thr 934 arrested in the dephosphorylated state. This double mutant showed a twofold increased transport activity in HEK293 cells, raising the hypothesis that auditory brainstem neurons show lower [Cl - ] i . and increased glycinergic inhibition. This was tested in a mouse model carrying the same KCC2 Thr 934Ala /Ser 937Asp mutation by the use of the CRISPR/Cas9 technology. Homozygous KCC2 Thr 934Ala /Ser 937Asp mice showed an earlier developmental onset of hyperpolarisation in the auditory brainstem. Mature neurons displayed stronger glycinergic inhibition due to hyperpolarized E Cl- . These data demonstrate that phospho-regulation of KCC2 Ser 937 is a potent way to interfere with the excitation-inhibition balance in neural circuits., (© 2023. The Author(s).)

Many functional aspects of the protein kinase p38α have been illustrated by more than three hundred structures determined in the presence of reducing agents. These structures correspond to free forms and complexes with activators, substrates, and inhibitors. Here we report the conformation of an oxidized state with an intramolecular disulfide bond between Cys119 and Cys162 that is conserved in vertebrates. The structure of the oxidized state does not affect the conformation of the catalytic site, but alters the docking groove by partially unwinding and displacing the short αD helix due to the movement of Cys119 towards Cys162. The transition between oxidized and reduced conformations provides a mechanism for fine-tuning p38α activity as a function of redox conditions, beyond its activation loop phosphorylation. Moreover, the conformational equilibrium between these redox forms reveals an unexplored cleft for p38α inhibitor design that we describe in detail., (© 2023. The Author(s).)

Synaptic plasticity plays a crucial role in memory formation by regulating the communication between neurons. Although actin polymerization has been linked to synaptic plasticity and dendritic spine stability, the causal link between actin polymerization and memory encoding has not been identified yet. It is not clear whether actin polymerization and structural changes in dendritic spines are a driver or a consequence of learning and memory. Using an extrinsically disordered form of the protein kinase LIMK1, which rapidly and precisely acts on ADF/cofilin, a direct modifier of actin, we induced long-term enlargement of dendritic spines and enhancement of synaptic transmission in the hippocampus on command. The activation of extrinsically disordered LIMK1 in vivo improved memory encoding and slowed cognitive decline in aged mice exhibiting reduced cofilin phosphorylation. The engineered memory by an extrinsically disordered LIMK1 supports a direct causal link between actin-mediated synaptic transmission and memory.

Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that are involved in synapse assembly and function. The NLGN gene family consists of 5 genes ( NLGN1-3 , 4X , and 4Y ). NLGN3 forms heterodimers with other NLGNs and is expressed at both excitatory and inhibitory synapses, although the distinct role at different synapses is not fully understood. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that targets various neuronal substrates to impact neuronal migration, neurite outgrowth, synaptic transmission, and plasticity. Both NLGNs and their presynaptic binding partners neurexins are highly associated with neurodevelopmental disorders. The NLGN3 gene is on the X chromosome and variants in NLGN3 have been linked to the pathophysiology in neurodevelopmental disorders. To better understand the endogenous modulation of NLGN3, we generated an HA-tagged knock-in mouse. We found that Cdk5 associates with NLGN3 in vivo and phosphorylates NLGN3 on serine 725 (S725) in the knock-in mouse of either sex. The phosphorylation affects the NLGN3 association with Kalirin-7, a postsynaptic guanine nucleotide exchange factors for Rho GTPase family proteins. We further observed that the phosphorylation modulates NLGN3 surface expression and NLGN3-mediated synaptic currents in cultured rat neurons. Thus, we characterized NLGN3 as a novel Cdk5 substrate and revealed the functional consequences of NLGN3 S725 phosphorylation in neurons. Our study provides a novel molecular mechanism underlying Cdk5-mediated regulation of postsynaptic cell adhesion molecules. SIGNIFICANCE STATEMENT NLGN3 is involved in synapse assembly and function at both excitatory and inhibitory synapses and has been associated with the pathophysiology of neurodevelopmental disorders. Cdk5 has brain-specific activity and is involved in neuronal transmission, synapse function, and plasticity. Here, we characterize NLGN3 as a Cdk5 substrate for the first time and show that Cdk5-mediated phosphorylation regulates NLGN3 function. We demonstrate that NLGN3 S725 is a Cdk5 phosphorylation site, and reveal that the site is important for NLGN3 association with Kalirin-7, NLGN3 surface expression, and NLGN3-mediated synaptic transmission., (Copyright © 2023 the authors.)

