Abstract: Neuronal inclusions of aggregated RNA‐binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS9s nearly uncharged, aggregation‐prone, yeast prion‐like, low sequence‐complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in‐cell phosphorylation sites across FUS LC. We show that both phosphorylation and phosphomimetic variants reduce its aggregation‐prone/prion‐like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self‐interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS‐associated cytotoxicity. Hence, post‐translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.

Abstract: Nature Communications 8: Article number:15308 (2017); Published: 18 May 2017; Updated: 16 June 2017 The original version of this Article contained an error in the spelling of the author Zemin Zhang, which was incorrectly given as Zeming Zhang. This has now been corrected in both the PDF and HTML versions of the Article.

Abstract: We previously reported that Parkinson’s disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.

Abstract: Glycogen Synthase Kinase-3 (GSK3 or GSK-3) is a promiscuous protein kinase and its phosphorylation of its diverse substrates has major influences on many areas of physiology and pathology, including cellular metabolism, lineage commitment and neuroscience. GSK3 was one of the first identified substrates of the heavily studied oncogenic kinase AKT, phosphorylation by which inhibits GSK3 activity via the formation of an autoinhibitory pseudosubstrate sequence. This has led to investigation of the role of GSK3 inhibition as a key component of the cellular responses to growth factors and insulin, which stimulate the class I PI 3-Kinases and in turn AKT activity and GSK3 phosphorylation. GSK3 has been shown to phosphorylate several upstream and downstream components of the PI3K/AKT/mTOR signalling network, including AKT itself, RICTOR, TSC1 and 2, PTEN and IRS1 and 2, with the potential to apply feedback control within the network. However, it has been clear for some time that functionally distinct, insulated pools of GSK3 exist which are regulated independently, so that for some GSK3 substrates such as β-catenin, phosphorylation by GSK3 is not controlled by input from PI3K and AKT. Instead, as almost all GSK3 substrates require a priming phosphorylated residue to be 4 amino acids C-terminal to the Ser/Thr phosphorylated by GSK3, the predominant form of regulation of the activity of GSK3 often appears to be through control over these priming events, specific to individual substrates. Therefore, a major role of GSK3 can be viewed as an amplifier of the electrostatic effects on protein function which are caused by these priming phosphorylation events. Here we discuss these different aspects to GSK3 regulation and function, and the functions of GSK3 as it integrates with signalling through the PI3K-AKT-mTOR signalling axis.

Abstract: As central nodes in cardiomyocyte signaling, nuclear AKT appears to play a cardio-protective role in cardiovascular disease. Here we describe a circular RNA, circ-Amotl1 that is highly expressed in neonatal human cardiac tissue, and potentiates AKT-enhanced cardiomyocyte survival. We hypothesize that circ-Amotl1 binds to PDK1 and AKT1, leading to AKT1 phosphorylation and nuclear translocation. In primary cardiomyocytes, epithelial cells, and endothelial cells, we found that forced circ-Amotl1 expression increased the nuclear fraction of pAKT. We further detected increased nuclear pAKT in circ-Amotl1-treated hearts. In vivo, circ-Amotl1 expression was also found to be protective against Doxorubicin (Dox)-induced cardiomyopathy. Putative PDK1- and AKT1-binding sites were then identified in silico. Blocking oligonucleotides could reverse the effects of exogenous circ-Amotl1. We conclude that circ-Amotl1 physically binds to both PDK1 and AKT1, facilitating the cardio-protective nuclear translocation of pAKT.

Abstract: Inhibitory Smads (I-Smads) have conserved carboxy-terminal MH2 domains but highly divergent amino-terminal regions when compared with receptor-regulated Smads (R-Smads) and common-partner Smads (co-Smads). Smad6 preferentially inhibits Smad signaling initiated by the bone morphogenetic protein (BMP) type I receptors ALK-3 and ALK-6, whereas Smad7 inhibits both transforming growth factor β (TGF-β)- and BMP-induced Smad signaling. I-Smads also regulate some non-Smad signaling pathways. Here, we discuss the vertebrate I-Smads, their roles as inhibitors of Smad activation and regulators of receptor stability, as scaffolds for non-Smad signaling, and their possible roles in the nucleus. We also discuss the posttranslational modification of I-Smads, including phosphorylation, ubiquitylation, acetylation, and methylation.

