Phosphorylation-deficient Mutant Research Articles

Phosphorylation of annexin A2 at serine 25 is required for endothelin-1 stimulated cell proliferation and AKT activation in melanoma cells.

Endothelin (ET)-1 contributes to melanoma progression via cell proliferation, invasion, and migration. We previously reported that annexin A2 (AnxA2) binds to ET receptors. In this study, we aimed to further investigate role of AnxA2 in melanoma cell proliferation after ET-1 stimulation. AnxA2 knockdown inhibited ET-1-stimulated cell proliferation and AKT activation in SK-MEL28 melanoma cells. ET-1 stimulation phosphorylated serine on AnxA2, and AnxA2 Ser25 phosphorylation-deficient mutant (AnxA2 S25A) cells showed lower ET-1-stimulated cell proliferation and AKT activation than the rescue AnxA2 knockdown (AnxA2 res) and AnxA2 Ser11 phosphorylation-deficient mutant (AnxA2 S11A) cells. Although AnxA2 S25A was localized to the plasma membrane, it exhibited lower colocalization with ET receptors than AnxA2 res and AnxA2 S11A on the plasma membrane. These results suggest that phosphorylation of AnxA2 Ser25 affects the colocalization of AnxA2 and ETRs and plays an important role in cell proliferation and AKT activation in ET-1 stimulated melanoma cells.

MEF2B C-terminal mutations enhance transcriptional activity and stability to drive B cell lymphomagenesis

The myocyte enhancer factor 2B (MEF2B) transcription factor is frequently mutated in germinal center (GC)-derived B-cell lymphomas. Its ammino (N)-terminal mutations drive lymphomagenesis by escaping interaction with transcriptional repressors, while the function of carboxy (C)-terminal mutations remains to be elucidated. Here, we show that MEF2B C-tail is physiologically phosphorylated at specific residues and phosphorylation at serine (S)324 is impaired by lymphoma-associated mutations. Lack of phosphorylation at S324 enhances the interaction of MEF2B with the SWI/SNF chromatin remodeling complex, leading to higher transcriptional activity. In addition, these mutants show an increased protein stability due to impaired interaction with the CUL3/KLHL12 ubiquitin complex. Mice expressing a phosphorylation-deficient lymphoma-associated MEF2B mutant display GC enlargement and develop GC-derived lymphomas, when crossed with Bcl2 transgenic mice. These results unveil converging mechanisms of action for a diverse spectrum of MEF2B mutations, all leading to its dysregulation and GC B-cell lymphomagenesis.

Abstract 6981: p38-mediated EZH2 phosphorylation at T367 regulates HER2 pathway in triple negative breast cancer