Background: O-GlcNAcylation is the enzymatic addition of a sugar, O-linked β-N-Acetylglucosamine, to the serine and threonine residues of proteins, and is abundant in diabetic conditions. We have previously shown that O-GlcNAcylation can trigger arrhythmias by indirectly increasing pathological Ca 2+ leak through the cardiac ryanodine receptor (RyR2) via Ca 2+ /calmodulin-dependent kinase II (CaMKII). However, RyR2 is well known to be directly regulated by other forms of serine and threonine modification, therefore, this study aimed to determine whether RyR2 is directly modified by O-GlcNAcylation and if this also alters the function of RyR2 and Ca 2+ leak., Methods: O-GlcNAcylation of RyR2 in diabetic human and animal hearts was determined using western blotting. O-GlcNAcylation of RyR2 was pharmacologically controlled and the propensity for Ca 2+ leak was determined using single cell imaging. The site of O-GlcNAcylation within RyR2 was determined using site-directed mutagenesis of RyR2., Results: We found that RyR2 is modified by O-GlcNAcylation in human, animal and HEK293 cell models. Under hyperglycaemic conditions O-GlcNAcylation was associated with an increase in Ca 2+ leak through RyR2 which persisted after CaMKII inhibition. Conversion of serine-2808 to alanine prevented an O-GlcNAcylation induced increase in Ca 2+ leak., Conclusions: These data suggest that the function of RyR2 can be directly regulated by O-GlcNAcylation and requires the presence of serine-2808., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Protein phosphorylation modification is crucial for signaling transduction in plant development and environmental adaptation. By precisely phosphorylating crucial components in signaling cascades, plants can switch on and off the specific signaling pathways needed for growth or defense. Here, we have summarized recent findings of key phosphorylation events in typical hormone signaling and stress responses. More interestingly, distinct phosphorylation patterns on proteins result in diverse biological functions of these proteins. Thus, we have also highlighted latest findings that show how the different phosphosites of a protein, also named phosphocodes, determine the specificity of downstream signaling in both plant development and stress responses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson's disease (PD), with growing importance also for Crohn's disease and cancer. LRRK2 is a large and complex protein possessing both GTPase and kinase activity. Moreover, LRRK2 activity and function can be influenced by its phosphorylation status. In this regard, many LRRK2 PD-associated mutants display decreased phosphorylation of the constitutive phosphorylation cluster S910/S935/S955/S973, but the role of these changes in phosphorylation status with respect to LRRK2 physiological functions remains unknown. Here, we propose that the S910/S935/S955/S973 phosphorylation sites act as key regulators of LRRK2-mediated autophagy under both basal and starvation conditions. We show that quadruple LRRK2 phosphomutant cells (4xSA; S910A/S935A/S955A/S973A) have impaired lysosomal functionality and fail to induce and proceed with autophagy during starvation. In contrast, treatment with the specific LRRK2 kinase inhibitors MLi-2 (100 nM) or PF-06447475 (150 nM), which also led to decreased LRRK2 phosphorylation of S910/S935/S955/S973, did not affect autophagy. In explanation, we demonstrate that the autophagy impairment due to the 4xSA LRRK2 phospho-dead mutant is driven by its enhanced LRRK2 kinase activity. We show mechanistically that this involves increased phosphorylation of LRRK2 downstream targets Rab8a and Rab10, as the autophagy impairment in 4xSA LRRK2 cells is counteracted by expression of phosphorylation-deficient mutants T72A Rab8a and T73A Rab10. Similarly, reduced autophagy and decreased LRRK2 phosphorylation at the constitutive sites were observed in cells expressing the pathological R1441C LRRK2 PD mutant, which also displays increased kinase activity. These data underscore the relation between LRRK2 phosphorylation at its constitutive sites and the importance of increased LRRK2 kinase activity in autophagy regulation and PD pathology., (© 2023. The Author(s).)

Understanding the molecular signaling mechanisms underlying cognition and neuronal plasticity would provide insights into the pathogenesis of neuronal disorders characterized by cognitive syndromes such as Alzheimer disease (AD). Phosphorylation of the mRNA translational factor eukaryotic elongation factor 2 (eEF2) by its specific kinase eEF2K is critically involved in protein synthesis regulation. In this review, we discussed recent studies on the roles of eEF2K/eEF2 signaling in the context of regulation/dysregulation of cognitive function and synaptic plasticity. We specifically focus on the discussion of recent evidence indicating suppression of eEF2K signaling as a potential novel therapeutic avenue for AD and related dementias (ADRDs)., (© 2021 International Society for Neurochemistry.)

A class-A GPCR dopamine D2 receptor (D2R) plays a critical role in the proper functioning of neuronal circuits through the downstream activation of both G-protein- and β-arrestin-dependent signaling pathways. Understanding the signaling pathways downstream of D2R is critical for developing effective therapies with which to treat dopamine (DA)-related disorders such as Parkinson's disease and schizophrenia. Extensive studies have focused on the regulation of D2R-mediated extracellular-signal-regulated kinase (ERK) 1/2 signaling; however, the manner in which ERKs are activated upon the stimulation of a specific signaling pathway of D2R remains unclear. The present study conducted a variety of experimental techniques, including loss-of-function experiments, site-directed mutagenesis, and the determination of protein interactions, in order to investigate the mechanisms underlying β-arrestin-biased signaling-pathway-mediated ERK activation. We found that the stimulation of the D2R β-arrestin signaling pathway caused Mdm2, an E3 ubiquitin ligase, to move from the nucleus to the cytoplasm and interact with tyrosine phosphorylated G-protein-coupled receptor kinase 2 (GRK2), which was facilitated by Src, a non-receptor tyrosine kinase. This interaction led to the ubiquitination of GRK2, which then moved to the plasma membrane and interacted with activated D2R, followed by the phosphorylation of D2R as well as the mediation of ERK activation. In conclusion, Mdm2-mediated GRK2 ubiquitination, which is selectively triggered by the stimulation of the D2R β-arrestin signaling pathway, is necessary for GRK2 membrane translocation and its interaction with D2R, which in turn mediates downstream ERK signaling. This study is primarily novel and provides essential information with which to better understand the detailed mechanisms of D2R-dependent signaling.

Arrestins bind active phosphorylated G protein-coupled receptors (GPCRs). Among the four mammalian subtypes, only arrestin-3 facilitates the activation of JNK3 in cells. In available structures, Lys-295 in the lariat loop of arrestin-3 and its homologue Lys-294 in arrestin-2 directly interact with the activator-attached phosphates. We compared the roles of arrestin-3 conformational equilibrium and Lys-295 in GPCR binding and JNK3 activation. Several mutants with enhanced ability to bind GPCRs showed much lower activity towards JNK3, whereas a mutant that does not bind GPCRs was more active. The subcellular distribution of mutants did not correlate with GPCR recruitment or JNK3 activation. Charge neutralization and reversal mutations of Lys-295 differentially affected receptor binding on different backgrounds but had virtually no effect on JNK3 activation. Thus, GPCR binding and arrestin-3-assisted JNK3 activation have distinct structural requirements, suggesting that facilitation of JNK3 activation is the function of arrestin-3 that is not bound to a GPCR.