Abstract: Pyruvate kinase M2 isoform (PKM2) catalyzes the last step of glycolysis and plays an important role in tumor cell proliferation. Recent studies have reported that PKM2 also regulates apoptosis. However, the mechanisms underlying such a role of PKM2 remain elusive. Here we show that PKM2 translocates to mitochondria under oxidative stress. In the mitochondria, PKM2 interacts with and phosphorylates Bcl2 at threonine (T) 69. This phosphorylation prevents the binding of Cul3-based E3 ligase to Bcl2 and subsequent degradation of Bcl2. A chaperone protein, HSP90α1, is required for this function of PKM2. HSP90α1's ATPase activity launches a conformational change of PKM2 and facilitates interaction between PKM2 and Bcl2. Replacement of wild-type Bcl2 with phosphorylation-deficient Bcl2 T69A mutant sensitizes glioma cells to oxidative stress-induced apoptosis and impairs brain tumor formation in an orthotopic xenograft model. Notably, a peptide that is composed of the amino acid residues from 389 to 405 of PKM2, through which PKM2 binds to Bcl2, disrupts PKM2-Bcl2 interaction, promotes Bcl2 degradation and impairs brain tumor growth. In addition, levels of Bcl2 T69 phosphorylation, conformation-altered PKM2 and Bcl2 protein correlate with one another in specimens of human glioblastoma patients. Moreover, levels of Bcl2 T69 phosphorylation and conformation-altered PKM2 correlate with both grades and prognosis of glioma malignancy. Our findings uncover a novel mechanism through which mitochondrial PKM2 phosphorylates Bcl2 and inhibits apoptosis directly, highlight the essential role of PKM2 in ROS adaptation of cancer cells, and implicate HSP90-PKM2-Bcl2 axis as a potential target for therapeutic intervention in glioblastoma.

Abstract: Stimulation of TNFR1 by TNFα can promote three distinct alternative mechanisms of cell death: necroptosis, RIPK1-independent and -dependent apoptosis. How cells decide which way to die is unclear. Here, we report that TNFα-induced phosphorylation of RIPK1 in the intermediate domain by TAK1 plays a key role in regulating this critical decision. Using phospho-Ser321 as a marker, we show that the transient phosphorylation of RIPK1 intermediate domain induced by TNFα leads to RIPK1-independent apoptosis when NF-κB activation is inhibited by cycloheximide. On the other hand, blocking Ser321 phosphorylation promotes RIPK1 activation and its interaction with FADD to mediate RIPK1-dependent apoptosis (RDA). Finally, sustained phosphorylation of RIPK1 intermediate domain at multiple sites by TAK1 promotes its interaction with RIPK3 and necroptosis. Thus, absent, transient and sustained levels of TAK1-mediated RIPK1 phosphorylation may represent distinct states in TNF-RSC to dictate the activation of three alternative cell death mechanisms, RDA, RIPK1-independent apoptosis and necroptosis.

Abstract: NLRP3 is a cytosolic pattern recognition receptor that senses microbes and endogenous danger signals. Upon activation, NLRP3 forms an inflammasome with the adapter ASC, resulting in caspase-1 activation, release of proinflammatory cytokines and cell death. How NLRP3 activation is regulated by transcriptional and posttranslational mechanisms to prevent aberrant activation remains incompletely understood. Here, we identify three conserved phosphorylation sites in NLRP3 and demonstrate that NLRP3 activation is controlled by phosphorylation of its pyrin domain (PYD). Phosphomimetic residues in NLRP3 PYD abrogate inflammasome activation and structural modeling indicates that phosphorylation of the PYD regulates charge-charge interaction between two PYDs that are essential for NLRP3 activation. Phosphatase 2A (PP2A) inhibition or knock-down drastically reduces NLRP3 activation, showing that PP2A can license inflammasome assembly via dephosphorylating NLRP3 PYD. These results propose that the balance between kinases and phosphatases acting on the NLRP3 PYD is critical for NLRP3 activation.

Abstract: MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.

