Direct Phosphorylation Research Articles

GSK-3β phosphorylation of DHX33 leads to its ubiquitination mediated protein degradation

DHX33 is a member of DEAD/H box protein family, and is involved in both RNA and DNA metabolism. It plays diverse roles in multiple cellular activities. DHX33 overexpression has been found to promote the development of many human cancers. However, the underlying mechanism to explain its high expression in cancer cells remains incompletely resolved. In this study, with both human cancer cell lines and normal fibroblasts, we found glycogen synthase kinase 3β (GSK-3β) regulates DHX33 protein stability. This is through its direct phosphorylation of DHX33 on T482, which triggers ubiquitination mediate protein degradation. We further identified one of the major ubiquitination sites of DHX33 to be on its N-terminal K94, a critical residue previously found to be important and highly conserved for ATP binding and helicase activity. Our study for the first time reveals an important upstream regulator, GSK-3β, as a critical kinase to phosphorylate DHX33 at the post-translational level leading to its degradation. Moreover, cancer cells have frequent GSK3β deactivation to disrupt this signaling cascade. Therefore, DHX33 is stabilized in many cancer cells as compared to normal cells. Our study unveils an important post-translational regulation of DHX33 in cells and further unveils a novel mechanism for DHX33 overexpression in cancer cells.

Synthetic Optimizations for Gram-Scale Preparation of 1-O-Methyl d-Glycero-α-d-gluco-heptoside 7-Phosphate from d-Glucose.

Heptose phosphates-unique linkers between endotoxic lipid A and O-antigen in the bacterial membrane-are pathogen-associated molecular patterns recognized by the receptors of the innate immune system. Understanding the mechanisms of immune system activation is important for the development of therapeutic agents to combat infectious diseases and overcome antibiotic resistance. However, in practice, it is difficult to obtain a substantial amount of heptose phosphates for biological studies due to the narrow scope of the reported synthetic procedures. We have optimized and developed an inexpensive and convenient synthesis for the first performed gram-scale production of 1-O-methyl d-glycero-α-d-gluco-heptoside 7-phosphate from readily available d-glucose. Scaling up to such amounts of the product, we have increased the efficiency of the synthesis and reduced the number of steps of the classical route through the direct phosphorylation of the O6,O7-unprotected heptose. The refined method could be of practical value for further biological screening of heptose phosphate derivatives.

Synthesis of alkyl-H-phosphinic acid alkyl esters from red phosphorus and alkyl bromides

Alkyl-H-phosphinic acid alkyl esters are synthesized in 65–71% yield via chemoselective reaction of alkyl bromides with available alkyl-H-phosphinic acids (60–65 °C, Et3N). The latter are prepared, in turn, by direct phosphorylation of alkyl bromides with red phosphorus under phase-transfer conditions.

Aspalathin alleviates skeletal muscle insulin resistance and mitochondrial dysfunction.

Natural compounds may bear promising therapeutic benefits against metabolic diseases such as type 2 diabetes mellitus (T2DM), which are characterized by a state of insulin resistance and mitochondrial dysfunction. Here, we examined the cellular mechanisms by which aspalathin, a dihydrochalcone C-glucoside unique to rooibos, may ameliorate palmitate-induced insulin resistance and mitochondrial dysfunction in cultured C2C12 myotubules. This current study demonstrated that aspalathin remains effective in improving glucose uptake in insulin-resistant skeletal muscle cells, supported by the upregulation of insulin-dependent signaling that involves the activation of insulin receptor (IR) and direct phosphorylation of protein kinase B (AKT). Interestingly, aspalathin also improved mitochondrial respiration and function, which was evident by an increased expression of carnitine palmitoyltransferase 1 (Cpt1), fatty acid transport protein 1 (Fatp1), sirtuin 1 (Sirt1), nuclear respiratory factor 1 (Nrf1), and transcription factor A, mitochondrial (Tfam). Importantly, our results showed that aspalathin treatment was effective in ameliorating the devastating outcomes of insulin resistance and mitochondrial dysfunction that are linked with an undesired pro-inflammatory response, by reducing the levels of well-known pro-inflammatory markers such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and protein kinase C-theta (PKC-theta). Thus, beyond improving glucose uptake and insulin signaling, the current study brings a new perspective in the therapeutic benefits of aspalathin in improving mitochondrial respiration and blocking inflammation to attenuate the detrimental effect of palmitate in skeletal muscle cells.