Plants respond to changing light intensity in the short term through regulation of light harvesting, electron transfer, and metabolism to mitigate redox stress. A sustained shift in light intensity leads to a long-term acclimation response (LTR). This involves adjustment in the stoichiometry of photosynthetic complexes through de novo synthesis and degradation of specific proteins associated with the thylakoid membrane. The light-harvesting complex II (LHCII) serine/threonine kinase STN7 plays a key role in short-term light harvesting regulation and was also suggested to be crucial to the LTR. Arabidopsis plants lacking STN7 (stn7) shifted to low light experience higher photosystem II (PSII) redox pressure than the wild type or those lacking the cognate phosphatase TAP38 (tap38), while the reverse is true at high light, where tap38 suffers more. In principle, the LTR should allow optimisation of the stoichiometry of photosynthetic complexes to mitigate these effects. We used quantitative label-free proteomics to assess how the relative abundance of photosynthetic proteins varied with growth light intensity in wild-type, stn7, and tap38 plants. All plants were able to adjust photosystem I, LHCII, cytochrome b 6 f, and ATP synthase abundance with changing white light intensity, demonstrating neither STN7 nor TAP38 is crucial to the LTR per se. However, stn7 plants grown for several weeks at low light (LL) or moderate light (ML) still showed high PSII redox pressure and correspondingly lower PSII efficiency, CO 2 assimilation, and leaf area compared to wild-type and tap38 plants, hence the LTR is unable to fully ameliorate these symptoms. In contrast, under high light growth conditions the mutants and wild type behaved similarly. These data are consistent with the paramount role of STN7-dependent LHCII phosphorylation in tuning PSII redox state for optimal growth in LL and ML conditions., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

This study investigated whether muscle contraction induces expression of regulated in development and DNA damage 1 (REDD1), a potent inhibitor of mTORC1, in mice muscle. Gastrocnemius muscle was unilaterally and isometrically contracted with electrical stimulation, and changes in muscle protein synthesis, mTORC1 signaling phosphorylation, and REDD1 protein, and mRNA were measured at time points of 0, 3, 6, 12, and 24 h after the contraction. At time point 0 and 3 h, muscle protein synthesis was blunted by the contraction, accompanied by a decrease in phosphorylation of 4E-BP1 at time point 0 h, suggesting suppression of mTORC1 was involved in blunting of muscle protein synthesis during and shortly after the contraction. REDD1 protein was not increased in the contracted muscle at these time points, but at time point 3 h, both REDD1 protein and mRNA were increased in the contralateral non-contracted muscle. The induction of REDD1 expression in the non-contracted muscle was attenuated by RU-486, an antagonist of the glucocorticoid receptor, suggesting that glucocorticoids are involved in this process. These findings suggest that muscle contraction induces temporal anabolic resistance in non-contracted muscle, potentially increasing the availability of amino acids for contracted muscle, allowing for the synthesis of muscle protein., (© 2023 The Author. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

The two members of the UBASH3/STS/TULA protein family have been shown to critically regulate key biological functions, including immunity and hemostasis, in mammalian biological systems. Negative regulation of signaling through immune receptor tyrosine-based activation motif (ITAM)- and hemITAM-bearing receptors mediated by Syk-family protein tyrosine kinases appears to be a major molecular mechanism of the down-regulatory effect of TULA-family proteins, which possess protein tyrosine phosphatase (PTP) activity. However, these proteins are likely to carry out some PTP-independent functions as well. Whereas the effects of TULA-family proteins overlap, their characteristics and their individual contributions to cellular regulation also demonstrate clearly distinct features. Protein structure, enzymatic activity, molecular mechanisms of regulation, and biological functions of TULA-family proteins are discussed in this review. In particular, the usefulness of the comparative analysis of TULA proteins in various metazoan taxa, for identifying potential roles of TULA-family proteins outside of their functions already established in mammalian systems, is examined.

Epithelial-mesenchymal transition (EMT) is associated with the invasive and metastatic phenotypes in colorectal cancer (CRC). However, the mechanisms underlying EMT in CRC are not completely understood. In this study, we find that HUNK inhibits EMT and metastasis of CRC cells via its substrate GEF-H1 in a kinase-dependent manner. Mechanistically, HUNK directly phosphorylates GEF-H1 at serine 645 (S645) site, which activates RhoA and consequently leads to a cascade of phosphorylation of LIMK-1/CFL-1, thereby stabilizing F-actin and inhibiting EMT. Clinically, the levels of both HUNK expression and phosphorylation S645 of GEH-H1 are not only downregulated in CRC tissues with metastasis compared with that without metastasis, but also positively correlated among these tissues. Our findings highlight the importance of HUNK kinase direct phosphorylation of GEF-H1 in regulation of EMT and metastasis of CRC., (© 2023. The Author(s).)