Abstract: Phosphofructokinase 1 (PFK1) plays a critical role in glycolysis; however, its role and regulation in tumorigenesis are not well understood. Here, we demonstrate that PFK1 platelet isoform (PFKP) is the predominant PFK1 isoform in human glioblastoma cells and its expression correlates with total PFK activity. We show that PFKP is overexpressed in human glioblastoma specimens due to an increased stability, which is induced by AKT activation resulting from phosphatase and tensin homologue (PTEN) loss and EGFR-dependent PI3K activation. AKT binds to and phosphorylates PFKP at S386, and this phosphorylation inhibits the binding of TRIM21 E3 ligase to PFKP and the subsequent TRIM21-mediated polyubiquitylation and degradation of PFKP. PFKP S386 phosphorylation increases PFKP expression and promotes aerobic glycolysis, cell proliferation, and brain tumor growth. In addition, S386 phosphorylation in human glioblastoma specimens positively correlates with PFKP expression, AKT S473 phosphorylation, and poor prognosis. These findings underscore the potential role and regulation of PFKP in human glioblastoma development. Phosphofructokinase 1 (PFK1) plays a critical role in glycolysis. Here the authors show that PFK1 platelet isoform is upregulated in Glioblastoma and is required for tumor growth mechanistically, such upregulation is due to an increased stability induced by AKT activation via phosphorylation on residue S386.

Abstract: Tumour cells secrete exosomes that are involved in the remodelling of the tumour-stromal environment and promoting malignancy. The mechanisms governing tumour exosome release, however, remain incompletely understood. Here we show that tumour cell exosomes secretion is controlled by pyruvate kinase type M2 (PKM2), which is upregulated and phosphorylated in tumours. During exosome secretion, phosphorylated PKM2 serves as a protein kinase to phosphorylate synaptosome-associated protein 23 (SNAP-23), which in turn enables the formation of the SNARE complex to allow exosomes release. Direct phosphorylation assay and mass spectrometry confirm that PKM2 phosphorylates SNAP-23 at Ser95. Ectopic expression of non-phosphorylated SNAP-23 mutant (Ser95→Ala95) significantly reduces PKM2-mediated exosomes release whereas expression of selective phosphomimetic SNAP-23 mutants (Ser95→Glu95 but not Ser20→Glu20) rescues the impaired exosomes release induced by PKM2 knockdown. Our findings reveal a non-metabolic function of PKM2, an enzyme associated with tumour cell reliance on aerobic glycolysis, in promoting tumour cell exosome release.

Abstract: The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.

Abstract: Heat shock protein 90 (HSP90) functions as a well-known onco-protein to regulate protein conformation, stability and degradation. Pyruvate kinase M2 (PKM2), a critical regulator of the metabolism, growth and metastasis of cancer cells, has been confirmed to be overexpressed in various human cancer including hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying the oncogenic functions of HSP90 and PKM2 overexpression in HCC remain unknown. The expression of HSP90 and PKM2 in HCC specimens and cells were detected by immunoblotting and immunostaining. The interaction between HSP90 and PKM2 was confirmed by tandem affinity purification, co-immunoprecipitation and Glutathione S transferase (GST)-pulldown assay. In this study, we found that HSP90 could bind to PKM2 and subsequently increased PKM2 abundance in HCC cells. Immunohistochemistry (IHC) staining showed that HSP90 level was positively correlated with PKM2 level in HCC tissues. Mechanistically, HSP90 was found to increase the phosphorylation of PKM2 at Thr-328. Protein kinase glycogen synthase kinase-3β (GSK-3β) formed a protein complex with HSP90 and PKM2, and directly mediated Thr-328 phosphorylation of PKM2 induced by HSP90. Thr-328 phosphorylation was critical for maintaining PKM2 stability and its biological functions in regulating glycolysis, mitochondria respiration, proliferation and apoptosis. Functionally, we found that HSP90 promoted the glycolysis and proliferation and inhibited apoptosis of HCC cells in a PKM2 dependent manner. In vivo experiments disclosed that PKM2 was required for the promoting effects of HSP90 on the growth of HCC cells in mice. Furthermore, we demonstrated that positive expression of HSP90 and PKM2 was correlated with poor clinicopathological features including high alpha fetoprotein (AFP) level, large tumor size, portal vein tumor thrombus (PVTT) and advanced tumor-node-metastasis (TNM) stage. Furthermore, we demonstrated that positive expression of HSP90 and PKM2, and a combination of these proteins could strongly predict the poor prognosis of HCC patients. We suggest that HSP90 potentiates the glycolysis and proliferation, reduces the apoptosis and thus enhances the growth of HCC cells through PKM2.