Convergent regulation of CaV1.2 channels by direct phosphorylation and by the small GTPase RAD in the cardiac fight-or-flight response

The L-type calcium currents conducted by the cardiac CaV1.2 calcium channel initiate excitation-contraction coupling and serve as a key regulator of heart rate, rhythm, and force of contraction. CaV1.2 is regulated by β-adrenergic/protein kinase A (PKA)-mediated protein phosphorylation, proteolytic processing, and autoinhibition by its carboxyl-terminal domain (CT). The small guanosine triphosphatase (GTPase) RAD (Ras associated with diabetes) has emerged as a potent inhibitor of CaV1.2, and accumulating evidence suggests a key role for RAD in mediating β-adrenergic/PKA upregulation of channel activity. However, the relative roles of direct phosphorylation of CaV1.2 channels and phosphorylation of RAD in channel regulation remain uncertain. Here, we investigated the hypothesis that these two mechanisms converge to regulate CaV1.2 channels. Both RAD and the proteolytically processed distal CT (dCT) strongly reduced CaV1.2 activity. PKA phosphorylation of RAD and phosphorylation of Ser-1700 in the proximal CT (pCT) synergistically reversed this inhibition and increased CaV1.2 currents. Our findings reveal that the proteolytically processed form of CaV1.2 undergoes convergent regulation by direct phosphorylation of the CT and by phosphorylation of RAD. These parallel regulatory pathways provide a flexible mechanism for upregulation of the activity of CaV1.2 channels in the fight-or-flight response.

Cyclin B3 implements timely vertebrate oocyte arrest for fertilization.

To ensure successful offspring ploidy, vertebrate oocytes must halt the cell cycle in meiosis II until sperm entry. Emi2 is essential to keep oocytes arrested until fertilization. However, how this arrest is implemented exclusively in meiosis II and not prematurely in meiosis I has until now remained enigmatic. Using mouse and frog oocytes, we show here that cyclin B3, an understudied B-type cyclin, is essential to keep Emi2 levels low in meiosis I. Direct phosphorylation of Emi2 at an evolutionarily highly conserved site by Cdk1/cyclin B3 targets Emi2 for degradation. In contrast, Cdk1/cyclin B1 is inefficient in Emi2 phosphorylation, and this provides a molecular explanation for the requirement of different B-type cyclins for oocyte maturation. Cyclin B3 degradation at exit from meiosis I enables Emi2 accumulation and thus timely arrest in meiosis II. Our findings illuminate the evolutionarily conserved mechanisms that control oocyte arrest for fertilization at the correct cell-cycle stage, which is essential for embryo viability.

Effect of caffeine on mitochondrial biogenesis in the skeletal muscle - A narrative review.

Caffeine is one of the most widely used substances as recreational drug for performance-enhancement in sport, underpinned by a strong evidence base. Although the effects of caffeine are widely investigated within the scope of performance physiology, the molecular effects of caffeine within skeletal muscle remain unclear. Evidence from invitro and invivo models suggest that caffeine regulates the glucose metabolism in the skeletal muscle. Moreover, caffeine seems to stimulate CaMKII, PPARδ/β, AMPK and PGC1α, classical markers of exercise-adaptations, including mitochondrial biogenesis and mitochondrial content. This review summarizes evidence to suggest caffeine-effects within skeletal muscle fibers, focusing on the putative role of caffeine on mitochondrial biogenesis to explore whether caffeine supplementation might be a strategy to enhance mitochondrial biogenesis.