The p38 MAP kinase (MAPK) signaling pathway is a key signal transduction cascade that cancer cells employ to sense and adapt to a plethora of environmental stimuli and has attracted much attention as a promising target for cancer therapy. Although the kinases that phosphorylate p38 have been extensively studied, the negative regulation of p38 phosphorylation remains to be elucidated. Here, we found that PPM1G was highly expressed in lung adenocarcinoma (LUAD) compared to normal tissues, and higher levels of PPM1G were observed in adverse staged LUAD. Furthermore, the higher levels of PPM1G were highly correlated with poor prognosis, according to the Cancer Genome Atlas cohort. Most importantly, we identified phospho-MEK6 as a direct substrate of PPM1G. PPM1G, a metal-dependent protein phosphatase family phosphatase, could reduce p38 phosphorylation via MEK6 dephosphorylation and contribute to the proliferation, invasion and metastasis of LUAD. Our study highlighted the essential role of PPM1G in LUAD and shed new light on unveiling the regulation of p38 activity via direct dephosphorylation of MEK6 in malignant transformation. Together, this study provides new insight into the complexity of regulating the versatile p38 signaling and suggests new directions in intervening in p38 MAPK signaling., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The hepatotoxicity of regorafenib is one of the most noteworthy concerns for patients, however the mechanism is poorly understood. Hence, there is a lack of effective intervention strategies. Here, by comparing the target with sorafenib, we show that regorafenib-induced liver injury is mainly due to its nontherapeutic target Eph receptor A2 (EphA2). EphA2 deficiency attenuated liver damage and cell apoptosis under regorafenib treatment in male mice. Mechanistically, regorafenib inhibits EphA2 Ser897 phosphorylation and reduces ubiquitination of p53 by altering the intracellular localization of mouse double minute 2 (MDM2) by affecting the extracellular signal-regulated kinase (ERK)/MDM2 axis. Meanwhile, we found that schisandrin C, which can upregulate the phosphorylation of EphA2 at Ser897 also has protective effect against the toxicity in vivo. Collectively, our findings identify the inhibition of EphA2 Ser897 phosphorylation as a key cause of regorafenib-induced hepatotoxicity, and chemical activation of EphA2 Ser897 represents a potential therapeutic strategy to prevent regorafenib-induced hepatotoxicity., (© 2023. The Author(s).)

The Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a central regulator of learning and memory, which poses a problem for targeting it therapeutically. Indeed, our study supports prior conclusions that long-term interference with CaMKII signaling can erase pre-formed memories. By contrast, short-term pharmacological CaMKII inhibition with the neuroprotective peptide tatCN19o interfered with learning in mice only mildly and transiently (for less than 1 h) and did not at all reverse pre-formed memories. These results were obtained with ≥500-fold of the dose that protected hippocampal neurons from cell death after a highly clinically relevant pig model of transient global cerebral ischemia: ventricular fibrillation followed by advanced life support and electrical defibrillation to induce the return of spontaneous circulation. Of additional importance for therapy development, our preliminary cardiovascular safety studies in mice and pig did not indicate any concerns with acute tatCN19o injection. Taken together, although prolonged interference with CaMKII signaling can erase memory, acute short-term CaMKII inhibition with tatCN19o did not cause such retrograde amnesia that would pose a contraindication for therapy., Competing Interests: Conflict of interest K. U. B. is co-founder and board member of Neurexis Therapeutics, a company that seeks to develop the tatCN19o inhibitor of CaMKII into a therapeutic drug for cerebral ischemia. O. R. B. is director of research and development at the same company., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Activation of platelet-derived growth factor (PDGF) receptors α and β (PDGFRα and PDGFRβ) at the cell surface by binding of PDGF isoforms leads to internalization of receptors, which affects the amplitude and kinetics of signaling. Ubiquitination of PDGF receptors in response to ligand stimulation is mediated by the Casitas b-lineage lymphoma (Cbl) family of ubiquitin ligases, promoting internalization and serving as a sorting signal for vesicular trafficking of receptors. We report here that another E3 ligase, i.e., tripartite motif-containing protein 21 (TRIM21), contributes to the ubiquitination of PDGFRβ in human primary fibroblasts AG1523 and the osteosarcoma cell line U2OS and regulates basal levels of PDGFRβ. We found that siRNA-mediated depletion of TRIM21 led to decreased ubiquitination of PDGFRβ in response to PDGF-BB stimulation, while internalization from the cell surface and the rate of ligand-induced degradation of the receptor were not affected. Moreover, induction of TRIM21 decreased the levels of PDGFRβ in serum-starved cells, and even more in growing cells, in the absence of PDGF stimulation. Consistently, siRNA knockdown of TRIM21 caused accumulation of the total amount of PDGFRβ, both in the cytoplasm and on the cell surface, without affecting mRNA levels of the receptor. We conclude that TRIM21 acts post-translationally and maintains basal levels of PDGFRβ, thus suggesting that ubiquitination of PDGFRβ by TRIM21 may direct a portion of receptor for degradation in growing cells in a ligand-independent manner.

Hypertrophic cardiomyopathy (HCM) is the leading genetic cause of heart disease. The heart comprises several proteins that work together to properly facilitate force production and pump blood throughout the body. Cardiac myosin binding protein-C (cMyBP-C) is a thick-filament protein, and mutations in cMyBP-C are frequently linked with clinical cases of HCM. Within the sarcomere, the N-terminus of cMyBP-C likely interacts with the myosin regulatory light chain (RLC); RLC is a subunit of myosin located within the myosin neck region that modulates contractile dynamics via its phosphorylation state. Phosphorylation of RLC is thought to influence myosin head position along the thick-filament backbone, making it more favorable to bind the thin filament of actin and facilitate force production. However, little is known about how these two proteins interact. We tested the effects of RLC phosphorylation on Ca2+-regulated contractility using biomechanical assays on skinned papillary muscle strips isolated from cMyBP-C KO mice and WT mice. RLC phosphorylation increased Ca2+ sensitivity of contraction (i.e., pCa50) from 5.80 ± 0.02 to 5.95 ± 0.03 in WT strips, whereas RLC phosphorylation increased Ca2+ sensitivity of contraction from 5.86 ± 0.02 to 6.15 ± 0.03 in cMyBP-C KO strips. These data suggest that the effects of RLC phosphorylation on Ca2+ sensitivity of contraction are amplified when cMyBP-C is absent from the sarcomere. This implies that cMyBP-C and RLC act in concert to regulate contractility in healthy hearts, and mutations to these proteins that lead to HCM (or a loss of phosphorylation with disease progression) may disrupt important interactions between these thick-filament regulatory proteins., (© 2023 Turner et al.)