Abstract Background: Triple negative breast carcinomas (TNBC) are highly aggressive tumors with limited effective treatments. Clinical studies showed that targeting low HER2 levels in TNBC with anti-HER2 agents has survival benefit. Enhancer of Zeste Homolog 2 (EZH2) is a lysine methyltransferase component of the Polycomb Repressive Complex2 that regulates differentiation genes in normal and neoplastic cells. EZH2 phosphorylation at T416 was shown to maintain the TNBC phenotype. We have reported that p38-induced EZH2-T367 phosphorylation enhances metastasis and that high pEZH2-T367 expression is significantly associated with human TNBC tumors compared to other breast cancer subtypes. In this study we tested the hypothesis that pEZH2-T367 may regulate the HER2 pathway in TNBC and that inhibition of pEZH2-T367 may upregulate HER2 signaling, with clinical implications. Methods: MDA-MB-231 (mesenchymal-like TNBC) and CCN6KO cells derived from MMTV-Cre;Ccn6fl/fl (CCN6KO) TNBC mouse model generated in our lab were transduced with vector or EZH2 shRNA knockdown (KD). MDA-MB-231 EZH2 KD cells were rescued with lentivirus containing EZH2-wild-type (WT) or T367A phosphorylation-deficient mutant. Gene and protein expression was analyzed by RNA sequencing, q-RT-PCR, immunoblot and immunohistochemistry. Vector and EZH2-KD rescue MDA-MB-231 cells were treated with anti-HER2 (trastuzumab or pertuzumab 100 ug/ml) followed by Hoestch proliferation assays. TNBC cells were treated with vehicle, EZH2 inhibitor (EPZ-6438, 20uM), p38 inhibitor (p38i, SB202190, 20uM), EPZ+p38i, anti-HER2 (100 ug/ml), and EPZ+p38i+anti-HER2, followed by Hoestch cell proliferation assays. Results: EZH2-T367A deregulates the expression of a 94-gene network of HER2 pathway genes including EREG, SERPINB5, MERTK, IL6R, SOCS3, and FASCN1 compared to EZH2-WT. EZH2-T367A increased protein levels of HER2, EREG, and PP2AB, a major HER2 regulator and was sufficient to sensitize TNBC cells to the anti-proliferative effects of anti-HER2. Supporting the role of pEZH2-T367, we found that the combination of p38i and EPZ significantly sensitizes TNBC cells to anti-HER2 therapies. Conclusions: We demonstrate that T367 phosphorylation mediates the effect of EZH2 on HER2 signaling in TNBC. Our data support a role for pEZH2-T367 in maintaining the HER2-negative phenotype in and suggest that blocking this phosphorylation event may render TNBC tumors amenable to anti-HER2 treatment strategies. Citation Format: Maria E. Gonzalez, Ahmad Eido, Celina G. Kleer. p38-mediated EZH2 phosphorylation at T367 regulates HER2 pathway in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6981.

Sumoylation and phosphorylation of PTEN boosts and curtails autophagy respectively by influencing cell membrane localisation

Autophagy is involved in the entirety of cellular survival, homeostasis and death which becomes more self-evident when its dysregulation is implicated in several pathological conditions. PTEN positively regulates autophagy and like other proteins undergo post-translational modifications. It is crucial to investigate the relationship between PTEN and autophagy as it is generally observed to be negligible in PTEN deficient cancer cells. Here, we have shown that such modifications of PTEN namely sumoylation and phosphorylation upregulates and downregulates autophagy respectively. Transfection of plasmid containing full length PTEN in PTEN-negative prostate cancer cell line PC3, induced autophagy on further starvation. When a sumoylation-deficient mutant of PTEN was transfected and cells were put under similar starvation, a decline in autophagy was observed. On the other hand, cells transfected with phosphorylation-deficient mutant of PTEN showed elevated expression of autophagy. Contrarily, transfection with phosphorylation-mimicking mutant caused reduced expression of autophagy. On further analysis, it was detected that PTEN's association with the plasma membrane was under positive and negative influence from its sumoylation and phosphorylation respectively. This association is integral as it is the foremost site for PTEN to oppose PI3K/AKT pathway and consequently upregulate autophagy. Thus, this study indicates that sumoylation and phosphorylation of PTEN can control autophagy via its cell membrane association.

A phosphorylation-deficient mutant of Sik3, a homolog of Sleepy, alters circadian sleep regulation by PDF neurons in Drosophila.

Sleep behavior has been observed from non-vertebrates to humans. Sleepy mutation in mice resulted in a notable increase in sleep and was identified as an exon-skipping mutation of the salt-inducible kinase 3 (Sik3) gene, conserved among animals. The skipped exon includes a serine residue that is phosphorylated by protein kinase A. Overexpression of a mutant gene with the conversion of this serine into alanine (Sik3-SA) increased sleep in both mice and the fruit fly Drosophila melanogaster. However, the mechanism by which Sik3-SA increases sleep remains unclear. Here, we found that Sik3-SA overexpression in all neurons increased sleep under both light-dark (LD) conditions and constant dark (DD) conditions in Drosophila. Additionally, overexpression of Sik3-SA only in PDF neurons, which are a cluster of clock neurons regulating the circadian rhythm, increased sleep during subjective daytime while decreasing the amplitude of circadian rhythm. Furthermore, suppressing Sik3-SA overexpression specifically in PDF neurons in flies overexpressing Sik3-SA in all neurons reversed the sleep increase during subjective daytime. These results indicate that Sik3-SA alters the circadian function of PDF neurons and leads to an increase in sleep during subjective daytime under constant dark conditions.