Glucose-driven TOR-FIE-PRC2 signalling controls plant development.

Nutrients and energy have emerged as central modulators of developmental programmes in plants and animals1-3. The evolutionarily conserved target of rapamycin (TOR) kinase is a master integrator of nutrient and energy signalling that controls growth. Despite its key regulatory roles in translation, proliferation, metabolism and autophagy2-5, little is known about how TOR shapes developmental transitions and differentiation. Here we show that glucose-activated TOR kinase controls genome-wide histone H3 trimethylation at K27 (H3K27me3) in Arabidopsis thaliana, which regulates cell fate and development6-10. We identify FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), an indispensable component of Polycomb repressive complex 2 (PRC2), which catalyses H3K27me3(refs.6-8,10-12), as a TOR target. Direct phosphorylation by TOR promotes the dynamic translocation of FIE from the cytoplasm to the nucleus. Mutation of the phosphorylation site on FIE abrogates the global H3K27me3 landscape, reprogrammes the transcriptome and disrupts organogenesis in plants. Moreover, glucose-TOR-FIE-PRC2 signalling modulates vernalization-induced floral transition. We propose that this signalling axis serves as a nutritional checkpoint leading to epigenetic silencing of key transcription factor genes that specify stem cell destiny in shoot and root meristems and control leaf, flower and silique patterning, branching and vegetative-to-reproduction transition. Our findings reveal a fundamental mechanism of nutrient signalling in direct epigenome reprogramming, with broad relevance for the developmental control of multicellular organisms.

Engineered kinases as a tool for phosphorylation of selected targets in vivo.

Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate interaction by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. We show the detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. We presume that after careful optimization, the novel approach we describe here can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors.

Glucocorticoids decrease thermogenic capacity and increase triacylglycerol synthesis by glycerokinase activation in the brown adipose tissue of rats.

Although it is well established that glucocorticoids inactivate thermogenesis and promote lipid accumulation in interscapular brown adipose tissue (IBAT), the underlying mechanisms remain unknown. We found that dexamethasone treatment (1 mg/kg) for 7 days in rats decreased the IBAT thermogenic activity, evidenced by its lower responsiveness to noradrenaline injection associated with reduced content of mitochondrial proteins, respiratory chain protein complexes, noradrenaline, and the β3 -adrenergic receptor. In parallel, to understand better how dexamethasone increases IBAT lipid content, we also investigated the activity of the ATP citrate lyase (ACL), a key enzyme of de novo fatty acid synthesis, glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway, and the three glycerol-3-P generating pathways: (1) glycolysis, estimated by 2-deoxyglucose uptake, (2) glyceroneogenesis, evaluated by phosphoenolpyruvate carboxykinase activity and pyruvate incorporation into triacylglycerol-glycerol, and (3) direct phosphorylation of glycerol, investigated by the content and activity of glycerokinase. Dexamethasone increased the mass and the lipid content of IBAT as well as plasma levels of glucose, insulin, non-esterified fatty acid, and glycerol. Furthermore, dexamethasone increased ACL and G6PD activities (79% and 48%, respectively). Despite promoting a decrease in the incorporation of U-[14 C]-glycerol into triacylglycerol (~54%), dexamethasone increased the content (~55%) and activity (~41%) of glycerokinase without affecting glucose uptake or glyceroneogenesis. Our data suggest that glucocorticoid administration reduces IBAT thermogenesis through sympathetic inactivation and stimulates glycerokinase activity and content, contributing to increased generation of glycerol-3-P, which is mostly used to esterify fatty acid and increase triacylglycerol content promoting IBAT whitening.