Bacterial phosphosignalling has been synonymous with two-component systems and their histidine kinases, but many bacteria, including Mycobacterium tuberculosis (Mtb), also code for Ser/Thr protein kinases (STPKs). STPKs are the main phosphosignalling enzymes in eukaryotes but the full extent of phosphorylation on protein Ser/Thr and Tyr (O-phosphorylation) in bacteria is untested. Here we explored the global signalling capacity of the STPKs in Mtb using a panel of STPK loss-of-function and overexpression strains combined with mass spectrometry-based phosphoproteomics. A deep phosphoproteome with >14,000 unique phosphosites shows that O-phosphorylation in Mtb is a vastly underexplored protein modification that affects >80% of the proteome and extensively interfaces with the transcriptional machinery. Mtb O-phosphorylation gives rise to an expansive, distributed and cooperative network of a complexity that has not previously been seen in bacteria and that is on par with eukaryotic phosphosignalling networks. A resource of >3,700 high-confidence direct substrate-STPK interactions and their transcriptional effects provides signalling context for >80% of Mtb proteins and allows the prediction and assembly of signalling pathways for mycobacterial physiology., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Purpose: Resistance exercise training (RET) attenuates age-related muscle and strength loss ("sarcopenia"). However, compared with machine-based RET, the efficacy of cost-effective, accessible elastic band RET (EB-RET) for muscle adaptive remodeling lacks supporting mechanistic evidence., Methods: Eight young (YM; 24 ± 4 yr) and eight older (OM; 68 ± 6 yr) untrained males consumed an oral stable isotope tracer (D 2 O) combined with serial vastus lateralis muscle biopsies to measure integrated myofibrillar protein synthesis (iMyoPS) and regulatory signaling over ~48 h before (habitual) and after an acute bout of EB-RET (6 × 12 repetitions at ~70% of one-repetition maximum). iMyoPS was determined via gas chromatography-pyrolysis-isotope ratio mass spectroscopy and regulatory signaling expression by immunoblot., Results: Habitual iMyoPS did not differ between YM and OM (1.62% ± 0.21% vs 1.43% ± 0.47%·d -1 , respectively, P = 0.128). There was a significant increase in iMyoPS after EB-RET in YM (2.23% ± 0.69%·d -1 , P = 0.02), but not OM (1.75% ± 0.54%·d -1 , P = 0.30). EB-RET increased the phosphorylation of key anabolic signaling proteins similarly in YM and OM at 1 h postexercise, including p-IRS-1 Ser636/639 , p-Akt Ser473 , p-4EBP-1 Thr37/46 , p-P70S6K Thr389 , and p-RPS6 Ser240/244 , whereas p-TSC2 Thr1462 and p-mTOR Ser2448 increased only in YM (all P < 0.05). There were no differences in the expression of amino acid transporters/sensors or proteolytic markers after EB-RET., Conclusions: iMyoPS was elevated after EB-RET in YM but not OM. However, the increase in acute anabolic signaling with EB-RET was largely similar between groups. In conclusion, the capacity for EB-RET to stimulate iMyoPS may be impaired in older age. Further work may be necessary to optimize prescriptive programming in YM and OM., (Copyright © 2022 by the American College of Sports Medicine.)

Phosphorylation is a ubiquitous mechanism by which signals are transduced in cells. Protein kinases, enzymes that catalyze the phosphotransfer reaction are, themselves, often regulated by phosphorylation. Paradoxically, however, a substantial fraction of more than 500 human protein kinases are capable of catalyzing their own activation loop phosphorylation. Commonly, these kinases perform this autophosphorylation reaction in trans , whereby transient dimerization leads to the mutual phosphorylation of the activation loop of the opposing protomer. In this study, we demonstrate that protein kinase D (PKD) is regulated by the inverse mechanism of dimerization-mediated trans -autoinhibition, followed by activation loop autophosphorylation in cis . We show that PKD forms a stable face-to-face homodimer that is incapable of either autophosphorylation or substrate phosphorylation. Dissociation of this trans -autoinhibited dimer results in activation loop autophosphorylation, which occurs exclusively in cis . Phosphorylation serves to increase PKD activity and prevent trans -autoinhibition, thereby switching PKD on. Our findings not only reveal the mechanism of PKD regulation but also have profound implications for the regulation of many other eukaryotic kinases.

Two-component systems typically consist of a paired histidine kinase and response regulator and couple environmental changes to adaptive responses. The response regulator VbrR from Vibrio parahaemolyticus , a member of the OmpR/PhoB family, regulates virulence and antibiotic resistance genes. The activation mechanism of VbrR remains unclear. Here, we report the crystal structures of full-length VbrR in complex with DNA in the active conformation and the N-terminal receiver domain (RD) and the C-terminal DNA-binding domain (DBD) in both active and inactive conformations. Structural and biochemical analyses suggest that unphosphorylated VbrR adopts mainly as inactive dimers through the DBD at the autoinhibitory state. The RD undergoes a monomer-to-dimer transition upon phosphorylation, which further induces the transition of DBD from an autoinhibitory dimer to an active dimer and enables its binding with target DNA. Our study suggests a new model for phosphorylation-induced activation of response regulators and sheds light on the pathogenesis of V . parahaemolyticus .