MLK3 localizes mainly to the cytoplasm and promotes oxidative stress injury via a positive feedback loop.

Activation of mixed lineage kinase 3 (MLK3) by phosphorylation at Thr277/Ser281 stimulates downstream apoptotic pathways and ultimately leads to cell injury. MLK3 is reported to localize to both the cytoplasm and nucleus in human ovarian cancer cells and immortalized ovarian epithelial cells (T80 and T90 cells), and phosphorylation at Thr477 is required for the cytoplasmic retention of MLK3 in T80 cells. However, the subcellular distribution of MLK3 in other cell types has rarely been reported, and whether phosphorylation of MLK3 at Thr277/Ser281 affects its subcellular distribution is unknown. Here, our bioinformatics analysis predicted that MLK3 was mainly distributed in the cytoplasm and nucleus. In the human HEK293T embryonic kidney cell line and murine HT22 hippocampal neuronal cell line, endogenous MLK3 was more abundant in the cytoplasm and less abundant in the nucleus. In addition, overexpressed Myc-tagged MLK3 and EGFP-tagged MLK3 were also observed to localize mainly to the cytoplasm. MLK3 that was activated by phosphorylation at Thr277/Ser281 was mainly distributed in the cytoplasm, and phosphorylation deficient (T277A/S281A) and mimic (T277E/S281E) mutants both showed distributions similar to that of wild type (wt) MLK3, further proving that phosphorylation at Thr277/Ser281 was not involved in regulating MLK3 subcellular localization. In HEK293T cells, H2O2 stimulation accelerated MLK3 phosphorylation (activation), and this phosphorylation was reduced by the antioxidant N-acetylcysteine in a dose-dependent manner. Overexpressing wt MLK3 promoted the production of intracellular reactive oxygen species and increased cell apoptosis, both of which were enhanced by the phosphorylation-mimic (T277E/S281E) MLK3 variant but not by the phosphorylation-deficient (T277A/S281A) MLK3 variant. These findings provided additional evidence for the cytoplasmic and nuclear distribution of MLK3 in HEK293T cells or HT22 cells and revealed the pivotal role of MLK3 in the positive feedback loop of oxidative stress injury.

LRRK2 phosphorylation status and kinase activity regulate (macro)autophagy in a Rab8a/Rab10-dependent manner

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.

MTORC2–NDRG1–CDC42 axis couples fasting to mitochondrial fission

Fasting triggers diverse physiological adaptations including increases in circulating fatty acids and mitochondrial respiration to facilitate organismal survival. The mechanisms driving mitochondrial adaptations and respiratory sufficiency during fasting remain incompletely understood. Here we show that fasting or lipid availability stimulates mTORC2 activity. Activation of mTORC2 and phosphorylation of its downstream target NDRG1 at serine 336 sustains mitochondrial fission and respiratory sufficiency. Time-lapse imaging shows that NDRG1, but not the phosphorylation-deficient NDRG1Ser336Ala mutant, engages with mitochondria to facilitate fission in control cells, as well as in those lacking DRP1. Using proteomics, a small interfering RNA screen, and epistasis experiments, we show that mTORC2-phosphorylated NDRG1 cooperates with small GTPase CDC42 and effectors and regulators of CDC42 to orchestrate fission. Accordingly, RictorKO, NDRG1Ser336Ala mutants and Cdc42-deficient cells each display mitochondrial phenotypes reminiscent of fission failure. During nutrient surplus, mTOR complexes perform anabolic functions; however, paradoxical reactivation of mTORC2 during fasting unexpectedly drives mitochondrial fission and respiration.