Cooperation of Striatin 3 and MAP4K4 promotes growth and tissue invasion

MAP4K4 is associated with increased motility and reduced proliferation in tumor cells, but the regulation of this dichotomous functionality remained elusive. We find that MAP4K4 interacts with striatin 3 and 4 (STRN3/4) and that STRN3 and MAP4K4 exert opposing functions in Hippo signaling and clonal growth. However, depletion of either STRN3 or MAP4K4 in medulloblastoma cells reduces invasion, and loss of both proteins abrogates tumor cell growth in the cerebellar tissue. Mechanistically, STRN3 couples MAP4K4 to the protein phosphatase 2A, which inactivates growth repressing activities of MAP4K4. In parallel, STRN3 enables growth factor-induced PKCθ activation and direct phosphorylation of VASPS157 by MAP4K4, which both are necessary for efficient cell invasion. VASPS157 directed activity of MAP4K4 and STRN3 requires the CNH domain of MAP4K4, which mediates its interaction with striatins. Thus, STRN3 is a master regulator of MAP4K4 function, and disruption of its cooperation with MAP4K4 reactivates Hippo signaling and represses tissue invasion in medulloblastoma.

Katacine Is a New Ligand of CLEC-2 that Acts as a Platelet Agonist.

Background CLEC-2 is a platelet receptor with an important role in thromboinflammation but a minor role in hemostasis. Two endogenous ligands of CLEC-2 have been identified, the transmembrane protein podoplanin and iron-containing porphyrin hemin, which is formed following hemolysis from red blood cells. Other exogenous ligands such as rhodocytin have contributed to our understanding of the role of CLEC-2. Objectives To identify novel CLEC-2 small-molecule ligands to aid therapeutic targeting of CLEC-2. Methods ALPHA screen technology has been used for the development of a high-throughput screening (HTS) assay recapitulating the podoplanin–CLEC-2 interaction. Light transmission aggregometry was used to evaluate platelet aggregation. Immunoprecipitation and western blot were used to evaluate direct phosphorylation of CLEC-2 and downstream protein phosphorylation. Autodock vina software was used to predict the molecular binding site of katacine and mass spectrometry to determine the polymeric nature of the ligand. Results and Conclusion We developed a CLEC-2–podoplanin interaction assay in a HTS format and screened 5,016 compounds from a European Union-open screen library. We identified katacine, a mixture of polymers of proanthocyanidins, as a novel ligand for CLEC-2 and showed that it induces platelet aggregation and CLEC-2 phosphorylation via Syk and Src kinases. Platelet aggregation induced by katacine is inhibited by the anti-CLEC-2 monoclonal antibody fragment AYP1 F(ab)′2. Katacine is a novel nonprotein ligand of CLEC-2 that could contribute to a better understanding of CLEC-2 activation in human platelets.

CDK5 Mediates Proinflammatory Effects of Microglia through Activated DRP1 Phosphorylation in Rat Model of Intracerebral Hemorrhage.

Introduction Cyclin-dependent kinase-5 (CDK5) is a key kinase involved in brain development and function and recently found to be involved in neuronal and astroglial apoptosis, neural stem/progenitor cell stemness, mitochondrial fission, and synaptic transmission. But the specific mechanism of CDK5-mediated anti-inflammatory remains unclear in ICH. The aim of the present study was to explore the role of CDK5 in mediating microglia activity through activated DRP1 phosphorylation in a rat ICH model. Methods We measured behavioral change after ICH; detected the expression of CDK5 in the rat brain using immunohistochemistry; and measured the protein levels of CDK5, p35, p25, p-histone H1, and p-DRP1 using Western blot analysis. Coimmunoprecipitation analysis indicated interaction of CDK5 and DRP1. Tumor necrosis factor-α, interleukin- (IL-) 1β, and IL-6 levels were measured using enzyme-linked immunosorbent assay (ELISA). Results After ICH, CDK5 protein level and kinase activity increased. Western blot data showed that CDK5 expression increased from 6 h and peaked at 2 d after ICH (p < 0.05), and the expression of p35 was lowest at 12 h, while the expression of p25 peaked at 2 d after ICH. Besides, p-DRP1 expression change follows with CDK5 kinase activity change. Coimmunoprecipitation showed that interaction between CDK5 and DRP1 certainly exists in microglia. Then, knockdown CDK5 or p35 expression by siRNA reduced the expression level of p-DRP1. ELISA data showed that the protein levels of proinflammatory mediators, such as TNF-α, IL-1β, and IL-6, were decreased by knockdown of CDK5. Conclusion CDK5 may regulate DRP1 by direct phosphorylation in microglia and further induce microglia secreting proinflammation factor.