The microtubule-associated conserved protein tau has attracted significant attention because of its essential role in the formation of pathological changes in the nervous system, which can reduce longevity. The study of the effects caused by tau dysfunction and the molecular mechanisms underlying them is complicated because different forms of tau exist in humans and model organisms, and the changes in protein expression can be multidirectional. In this article, we show that an increase in the expression of the main isoform of the Drosophila melanogaster tau protein in the nervous system has differing effects on lifespan depending on the sex of individuals but has no effect on the properties of the nervous system, in particular, the synaptic activity and distribution of another microtubule-associated protein, Futsch, in neuromuscular junctions. Reduced expression of tau in the nervous system does not affect the lifespan of wild-type flies, but it does increase the lifespan dramatically shortened by overexpression of the shaggy gene encoding the GSK3 (Glycogen Synthase Kinase 3) protein kinase, which is one of the key regulators of tau phosphorylation levels. This effect is accompanied by the normalization of the Futsch protein distribution impaired by shaggy overexpression. The results presented in this article demonstrate that multidirectional changes in tau expression can lead to effects that depend on the sex of individuals and the expression level of GSK3., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Maintenance of stemness is tightly linked to cell cycle regulation through protein phosphorylation by cyclin-dependent kinases (CDKs). However, how this process is reversed during differentiation is unknown. We report here that exit from stemness and differentiation of pluripotent cells along the neural lineage are controlled by CDC14, a CDK-counteracting phosphatase whose function in mammals remains obscure. Lack of the two CDC14 family members, CDC14A and CDC14B, results in deficient development of the neural system in the mouse and impairs neural differentiation from embryonic stem cells (ESCs). Mechanistically, CDC14 directly dephosphorylates specific proline-directed Ser/Thr residues of undifferentiated embryonic transcription Factor 1 (UTF1) during the exit from stemness, triggering its proteasome-dependent degradation. Multiomic single-cell analysis of transcription and chromatin accessibility in differentiating ESCs suggests that increased UTF1 levels in the absence of CDC14 prevent the proper firing of bivalent promoters required for differentiation. CDC14 phosphatases are dispensable for mitotic exit, suggesting that CDC14 phosphatases have evolved to control stemness rather than cell cycle exit and establish the CDK-CDC14 axis as a critical molecular switch for linking cell cycle regulation and self-renewal., (© 2022 The Authors.)

The transcription factor FoxO1 (forkhead box O1) regulates genes that are involved in development, metabolism, cellular innovation, longevity, and stress responses. Assessment of FoxO1 activity is therefore critical to understand the regulatory network of this transcription factor. FoxO1 transactivation activity relies on its ability to bind to the promoters of target genes, which is controlled by posttranslational modifications (e.g., dephosphorylation or phosphorylation) that may promote nuclear translocation or exclusion of FoxO1. In this chapter we describe the protocols for FoxO1 activity assessment using Western blotting analysis of the posttranslational modification of FoxO1 in whole cell lysates and ELISA of DNA binding activity of FoxO1 in nuclear extracts., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Protein kinase C epsilon (PKCε) belongs to a family of serine/threonine kinases that control cell proliferation, differentiation and survival. Aberrant PKCε activation and overexpression is a frequent feature of numerous cancers. However, its role in regulation of lipid metabolism in cancer cells remains elusive. Here we report a novel function of PKCε in regulating of prostate cancer cell proliferation by modulation of PKM2-mediated de novo lipogenesis. We show that PKCε promotes de novo lipogenesis and tumor cell proliferation via upregulation of lipogenic enzymes and lipid contents in prostate cancer cells. Mechanistically, PKCε interacts with NABD (1-388) domain of C-terminal deletion on pyruvate kinase isoform M2 (PKM2) and enhances the Tyr105 phosphorylation of PKM2, leading to its nuclear localization. Moreover, forced expression of mutant Tyr105 (Y105F) or PKM2 inhibition suppressed de novo lipogenesis and cell proliferation induced by overexpression of PKCε in prostate cancer cells. In a murine tumor model, inhibitor of PKM2 antagonizes lipogenic enzymes expression and prostate cancer growth induced by overexpression of PKCε in vivo. These data indicate that PKCε is a critical regulator of de novo lipogenesis, which may represent a potential therapeutic target for the treatment of prostate cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The two members of the UBASH3/TULA/STS-protein family have been shown to critically regulate cellular processes in multiple biological systems. The regulatory function of TULA-2 (also known as UBASH3B or STS-1) in platelets is one of the best examples of the involvement of UBASH3/TULA/STS proteins in cellular regulation. TULA-2 negatively regulates platelet signaling mediated by ITAM- and hemITAM-containing membrane receptors that are dependent on the protein tyrosine kinase Syk, which currently represents the best-known dephosphorylation target of TULA-2. The biological responses of platelets to collagen and other physiological agonists are significantly downregulated as a result. The protein structure, enzymatic activity and regulatory functions of UBASH3/TULA/STS proteins in the context of platelet responses and their regulation are discussed in this review.

During meiotic maturation, accurate progression of meiosis is ensured by multiple protein kinases and by signal transduction pathways they are involved in. However, the mechanisms regulating the functions of phosphorylated proteins are unclear. Herein, we investigated the role of Pin1, a peptidyl-prolyl cis-trans isomerase family member that regulates protein functions by altering the structure of the peptide bond of proline in phosphorylated proteins in meiosis. First, we analyzed changes in the expression of Pin1 during meiotic maturation and found that although its levels were constant, its localization was dynamic in different stages of meiosis. Furthermore, we confirmed that the spindle rotates near the cortex when Pin1 is inhibited by juglone during meiotic maturation, resulting in an error in the extrusion of the first polar body. In Pin1 -/- mice, frequent polar body extrusion errors were observed in ovulation, providing insights into the mechanism underlying the errors in the extrusion of the polar body. Although multiple factors and mechanisms might be involved, Pin1 functions in meiosis progression via actin- and microtubule-associated phosphorylated protein targets. Our results show that functional regulation of Pin1 is indispensable in oocyte production and should be considered while developing oocyte culture technologies for reproductive medicine and animal breeding.