Phosphorylation barcodes direct biased chemokine signaling at CXCR3

G protein-coupled receptor (GPCR)-biased agonism, selective activation of certain signaling pathways relative to others, is thought to be directed by differential GPCR phosphorylation "barcodes." At chemokine receptors, endogenous chemokines can act as "biased agonists", which may contribute to the limited success when pharmacologically targeting these receptors. Here, mass spectrometry-based global phosphoproteomics revealed that CXCR3 chemokines generate different phosphorylation barcodes associated with differential transducer activation. Chemokine stimulation resulted in distinct changes throughout the kinome in global phosphoproteomics studies. Mutation of CXCR3 phosphosites altered β-arrestin 2 conformation in cellular assays and was consistent with conformational changes observed in molecular dynamics simulations. Tcells expressing phosphorylation-deficient CXCR3 mutants resulted in agonist- and receptor-specific chemotactic profiles. Our results demonstrate that CXCR3 chemokines are non-redundant and act as biased agonists through differential encoding of phosphorylation barcodes, leading to distinct physiological processes.

Rhizobia induce SYMRK endocytosis in Phaseolus vulgaris root hair cells

Main conclusionPvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis, which rely on the phosphorylation status of T589, the endocytic YXXØ motif and the kinase activity of the receptor.Legume-rhizobia nodulation is a complex developmental process. It initiates when the rhizobia-produced Nod factors are perceived by specific LysM receptors present in the root hair apical membrane. Consequently, SYMRK (Symbiosis Receptor-like Kinase) becomes active in the root hair and triggers an extensive signaling network essential for the infection process and nodule organogenesis. Despite its relevant functions, the underlying cellular mechanisms involved in SYMRK signaling activity remain poorly characterized. In this study, we demonstrated that PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis. We found that in uninoculated roots, PvSYMRK-EGFP is mainly associated with the plasma membrane, although intracellular puncta labelled with PvSymRK-EGFP were also observed in root hair and nonhair-epidermal cells. Inoculation with Rhizobium etli producing Nod factors induces in the root hair a redistribution of PvSYMRK-EGFP from the plasma membrane to intracellular puncta. In accordance, deletion of the endocytic motif YXXØ (YKTL) and treatment with the endocytosis inhibitors ikarugamycin (IKA) and tyrphostin A23 (TyrA23), as well as brefeldin A (BFA), drastically reduced the density of intracellular PvSYMRK-EGFP puncta. A similar effect was observed in the phosphorylation-deficient (T589A) and kinase-dead (K618E) mutants of PvSYMRK-EGFP, implying these structural features are positive regulators of PvSYMRK-EGFP endocytosis. Our findings lead us to postulate that rhizobia-induced endocytosis of SYMRK modulates the duration and amplitude of the SYMRK-dependent signaling pathway.

Proper phosphorylation of septin 12 regulates septin 4 and soluble adenylyl cyclase expression to induce sperm capacitation.

Septin-based ring complexes maintain the sperm annulus. Defective annular structures are observed in the sperm of Sept12- and Sept4-null mice. In addition, sperm capacitation, a process required for proper fertilization, is inhibited in Sept4-null mice, implying that the sperm annulus might play a role in controlling sperm capacitation. Hence, we analyzed sperm capacitation of sperm obtained from SEPT12 Ser196 phosphomimetic (S196E), phosphorylation-deficient (S196A), and SEPT4-depleted mutant mice. Capacitation was reduced in the sperm of both the Sept12 S196E- and Sept12 S196A-knock-in mice. The protein levels of septins, namely, SEPT4 and SEPT12, were upregulated, and these proteins were concentrated in the sperm annulus during capacitation. Importantly, the expression of soluble adenylyl cyclase (sAC), a key enzyme that initiates capacitation, was upregulated, and sAC was recruited to the sperm annulus following capacitation stimulation. We further found that SEPT12, SEPT4, and sAC formed a complex and colocalized to the sperm annulus. Additionally, sAC expression was reduced and disappeared in the annulus of the SEPT12 S196E- and S196A-mutant mouse sperm. In the sperm of the SEPT4-knockout mice, sAC did not localize to the annulus. Thus, our data demonstrate that SEPT12 phosphorylation status and SEPT4 activity jointly regulate sAC protein levels and annular localization to induce sperm capacitation.