Abstract 2955: SMYD2 regulates chromatin modifier KMT2D in ER+/PIK3CA mutant breast cancer

Abstract Breast cancer leads current cancer diagnoses worldwide, accounting for 1 in 4 cancer cases and 1 in 6 cancer deaths in women. Over 70% of breast tumors are hormone receptor positive and can be treated with endocrine therapy, however tumor recurrence remains a major obstacle. Oncogenic mutations in the catalytic subunit of PI3K occur in up to 40% of ER+/HER2- negative breast tumors, making it a promising therapeutic target. Clinically, PI3K inhibitors have shown limited efficacy as monotherapy due to a number of resistance mechanisms, including an upregulation of ER-dependent signaling. Identification of the ER-PI3K signaling crosstalk led to clinical trials which resulted in the approval of the PI3K inhibitor alpelisib in combination with fulvestrant in patients with hormone-refractory ER+/PIK3CA-mutant breast cancer. Further work uncovered a key role for the chromatin modifier KMT2D in regulating ER-dependent transcription upon PI3K inhibition. PI3K effectors AKT1/SGK1 negatively regulate KMT2D through direct S1331 phosphorylation. When PI3K is inhibited, this downstream regulatory event is lost, leading to increased chromatin recruitment of KMT2D and an upregulation of ER-dependent transcription. We have identified a novel methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. Knockdown of SMYD2 sensitizes ER+/PIK3CA mutant breast cancer cell lines to PI3K inhibition. The combination of SMYD2 knockdown and alpelisib leads to a significant reduction in tumor growth compared to alpelisib alone in a xenograft model. Mechanistically, SMYD2 knockdown is able to attenuate alpelisib-induced KMT2D chromatin binding globally and at ER loci. Accordingly, loss of SMYD2 abrogates alpelisib-induced changes in gene expression, including ER-dependent transcription. Altogether our results demonstrate a role for epigenetic therapy using SMYD2 inhibitors in combination with alpelisib for the treatment of ER+/PIK3CA mutant breast cancer. Citation Format: Ryan Blawski, Mirna Sallaku, Xinyu Guo, Srushti Kittane, Maurizio Scaltriti, Minkui Luo, Eneda Toska. SMYD2 regulates chromatin modifier KMT2D in ER+/PIK3CA mutant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2955.

Abstract 5848: Inhibition of the CRM1 nuclear export protein can treat mutant KRAS tumors through stabilizing the DLC1 tumor suppressor protein