β-arrestins mediate regulatory processes for over 800 different G protein-coupled receptors (GPCRs) by adopting specific conformations that result from the geometry of the GPCR-β-arrestin complex. However, whether β-arrestin1 and 2 respond differently for binding to the same GPCR is still unknown. Employing GRK knockout cells and β-arrestins lacking the finger-loop-region, we show that the two isoforms prefer to associate with the active parathyroid hormone 1 receptor (PTH1R) in different complex configurations ("hanging" and "core"). Furthermore, the utilisation of advanced NanoLuc/FlAsH-based biosensors reveals distinct conformational signatures of β-arrestin1 and 2 when bound to active PTH1R (P-R*). Moreover, we assess β-arrestin conformational changes that are induced specifically by proximal and distal C-terminal phosphorylation and in the absence of GPCR kinases (GRKs) (R*). Here, we show differences between conformational changes that are induced by P-R* or R* receptor states and further disclose the impact of site-specific GPCR phosphorylation on arrestin-coupling and function., (© 2022. The Author(s).)

The synthetase 3β-hydroxysterol-Δ24 reductase (DHCR24) is a key regulator involved in cholesterol synthesis and homeostasis. A growing body of evidence indicates that DHCR24 is downregulated in the brain of various models of Alzheimer's disease (AD), such as astrocytes isolated from AD mice. For the past decades, astrocytic tau pathology has been found in AD patients, while the origin of phosphorylated tau in astrocytes remains unknown. A previous study suggests that downregulation of DHCR24 is associated with neuronal tau hyperphosphorylation. Herein, the present study is to explore whether DHCR24 deficiency can also affect tau phosphorylation in astrocytes. Here, we showed that DHCR24 knockdown could induce tau hyperphosphorylation at Thr181, Ser199, Thr231, Ser262, and Ser396 sites in C8D1A astrocytes. Meanwhile, we found that DHCR24-silencing cells had reduced the level of free cholesterol in the plasma membrane and intracellular organelles, as well as cholesterol esters. Furthermore, reduced cellular cholesterol level caused a decreased level of the caveolae-associated protein, cavin1, which disrupted lipid rafts/caveolae and activated rafts/caveolae-dependent Ras/MEK/ERK signaling pathway. In contrast, overexpression of DHCR24 prevented the overactivation of Ras/MEK/ERK signaling by increasing cellular cholesterol content, therefore decreasing tau hyperphosphorylation in C8D1A astrocytes. Herein, we firstly found that DHCR24 knockdown can lead to tau hyperphosphorylation in the astrocyte itself by activating lipid raft-dependent Ras/MEK/ERK signaling, which might contribute to the pathogenesis of AD and other degenerative tauopathies., (© 2022. The Author(s).)

G protein-coupled receptor (GPCR) kinases (GRKs) and arrestins mediate GPCR desensitization, internalization, and signaling. The spatial pattern of GPCR phosphorylation is predicted to trigger these discrete GRK and arrestin-mediated functions. Here, we provide evidence that distal carboxyl-terminal tail (C-tail), but not proximal, phosphorylation of the chemokine receptor CXCR4 specifies βarrestin1 (βarr1)-dependent signaling. We demonstrate by pharmacologic inhibition of GRK2/3-mediated phosphorylation of the chemokine receptor CXCR4 coupled with site-directed mutagenesis and bioluminescence resonance energy transfer approaches that distal, not proximal, C-tail phosphorylation sites are required for recruitment of the adaptor protein STAM1 (signal-transducing adaptor molecule) to βarr1 and focal adhesion kinase phosphorylation but not extracellular signal-regulated kinase 1/2 phosphorylation. In addition, we show that GPCRs that have similarly positioned C-tail phosphoresidues are also able to recruit STAM1 to βarr1. However, although necessary for some GPCRs, we found that distal C-tail sites might not be sufficient to specify recruitment of STAM1 to βarr1 for other GPCRs. In conclusion, this study provides evidence that distal C-tail phosphorylation sites specify GRK-βarrestin-mediated signaling by CXCR4 and other GPCRs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Arrestins were first discovered as suppressors of G protein-mediated signaling by G protein-coupled receptors. It was later demonstrated that arrestins also initiate several signaling branches, including mitogen-activated protein kinase cascades. Arrestin-3-dependent activation of the JNK family can be recapitulated with peptide fragments, which are monofunctional elements distilled from this multi-functional arrestin protein. Here, we use maltose-binding protein fusions of arrestin-3-derived peptides to identify arrestin elements that bind kinases of the ASK1-MKK4/7-JNK3 cascade and the shortest peptide facilitating JNK signaling. We identified a 16-residue arrestin-3-derived peptide expressed as a Venus fusion that leads to activation of JNK3α2 in cells. The strength of the binding to the kinases does not correlate with peptide activity. The ASK1-MKK4/7-JNK3 cascade has been implicated in neuronal apoptosis. While inhibitors of MAP kinases exist, short peptides are the first small molecule tools that can activate MAP kinases.