Phosphorylation of TGIF2 represents a therapeutic target that drives EMT and metastasis of lung adenocarcinoma

BackgroundTGF-β-induced factor homeobox 2 (TGIF2) is a transcription regulator that is phosphorylated by EGFR/ERK signaling. However, the functions of phosphorylated (p)-TGIF2 in cancer are largely unknown. Here, we investigated the roles of p-TGIF2 in promoting epithelial–mesenchymal transition (EMT) and metastasis in lung adenocarcinoma (LUAD).MethodsIn vitro and in vivo experiments were conducted to investigate the role of TGIF2 in LUAD EMT and metastasis. Dual-luciferase reporter and ChIP assays were employed to observe the direct transcriptional regulation of E-cadherin by TGIF2 and HDAC1. Co-immunoprecipitation was performed to identify the interaction between TGIF2 and HDAC1.ResultsDownregulating the expression of TGIF2 inhibited LUAD cell migration, EMT and metastasis in vitro and in vivo. Phosphorylation of TGIF2 by EGFR/ERK signaling was required for TGIF2-promoted LUAD EMT and metastasis since phosphorylation-deficient TGIF2 mutant lost these functions. Phosphorylation of TGIF2 was necessary to recruit HDAC1 to the E-cadherin promoter sequence and subsequently suppress E-cadherin transcription. Meanwhile, inhibition of HDAC1 repressed the TGIF2 phosphorylation-induced migration and EMT of LUAD cells. In xenograft mouse models, both inhibition of ERK and HDAC1 could significantly inhibited TGIF2-enhanced metastasis. Furthermore, TGIF2-positive staining was significantly correlated with E-cadherin-negative staining in human lung cancer specimens.ConclusionsOur study reveals the novel function of p-TGIF2 in promoting EMT and metastasis in LUAD; p-TGIF2 could be a potential therapeutic target to inhibit LUAD metastasis.

Biomolecular condensate assembly of nArgBP2 tunes its functionality to manifest the structural plasticity of dendritic spines

nArgBP2, a candidate gene for intellectual disability, is a postsynaptic protein critical for dendritic spine development and morphogenesis, and its knockdown (KD) in developing neurons severely impairs spine-bearing excitatory synapse formation. Surprisingly, nArgBP2 KD in mature neurons did not cause morphological defects in the existing spines at rest, raising questions of how it functions in mature neurons. We found that unlike its inaction at rest, nArgBP2 KD completely inhibited the enlargement of dendritic spines during chemically induced long-term potentiation (cLTP) in mature neurons. We further found that nArgBP2 forms condensates in dendritic spines and that these condensates are dispersed by cLTP, which spatiotemporally coincides with spine head enlargement. Condensates with CaMKII phosphorylation-deficient mutant or CaMKII inhibition are neither dispersed nor accompanied by spine enlargement during cLTP. We found that nArgBP2 condensates in spines exhibited liquid-like properties, and in heterologous and in vitro expression systems, nArgBP2 undergoes liquid-liquid phase separation via multivalent intermolecular interactions between SH3 domains and proline-rich domains. It also forms coacervates with CaMKIIα, which is rapidly dissembled by calcium/CaMKIIα-dependent phosphorylation. We further showed that the interaction between nArgBP2 and WAVE1 competes with nArgBP2 phase separation and that blocking the nArgBP2-WAVE1 interaction prevents spine enlargement during cLTP. Together, our results suggest that nArgBP2 at rest is confined to the condensates but is released by CaMKIIα-mediated phosphorylation during synaptic plasticity, which regulates its timely interaction with WAVE1 to induce spine head enlargement in mature neurons.

Impact of ATP-citrate lyase catalytic activity and serine 455 phosphorylation on histone acetylation and inflammatory responses in human monocytic THP-1 cells.