Abstract: The bioactive lipid, sphingosine 1-phosphate (S1P) is produced by phosphorylation of sphingosine and this is catalysed by two sphingosine kinase isoforms (SK1 and SK2). Here we discuss structural functional aspects of SK1 (which is a dimeric quaternary enzyme) that relate to coordinated coupling of membrane association with phosphorylation of Ser225 in the 'so-called' R-loop, catalytic activity and protein-protein interactions (e.g. TRAF2, PP2A and Gq). S1P formed by SK1 at the plasma-membrane is released from cells via S1P transporters to act on S1P receptors to promote tumorigenesis. We discuss here an additional novel mechanism that can operate between cancer cells and fibroblasts and which involves the release of the S1P receptor, S1P2 in exosomes from breast cancer cells that regulates ERK-1/2 signalling in fibroblasts. This novel mechanism of signalling might provide an explanation for the role of S1P2 in promoting metastasis of cancer cells and which is dependent on the micro-environmental niche.

Abstract: The conversion from estrogen-dependent to estrogen-independent state of ER+ breast cancer cells is the key step to promote resistance to endocrine therapies. Although the crucial role of MAPK/ERK signaling pathway in estrogen-independent breast cancer cell growth is well established, the underlying mechanism is not fully understood. In this study, we profiled lncRNA expression against a focused group of lncRNAs selected from lncRNA database. CRISPR/Cas9 was employed to knockout (KO) linc-RoR in MCF-7 cells, while rescue experiments were carried out to re-express linc-RoR in KO cells. Colony formation and MTT assays were used to examine the role of linc-RoR in estrogen-independent growth and tamoxifen resistance. Western blot and qRT-PCR were used to determine the change of protein and lncRNA levels, respectively. The expression of DUSP7 in clinical specimens was downloaded from Oncomine ( www.oncomine.org ) and the dataset from Kaplan-Meier Plotter ( http://kmplot.com ) was used to analyze the clinical outcomes in relation to DUSP7. We identified that linc-RoR functions as an onco-lncRNA to promote estrogen-independent growth of ER+ breast cancer. Under estrogen deprivation, linc-RoR causes the upregulation of phosphorylated MAPK/ERK pathway which in turn activates ER signaling. Knockout of linc-RoR abrogates estrogen deprivation-induced ERK activation as well as ER phosphorylation, whereas re-expression of linc-RoR restores all above phenotypes. Moreover, we show that the ERK-specific phosphatase Dual Specificity Phosphatase 7 (DUSP7), also known as MKP-X, is involved in linc-RoR KO-induced repression of MAPK/ERK signaling. Interestingly, linc-RoR KO increases the protein stability of DUSP7, resulting in repression of ERK phosphorylation. Clinical data analysis reveal that DUSP7 expression is lower in ER+ breast cancer samples than that in ER- breast cancer. Moreover, downregulation of DUSP7 expression is associated with poor patient survival. Taken together, these results suggest that linc-RoR promotes estrogen-independent growth and activation of MAPK/ERK pathway of breast cancer cells by regulating the ERK-specific phosphatase DUSP7. Thus, this study might help not only in establishing a role for linc-RoR in estrogen-independent and tamoxifen resistance of ER+ breast cancer, but also suggesting a link between linc-RoR and MAPK/ERK pathway.

Abstract: Determining the functional relationship between Tau phosphorylation and aggregation has proven a challenge owing to the multiple potential phosphorylation sites and their clustering in the Tau sequence. We use here in vitro kinase assays combined with NMR spectroscopy as an analytical tool to generate well-characterized phosphorylated Tau samples and show that the combined phosphorylation at the Ser202/Thr205/Ser208 sites, together with absence of phosphorylation at the Ser262 site, yields a Tau sample that readily forms fibers, as observed by thioflavin T fluorescence and electron microscopy. On the basis of conformational analysis of synthetic phosphorylated peptides, we show that aggregation of the samples correlates with destabilization of the turn-like structure defined by phosphorylation of Ser202/Thr205.