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Insulin Receptor Substrate 2 (IRS2) is a signaling adaptor protein for the insulin (IR) and Insulin-like Growth Factor-1 (IGF-1R) receptors. In breast cancer, IRS2 contributes to both the initiation of primary tumor growth and the establishment of secondary metastases through regulation of cancer stem cell (CSC) function and invasion. However, how IRS2 mediates its diverse functions is not well understood. We used CRISPR/Cas9-mediated gene editing to modify endogenous IRS2 to study the expression, localization, and function of this adaptor protein. A cassette containing an auxin-inducible degradation (AID) sequence, 3x-FLAG tag, and mNeon-green was introduced at the N-terminus of the IRS2 protein to provide rapid and reversible control of IRS2 protein degradation and analysis of endogenous IRS2 expression and localization. Live fluorescence imaging of these cells revealed that IRS2 shuttles between the cytoplasm and nucleus in response to growth regulatory signals in a PI3K-dependent manner. Inhibition of nuclear export or deletion of a putative nuclear export sequence in the C-terminal tail promotes nuclear retention of IRS2, implicating nuclear export in the mechanism by which IRS2 intracellular localization is regulated. Moreover, the acute induction of IRS2 degradation reduces tumor cell invasion, demonstrating the potential for therapeutic targeting of this adaptor protein. Our data highlight the value of our model of endogenously tagged IRS2 as a tool to study IRS2 localization and function., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Raf kinase inhibitor protein (RKIP) is a multifunctional modulator of intracellular signal transduction. Although most of its functions have been considered cytosolic, we show here that the localization of RKIP is primarily nuclear in both growing and quiescent Madin-Darby canine kidney epithelial cells and in Cal-51 and BT-20 human breast cancer cells. We have identified a putative bipartite nuclear localization signal (NLS) in RKIP that maps to the surface of the protein surrounding a known regulatory region. Like classical NLS sequences, the putative NLS of RKIP is rich in arginine and lysine residues. Deletion of and point mutations in the putative NLS lead to decreased nuclear localization. Point mutation of all the basic residues in the putative NLS of RKIP particularly strongly reduces nuclear localization. We found consistent results in reexpression experiments with wildtype or mutant RKIP in RKIP-silenced cells. A fusion construct of the putative NLS of RKIP alone to a heterologous reporter protein leads to nuclear localization of the fusion protein, demonstrating that this sequence alone is sufficient for import into the nucleus. We found that RKIP interacts with the nuclear transport factor importin α in BT-20 and MDA-MB-231 human breast cancer cells, suggesting importin-mediated active nuclear translocation. Evaluating the biological function of nuclear localization of RKIP, we found that the presence of the putative NLS is important for the role of RKIP in mitotic checkpoint regulation in MCF-7 human breast cancer cells. Taken together, these findings suggest that a bipartite NLS in RKIP interacts with importin α for active transport of RKIP into the nucleus and that this process may be involved in the regulation of mitotic progression., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is activated by intracellular nutritional sufficiency and extracellular growth signals. It has been reported that mTORC1 acts as a hub that integrates these inputs to orchestrate a number of cellular responses, including translation, nucleotide synthesis, lipid synthesis, and lysosome biogenesis. However, little is known about specific control of mTORC1 signaling downstream of this complex. Here, we demonstrate that Ragulator, a heteropentameric protein complex required for mTORC1 activation in response to amino acids, is critical for inhibiting the nuclear translocation of transcription factor EB (TFEB). We established a unique RAW264.7 clone that lacked Ragulator but retained total mTORC1 activity. In a nutrition-sufficient state, the nuclear translocation of TFEB was markedly enhanced in the clone despite total mTORC1 kinase activity. In addition, as a cellular phenotype, the number of lysosomes was increased by tenfold in the Ragulator-deficient clone compared with that of control cells. These findings indicate that mTORC1 essentially requires the Ragulator complex for regulating the subcellular distribution of TFEB. Our findings also suggest that other scaffold proteins may be associated with mTORC1 for the specific regulation of downstream signaling., 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.)

Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JH receptor [JHR]), a ligand-activated complex of transcription factors consisting of the JH-binding protein methoprene-tolerant (MET) and its partner taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly because of the difficulty of obtaining purified and functional JHR proteins. Here, we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle T. castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were coexpressed in an insect cell line using a baculovirus system. MET-TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex in greater detail. Biochemical analyses and MS confirmed that T. castaneum JHR was a 1:1 heterodimer consisting of MET and Taiman proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the MET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of MET. Our present characterization of the recombinant JHR is an initial step toward understanding JHR structure and function., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The ATR pathway is one of the major DNA damage checkpoints, and Rad17 is a DNA-binding protein that is phosphorylated upon DNA damage by ATR kinase. Rad17 recruits the 9-1-1 complex that mediates the checkpoint activation, and proteasomal degradation of Rad17 is important for recovery from the ATR pathway. Here, we identified several Rad17 mutants deficient in nuclear localization and resistant to proteasomal degradation. The nuclear localization signal was identified in the central basic domain of Rad17. Rad17 Δ230-270 and R240A/L243A mutants that were previously postulated to lack the destruction box, a sequence that is recognized by the ubiquitin ligase/anaphase-promoting complex that mediates degradation of Rad17, also showed cytoplasmic localization. Our data indicate that the nuclear translocation of Rad17 is functionally linked to the proteasomal degradation. The ATP-binding activity of Rad17, but not hydrolysis, is essential for the nuclear translocation, and the ATPase domain orchestrates the nuclear translocation, the proteasomal degradation, as well as the interaction with the 9-1-1 complex. The Rad17 mutant that lacked a nuclear localization signal was proficient in the interaction with the 9-1-1 complex, suggesting cytosolic association of Rad17 and the 9-1-1 complex. Finally, we identified two tandem canonical and noncanonical destruction boxes in the N-terminus of Rad17 as the bona fide destruction box, supporting the role of anaphase-promoting complex in the degradation of Rad17. We propose a model in which Rad17 is activated in the cytoplasm for translocation into the nucleus and continuously degraded in the nucleus even in the absence of exogenous DNA damage., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

A year ago, when I first contemplated writing this article, my intent was to provide a detailed review of the contributions of the diverse community of talented scientists in my lab to the nuclear receptor research field. In the throes of a deadly pandemic, political turmoil, and Black Lives Matter, however, I found myself compelled to tell a more personal story. While I will still cover milestones in our understanding of the intracellular trafficking of the thyroid hormone receptor, now these will be set against the backdrop of my path as a woman in STEM and on being intentionally inclusive. By sharing reflections on my journey, I hope to encourage young investigators to persist in their pursuit of a career in science., Competing Interests: Conflict of interest The author declares that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

The human genome contains vast genetic diversity as naturally occurring coding variants, yet the impact of these variants on protein function and physiology is poorly understood. RGS14 is a multifunctional signaling protein that suppresses synaptic plasticity in dendritic spines of hippocampal neurons. RGS14 also is a nucleocytoplasmic shuttling protein, suggesting that balanced nuclear import/export and dendritic spine localization are essential for RGS14 functions. We identified genetic variants L505R (LR) and R507Q (RQ) located within the nuclear export sequence (NES) of human RGS14. Here we report that RGS14 encoding LR or RQ profoundly impacts protein functions in hippocampal neurons. RGS14 membrane localization is regulated by binding Gαi-GDP, whereas RGS14 nuclear export is regulated by Exportin 1 (XPO1). Remarkably, LR and RQ variants disrupt RGS14 binding to Gαi1-GDP and XPO1, nucleocytoplasmic equilibrium, and capacity to inhibit long-term potentiation (LTP). Variant LR accumulates irreversibly in the nucleus, preventing RGS14 binding to Gαi1, localization to dendritic spines, and inhibitory actions on LTP induction, while variant RQ exhibits a mixed phenotype. When introduced into mice by CRISPR/Cas9, RGS14-LR protein expression was detected predominantly in the nuclei of neurons within hippocampus, central amygdala, piriform cortex, and striatum, brain regions associated with learning and synaptic plasticity. Whereas mice completely lacking RGS14 exhibit enhanced spatial learning, mice carrying variant LR exhibit normal spatial learning, suggesting that RGS14 may have distinct functions in the nucleus independent from those in dendrites and spines. These findings show that naturally occurring genetic variants can profoundly alter normal protein function, impacting physiology in unexpected ways., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3' terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys 382 -p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G 1 -S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (Published in the U.S.A.)