ATP-citrate lyase (ACLY) is a key enzyme provoking metabolic and epigenetic gene regulation. Molecularly, these functions are exerted by the provision of acetyl-coenzyme A, which is then used as a substrate for de novo lipogenesis or as an acetyl-group donor in acetylation reactions. It has been demonstrated that ACLY activity can be positively regulated via phosphorylation at serine 455 by Akt and protein kinase A. Nonetheless, the impact of phosphorylation on ACLY function in human myeloid cells is poorly understood. In this study we reconstituted ACLY knockout human monocytic THP-1 cells with a wild type ACLY as well as catalytically inactive H760A, and phosphorylation-deficient S455A mutants. Using these cell lines, we determined the impact of ACLY activity and phosphorylation on histone acetylation and pro-inflammatory gene expression in response to lipopolysaccharide (LPS). Our results show that ACLY serine 455 phosphorylation does not influence the proper enzymatic function of ACLY, since both, wild type ACLY and phosphorylation-deficient mutant, exhibited increased cell growth and histone acetylation as compared to cells with a loss of ACLY activity. Transcriptome analysis revealed enhanced expression of pro-inflammatory and interferon response genes in ACLY knockout and H760A THP-1 cells under unstimulated or LPS-treated conditions. At the same time, S455A ACLY-expressing cells showed a phenotype very similar to wild type cells. Contrary to ACLY knockout, pharmacological inhibition of ACLY in THP-1 cells or in primary human macrophages does not enhance LPS-triggered pro-inflammatory gene expression. Our data thus suggest that ACLY retains functionality in the absence of Akt/PKA-mediated phosphorylation in human myeloid cells. Furthermore, loss of ACLY activity may elicit long-term adaptive mechanisms, increasing inflammatory responses.

Mitotic phosphorylation inhibits the Golgi mannosidase MAN1A1.

N-glycans are processed mainly in the Golgi, and a well-organized Golgi structure is required for accurate glycosylation. However, during mitosis the Golgi undergoes severe fragmentation. The resulting trafficking block leads to an extended exposure of cargo molecules to Golgi enzymes. It is unclear how cells avoid glycosylation defects during mitosis. In this study, we report that Golgi α-1,2-mannosidase IA (MAN1A1), the first enzyme that cargo proteins encounter once arriving the Golgi, is phosphorylated at serine 12 by CDK1 in mitosis, which attenuates its activity, affects the production of glycan isomers, and reduces its interaction with the subsequent glycosyltransferase, MGAT1. Expression of wild-type MAN1A1, but not its phosphomimetic mutant, rescues the glycosylation defects in mannosidase I-deficient cells, whereas expression of its phosphorylation-deficient mutant in mitosis increases the formation of complex glycans. Our study reveals that glycosylation is regulated by cytosolic signaling during the cell cycle.

Blocking ERK-DAPK1 Axis Attenuates Glutamate Excitotoxicity in Epilepsy.

Glutamate excitotoxicity induces neuronal cell death during epileptic seizures. Death-associated protein kinase 1 (DAPK1) expression is highly increased in the brains of epilepsy patients; however, the underlying mechanisms by which DAPK1 influences neuronal injury and its therapeutic effect on glutamate excitotoxicity have not been determined. We assessed multiple electroencephalograms and seizure grades and performed biochemical and cell death analyses with cellular and animal models. We applied small molecules and peptides and knocked out and mutated genes to evaluate the therapeutic efficacy of kainic acid (KA), an analog of glutamate-induced neuronal damage. KA administration increased DAPK1 activity by promoting its phosphorylation by activated extracellular signal-regulated kinase (ERK). DAPK1 activation increased seizure severity and neuronal cell death in mice. Selective ERK antagonist treatment, DAPK1 gene ablation, and uncoupling of DAPK1 and ERK peptides led to potent anti-seizure and anti-apoptotic effects in vitro and in vivo. Moreover, a DAPK1 phosphorylation-deficient mutant alleviated glutamate-induced neuronal apoptosis. These results provide novel insight into the pathogenesis of epilepsy and indicate that targeting DAPK1 may be a potential therapeutic strategy for treating epilepsy.