Abstract Mutant KRAS is found in &amp;gt;30% of lung adenocarcinomas, and tumors with mutant KRAS usually have poor outcomes with current therapies. The rationale for the new treatment approach described here for tumors with mutant KRAS arises from the critical antitumor role that we have uncovered for reactivating the DLC1 tumor suppressor protein, which is frequently downregulated in a wide range of human cancers. Although several mechanisms account for DLC1 downregulation, here we have focused on a subset of mutant KRAS lung tumor lines that express wild type DLC1 mRNA but lack detectable DLC1 protein. We have recently reported that cytoplasmic EZH2 protein directly methylates Lysine-678 of DLC1 protein leading to its ubiquitination by CUL-4A and consequently, proteasomal degradation. In this study, we have found a specific inhibitor of CRM1 protein (Chromosomal Region Maintenance 1, also known as Exportin 1 [XPO1]) substantially reduces cytoplasmic EZH2 level in several mutant KRAS lung cancer cell lines demonstrating that cytoplasmic EZH2 depends on its nuclear export by CRM1. CRM1, which is the major mammalian nuclear export regulator, is frequently overexpressed and/or mutated in human cancer, including lung adenocarcinomas, and these phenotypes are associated with poor prognosis. Endogenous CRM1 and EZH2 protein colocalized and formed a complex in several lung cancer lines and patient-derived tumor xenografts implying the cancer relevance of this interaction. In addition to blocking the cytoplasmic export of EZH2, pharmacologic inhibition of CRM1 increased the steady-state level of DLC1 protein in several mutant KRAS lung cancer lines and patient-derived xenografts. The tumor suppressor activity of the post-translationally stabilized DLC1 protein was further increased by kinase inhibitors of AKT and SRC, which reverse the direct phosphorylation and attenuation of DLC1 tumor suppressor functions by these kinases. The three-drug combination of inhibitors of CRM1 (Selinexor), AKT (MK-2206), and SRC (Saracatinib), which is well-tolerated in mice, induced potent inhibition of tumor xenograft growth and was associated with induction of markers of apoptosis and senescence. Although each of these inhibitors has multiple targets, CRISPR/Cas9 knockout of DLC1 abolished most of the tumor growth inhibitory activity of the three-drug combination, strongly suggesting that DLC1 is the predominant target for the antitumor activity of the drug combination. Interestingly, KRAS siRNA knockdown phenocopied CRM1 inhibition, as it reduced cytoplasmic EZH2, DLC1 methylation, and DLC1 degradation. Our findings suggest CRM1 inhibitors, when given in conjunction with AKT and/or SRC kinase inhibitors, may have clinical efficacy against lung tumors with mutant KRAS. Citation Format: Brajendra K. Tripathi, Nicole Hirsh, Xiaolan Qian, Dunrui Wang, Marian E. Durkin, Fernando J. de Miguel, Katerina Politi, James H. Doroshow, Douglas R. Lowy. Inhibition of the CRM1 nuclear export protein can treat mutant KRAS tumors through stabilizing the DLC1 tumor suppressor protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5848.

Serine 663-dependent phosphorylation of SERCA2 in ischemic heart disease: Regulation of calcium homeostasis, cell death and myocardial infarct size

Ischemic heart disease is one of the leading cause of death and disability worldwide. Calcium handling in the post-ischemic myocardium is a prime target to treat patients with acute myocardial infarction. Although SERCA2 is a promising target for the treatment of Ca2+ dyshomeostasis and contractile dysfunction following ischemia-reperfusion, the mechanisms through which SERCA2 is regulated remains still unclear. Post-translational modifications of SERCA2 have been recently shown to modulate its activity, however specific phosphorylation sites of SERCA2 have not yet been identified. We hypothesized that a direct phosphorylation of SERCA2 controls SERCA2 activity, Ca2+ homeostasis and subsequent cell death in heart. We combined in silico analysis with in vitro and genetic manipulations to determine potential SERCA2 phosphorylation sites and their effect on Ca2+ homeostasis and cell death in heart. Importantly, we developed an in vivo AAV-myocardial rescue strategy to assess the effect of the SERCA2 phosphorylation event on infarct size and we investigated the potential application of our finding in patient hearts. Our results show for the first time that SERCA2 is a potential substrate for the enzyme GSK3β, whose interaction is improved during an ischemic stress, associated with an increase of the phosphorylation level of SERCA2 at S663 in human hearts. Using a CRISPR/Cas9 approaches, we demonstrated that when SERCA2 cannot be phosphorylated at serine 663, the activity of SERCA2 is significantly increased. As a consequence, we provided evidences that when SERCA2 is resistant to serine-663 phosphorylation, cells are protected cells against reperfusion injury, through a cellular detoxification of calcium into reticulum lumen. As in vivo proof of concept, using AAV-myocardial strategy, phosphoresistant S663 mutant showed an improved excitation-contraction coupling and developed significantly smaller infarct as compared to WT, whereas infarct size was drastically increased in phosphomimetic S663 rescued mice. We conclude that the increased phosphorylation of SERCA2 at serine 663 is a contemporary event of cardiovascular diseases in human hearts, and that avoiding the phosphorylation of SERCA2 at S663 offers a novel therapeutic strategy against acute myocardial infarction, through detoxification of cellular Ca2+ excess into reticulum lumen.