AMP-activated protein kinase (AMPK) is a conserved energy sensor that plays roles in diverse biological processes via phosphorylating various substrates. Emerging studies have demonstrated the regulatory roles of AMPK in DNA repair, but the underlying mechanisms remain to be fully understood. Herein, using mass spectrometry-based proteomic technologies, we systematically investigate the regulatory network of AMPK in DNA damage response (DDR). Our system-wide phosphoproteome study uncovers a variety of newly-identified potential substrates involved in diverse biological processes, whereas our system-wide histone modification analysis reveals a link between AMPK and histone acetylation. Together with these findings, we discover that AMPK promotes apoptosis by phosphorylating apoptosis-stimulating of p53 protein 2 (ASPP2) in an irradiation (IR)-dependent manner and regulates histone acetylation by phosphorylating histone deacetylase 9 (HDAC9) in an IR-independent manner. Besides, we reveal that disrupting the histone acetylation by the bromodomain BRD4 inhibitor JQ-1 enhances the sensitivity of AMPK-deficient cells to IR. Therefore, our study has provided a resource to investigate the interplay between phosphorylation and histone acetylation underlying the regulatory network of AMPK, which could be beneficial to understand the exact role of AMPK in DDR., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Prostate cancer ranks among the most commonly diagnosed malignancies for men and has become a non-negligible threat for public health. Interplay between inflammatory factors and cancer cells renders inflammatory tissue environment as a predisposing condition for cancer development. The Hippo pathway is a conserved signaling pathway across multiple species during evolution that regulates tissue homeostasis and organ development. Nevertheless, whether Hippo pathway regulates cancer-related inflammatory factors remains elusive. Here, we show that high cell density-mediated activation of the Hippo pathway blunts STAT3 activity in prostate cancer cells. Hippo pathway component MST2 kinase phosphorylates STAT3 at T622, which is located in the SH2 domain of STAT3. This phosphorylation blocks the SH2 domain in one STAT3 molecule to bind with the phosphorylated Y705 site in another STAT3 molecule, which further counteracts IL6-induced STAT3 dimerization and activation. Expression of a nonphosphorylatable STAT3 T622A mutant enhances STAT3 activity and IL6 expression at high cell density and promotes tumor growth in a mice xenograft model. Our findings demonstrate that STAT3 is a novel phosphorylation substrate for MST2 and thereby highlight a regulatory cascade underlying the crosstalk between inflammation and the Hippo pathway in prostate cancer cells., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Emerging evidence suggests that G protein-coupled receptor (GPCR) kinases (GRKs) are associated with the pathophysiology of Alzheimer's disease (AD). However, GRKs have not been directly implicated in regulation of the amyloid-β (Aβ) pathogenic cascade in AD. Here, we determine that GRKs phosphorylate a non-canonical substrate, anterior pharynx-defective 1A (APH1A), an integral component of the γ-secretase complex. Significantly, we show that GRKs generate distinct phosphorylation barcodes in intracellular loop 2 (ICL2) and the C terminus of APH1A, which differentially regulate recruitment of the scaffolding protein β-arrestin 2 (βarr2) to APH1A and γ-secretase-mediated Aβ generation. Further molecular dynamics simulation studies reveal an interaction between the βarr2 finger loop domain and ICL2 and ICL3 of APH1A, similar to a GPCR-β-arrestin complex, which regulates γ-secretase activity. Collectively, these studies provide insight into the molecular and structural determinants of the APH1A-βarr2 interaction that critically regulate Aβ generation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Checkpoint kinase 2 (CHK2) plays an important role in safeguarding the mitotic progression, specifically the spindle assembly, though the mechanism of regulation remains poorly understood. Here, we identified a novel mitotic phosphorylation site on CHK2 Tyr156, and its responsible kinase JAK2. Expression of a phospho-deficient mutant CHK2 Y156F or treatment with JAK2 inhibitor IV compromised mitotic spindle assembly, leading to genome instability. In contrast, a phospho-mimicking mutant CHK2 Y156E restored mitotic normalcy in JAK2-inhibited cells. Mechanistically, we show that this phosphorylation is required for CHK2 interaction with and phosphorylation of the spindle assembly checkpoint (SAC) kinase Mps1, and failure of which results in impaired Mps1 kinetochore localization and defective SAC. Concordantly, analysis of clinical cancer datasets revealed that deletion of JAK2 is associated with increased genome alteration; and alteration in CHEK2 and JAK2 is linked to preferential deletion or amplification of cancer-related genes. Thus, our findings not only reveal a novel JAK2-CHK2 signaling axis that maintains genome integrity through SAC but also highlight the potential impact on genomic stability with clinical JAK2 inhibition., (© 2022. The Author(s).)

Aberrant cortisol and activation of the glucocorticoid receptor (GR) play an essential role in age-related progression of Alzheimer's disease (AD). However, the GR pathways required for influencing the pathobiology of AD dementia remain unknown. To address this, we studied an early phase of AD-like progression in the well-established APP/PS1 mouse model combined with targeted mutations in the BDNF-dependent GR phosphorylation sites (serines 134/267) using molecular, behavioral and neuroimaging approaches. We found that disrupting GR phosphorylation (S134A/S267A) in mice exacerbated the deleterious effects of the APP/PS1 genotype on mortality, neuroplasticity and cognition, without affecting either amyloid-β deposition or vascular pathology. The dynamics, maturation and retention of task-induced new dendritic spines of cortical excitatory neurons required GR phosphorylation at the BDNF-dependent sites that amyloid-β compromised. Parallel studies in postmortem human prefrontal cortex revealed AD subjects had downregulated BDNF signaling and concomitant upregulated cortisol pathway activation, which correlated with cognitive decline. These results provide key evidence that the loss of neurotrophin-mediated GR phosphorylation pathway promotes the detrimental effects of the brain cortisol response that contributes to the onset and/or progression of AD dementia. These findings have important translational implications as they provide a novel approach to treating AD dementia by identifying drugs that increase GR phosphorylation selectively at the neurotrophic sites to improve memory and cognition., (© 2022. The Author(s).)

Protein phosphorylation is the most frequent post-translational modification (PTM) that plays important regulatory roles in a wide range of biological processes. Phosphorylation mainly occurs on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues, with the phosphorylated Tyr sites accounting for ~1-2% of all phosphorylated residues. Tyr phosphorylation was initially believed to be less common in plants compared to animals; however, recent investigation indicates otherwise. Although they lack typical protein Tyr kinases, plants possess many dual-specificity protein kinases that were implicated in diverse cellular processes by phosphorylating Ser, Thr, and Tyr residues. Analyses of sequenced plant genomes also identified protein Tyr phosphatases and dual-specificity protein phosphatases. Recent studies have revealed important regulatory roles of Tyr phosphorylation in many different aspects of plant growth and development and plant interactions with the environment. This short review summarizes studies that implicated the Tyr phosphorylation in biosynthesis and signaling of plant hormones.