STAT3 suppresses the AMPKα/ULK1-dependent induction of autophagy in glioblastoma cells.

Despite advances in molecular characterization, glioblastoma (GBM) remains the most common and lethal brain tumour with high mortality rates in both paediatric and adult patients. The signal transducer and activator of transcription 3 (STAT3) is an important oncogenic driver of GBM. Although STAT3 reportedly plays a role in autophagy of some cells, its role in cancer cell autophagy remains unclear. In this study, we found Serine‐727 and Tyrosine‐705 phosphorylation of STAT3 was constitutive in GBM cell lines. Tyrosine phosphorylation of STAT3 in GBM cells suppresses autophagy, whereas knockout (KO) of STAT3 increases ULK1 gene expression, increases TSC2‐AMPKα‐ULK1 signalling, and increases lysosomal Cathepsin D processing, leading to the stimulation of autophagy. Rescue of STAT3‐KO cells by the enforced expression of wild‐type (WT) STAT3 reverses these pathways and inhibits autophagy. Conversely, expression of Y705F‐ and S727A‐STAT3 phosphorylation deficient mutants in STAT3‐KO cells did not suppress autophagy. Inhibition of ULK1 activity (by treatment with MRT68921) or its expression (by siRNA knockdown) in STAT3‐KO cells inhibits autophagy and sensitizes cells to apoptosis. Taken together, our findings suggest that serine and tyrosine phosphorylation of STAT3 play critical roles in STAT3‐dependent autophagy in GBM, and thus are potential targets to treat GBM.

Phosphorylation barcode ensembles encoded by biased CXCR3 agonists direct non‐redundant chemokine signaling

Chemokine receptors, a group of G protein‐coupled receptors (GPCRs), interact with transducers such as G proteins, β‐arrestins, and GPCR kinases (GRKs). In the chemokine system, many chemokine agonists act as “biased agonists” that preferentially activate distinct signaling effectors when binding to the same receptor, resulting in distinct physiological effects. Although one third of FDA‐approved drugs target GPCRs, there has been limited success in targeting the chemokine system. Currently, there is little evidence that differential receptor phosphorylation, or “phosphorylation barcodes,” direct these biased responses at chemokine receptors. To address this knowledge gap, we used mass spectrometry to demonstrate that chemokines of CXCR3 promote different ensembles of phosphorylation barcodes that are associated with differential activation of G proteins, β‐arrestins and GRKs. Chemokine stimulation also resulted in distinct changes throughout the kinome in global phosphoproteomic studies. Mutation of specific CXCR3 phosphosites altered β‐arrestin conformation and impacted β‐arrestin activation in molecular dynamics simulations. T‐cells expressing phosphorylation‐deficient CXCR3 mutants resulted in distinct agonist‐ and receptor‐specific chemotactic and signaling profiles that were not completely explained by engagement of G proteins, β‐arrestins, and GRKs alone. Our results directly link distinct GPCR phosphorylation patterns with non‐redundant chemokine signaling (Figure 1).

G protein‐coupled receptor kinases regulate &[beta]‐arrestin interactions with the D2 dopamine receptor in an isoform‐specific manner and in the absence of direct receptor phosphorylation