G protein‐coupled receptor kinases regulate &amp;[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.

Disruption of β-catenin-mediated negative feedback reinforces cAMP-induced neuronal differentiation in glioma stem cells

Accumulating evidence supports the existence of glioma stem cells (GSCs) and their critical role in the resistance to conventional treatments for glioblastoma multiforme (GBM). Differentiation therapy represents a promising alternative strategy against GBM by forcing GSCs to exit the cell cycle and reach terminal differentiation. In this study, we demonstrated that cAMP triggered neuronal differentiation and compromised the self-renewal capacity in GSCs. In addition, cAMP induced negative feedback to antagonize the differentiation process by activating β-catenin pathway. Suppression of β-catenin signaling synergized with cAMP activators to eliminate GSCs in vitro and extended the survival of animals in vivo. The cAMP/PKA pathway stabilized β-catenin through direct phosphorylation of the molecule and inhibition of GSK-3β. The activated β-catenin translocated into the nucleus and promoted the transcription of APELA and CARD16, which were found to be responsible for the repression of cAMP-induced differentiation in GSCs. Overall, our findings identified a negative feedback mechanism for cAMP-induced differentiation in GSCs and provided potential targets for the reinforcement of differentiation therapy for GBM.

Regulation of Mus81-Eme1 structure-specific endonuclease by Eme1 SUMO-binding and Rad3ATR kinase is essential in the absence of Rqh1BLM helicase.

The Mus81-Eme1 structure-specific endonuclease is crucial for the processing of DNA recombination and late replication intermediates. In fission yeast, stimulation of Mus81-Eme1 in response to DNA damage at the G2/M transition relies on Cdc2CDK1 and DNA damage checkpoint-dependent phosphorylation of Eme1 and is critical for chromosome stability in absence of the Rqh1BLM helicase. Here we identify Rad3ATR checkpoint kinase consensus phosphorylation sites and two SUMO interacting motifs (SIM) within a short N-terminal domain of Eme1 that is required for cell survival in absence of Rqh1BLM. We show that direct phosphorylation of Eme1 by Rad3ATR is essential for catalytic stimulation of Mus81-Eme1. Chk1-mediated phosphorylation also contributes to the stimulation of Mus81-Eme1 when combined with phosphorylation of Eme1 by Rad3ATR. Both Rad3ATR- and Chk1-mediated phosphorylation of Eme1 as well as the SIMs are critical for cell fitness in absence of Rqh1BLM and abrogating bimodal phosphorylation of Eme1 along with mutating the SIMs is incompatible with rqh1Δ cell viability. Our findings unravel an elaborate regulatory network that relies on the poorly structured N-terminal domain of Eme1 and which is essential for the vital functions Mus81-Eme1 fulfills in absence of Rqh1BLM.

The spindle assembly checkpoint and the spatial activation of Polo kinase determine the duration of cell division and prevent tumor formation.

The maintenance of a restricted pool of asymmetrically dividing stem cells is essential for tissue homeostasis. This process requires the control of mitotic progression that ensures the accurate chromosome segregation. In addition, this event is coupled to the asymmetric distribution of cell fate determinants in order to prevent stem cell amplification. How this coupling is regulated remains poorly described. Here, using asymmetrically dividing Drosophila neural stem cells (NSCs), we show that Polo kinase activity levels determine timely Cyclin B degradation and mitotic progression independent of the spindle assembly checkpoint (SAC). This event is mediated by the direct phosphorylation of Polo kinase by Aurora A at spindle poles and Aurora B kinases at centromeres. Furthermore, we show that Aurora A-dependent activation of Polo is the major event that promotes NSC polarization and together with the SAC prevents brain tumor growth. Altogether, our results show that an Aurora/Polo kinase module couples NSC mitotic progression and polarization for tissue homeostasis.