The D2 dopamine receptor (D2R) regulates numerous CNS functions including movement, cognition, emotion, and reward circuitry. Agonist binding to the D2R promotes the activation of G protein‐mediated signaling and the recruitment of β‐arrestin to the receptor. Activated D2Rs are also phosphorylated by members of the G protein‐coupled receptor kinase (GRK) family. GRK‐mediated receptor phosphorylation is canonically thought to promote β‐arrestin recruitment to GPCRs, however, previous research has shown that receptor phosphorylation is not required for β‐arrestin recruitment to the D2R. Thus, the role of GRKs in D2R‐β‐arrestin interactions remains unclear. In the current study, we examined the relationship between GRKs and β‐arrestin recruitment using GRK overexpression and GRK knockout cells engineered using CRISPR‐Cas9 technology. Overexpression of individual GRK isoforms in parental HEK293 cells potentiated D2R‐β‐arrestin interactions to varying degrees depending on the isoform. Complete knockout of all GRK isoforms in HEK293 cells (GRK‐KO) significantly abrogated, but did not eliminate, β‐arrestin recruitment to the D2R. Further, knockout of individual GRK isoforms had variable effects on D2R‐β‐arrestin interactions ranging from no effect to significant inhibition. Expression of individual GRK isoforms in the GRK‐KO cells rescued D2R‐mediated β‐arrestin recruitment to levels seen in the parental HEK293 cells. Overall, in these experiments, GRK2 seemed to exhibit the greatest modulatory effects on D2R‐β‐arrestin interactions. We next evaluated the role of receptor phosphorylation in GRK‐mediated effects using a phosphorylation‐deficient (PO4‐null) D2R mutant (Namkung et. al., JBC 284:34103, 2009). The WT‐D2R and PO4‐null‐D2R exhibited identical levels of agonist‐stimulated β‐arrestin interactions as well as receptor internalization. Notably, inhibition of GRK catalytic activity attenuated agonist‐stimulated β‐arrestin recruitment to both the WT‐D2R and PO4‐null‐D2R, suggesting the existence of an unknown GRK substrate. Upon recruitment to GPCRs, β‐arrestins undergo conformational changes that correlate with their downstream signaling effects. Consequently, we sought to investigate if GRKs play a role in β‐arrestin conformational changes seen upon D2R activation. To probe these phenomena, we used intramolecular fluorescent arsenical hairpin (FlAsH) nanoBRET β‐arrestin biosensors. We found that GRK2 increased β‐arrestin conformational changes induced by the WT‐D2R and PO4‐null‐D2R. Further, activation of the PO4‐null‐D2R induced greater conformational changes in β‐arrestin than with the WT‐D2R, suggesting that receptor phosphorylation does not mediate β‐arrestin activation by this GPCR. Taken together, these data suggest that GRKs function noncanonically to modulate D2R‐β‐arrestin interactions and highlight the need to identify additional GRK substrates to better inform our understanding of GPCR signaling.

The phosphatase CTDSPL2 is phosphorylated in mitosis and a target for restraining tumor growth and motility in pancreatic cancer.

Carboxy-terminal domain (CTD) small phosphatase like 2 (CTDSPL2), also known as SCP4 or HSPC129, is a new member of the small CTD phosphatase (SCP) family and its role in cancers remains unclear. Here, we used a Phos-tag technique to screen a series of phosphatases and identified CTDSPL2 as a mitotic regulator. We demonstrated that CTDSPL2 was phosphorylated at T86, S104, and S134 by cyclin-dependent kinase 1 (CDK1) in mitosis. Depletion of CTDSPL2 led to mitotic defects and prolonged mitosis. Resultantly, CTDSPL2 deletion restrained proliferation, migration, and invasion in pancreatic cancer cells. We further confirmed the dominant negative effects of a phosphorylation-deficient mutant form of CTDSPL2, implying the biological significance of CTDSPL2 mitotic phosphorylation. Moreover, RT2 cell cycle array analysis revealed p21 and p27 as downstream regulators of CTDSPL2, and inhibition of p21 and/or p27 partially rescued the phenotype in CTDSPL2-deficient cell lines. Importantly, both CTDSPL2 depletion and phosphorylation-deficient mutant CTDSPL2 hindered tumor growth in xenograft models. Together, our findings for the first time highlight the novel role of CTDSPL2 in regulating cell mitosis, proliferation and motility in pancreatic cancer and point out the implications of CTDSPL2 in regulating two critical cell cycle participants (p21 and p27), providing an alternative molecular target for pancreatic cancer treatment.