Muscle atrophy is regulated by the balance between protein degradation and synthesis. FOXO1, a transcription factor, helps to determine this balance by activating pro-atrophic gene transcription when present in muscle fiber nuclei. Foxo1 nuclear efflux is promoted by AKT-mediated Foxo1 phosphorylation, eliminating FOXO1's atrophy-promoting effect. AKT activation can be promoted by insulin-like growth factor 1 (IGF1) or insulin via a pathway including IGF1 or insulin, phosphatidylinositol 3-kinase, and AKT. We used confocal fluorescence time-lapse imaging of FOXO1-GFP in adult isolated living muscle fibers maintained in culture to explore the effects of IGF1 and insulin on FOXO1-GFP nuclear efflux with and without pharmacological inhibitors. We observed that although AKT inhibitor blocks the IGF1- or insulin-induced effect on FOXO1 nuclear efflux, phosphatidylinositol 3-kinase inhibitors, which we show to be effective in these fibers, do not. We also found that inhibition of the protein kinase ACK1 or ATM contributes to the suppression of FOXO1 nuclear efflux after IGF1. These results indicate a novel pathway that has been unexplored in the IGF1- or insulin-induced regulation of FOXO1 and present information useful both for therapeutic interventions for muscle atrophy and for further investigative areas into insulin insensitivity and type 2 diabetes., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Russell and Schneider.)

Despite the continued analysis of HDAC inhibitors in clinical trials, the heterogeneous nature of the protein complexes they target limits our understanding of the beneficial and off-target effects associated with their application. Among the many HDAC protein complexes found within the cell, Sin3 complexes are conserved from yeast to humans and likely play important roles as regulators of transcriptional activity. The presence of two Sin3 paralogs in humans, SIN3A and SIN3B, may result in a heterogeneous population of Sin3 complexes and contributes to our poor understanding of the functional attributes of these complexes. Here, we profile the interaction networks of SIN3A and SIN3B to gain insight into complex composition and organization. In accordance with existing data, we show that Sin3 paralog identity influences complex composition. Additionally, chemical cross-linking MS identifies domains that mediate interactions between Sin3 proteins and binding partners. The characterization of rare SIN3B proteoforms provides additional evidence for the existence of conserved and divergent elements within human Sin3 proteins. Together, these findings shed light on both the shared and divergent properties of human Sin3 proteins and highlight the heterogeneous nature of the complexes they organize., Competing Interests: Conflict of interest—Authors declare no competing interests., (© 2020 Adams et al.)

Disorders that disrupt myelin formation during development or in adulthood, such as multiple sclerosis and peripheral neuropathies, lead to severe pathologies, illustrating myelin's crucial role in normal neural functioning. However, although our understanding of glial biology is increasing, the signals that emanate from axons and regulate myelination remain largely unknown. To identify the core components of the myelination process, here we adopted a microarray analysis approach combined with laser-capture microdissection of spinal motoneurons during the myelinogenic phase of development. We identified neuronal genes whose expression was enriched during myelination and further investigated hepatoma-derived growth factor-related protein 3 (HRP3 or HDGFRP3). HRP3 was strongly expressed in the white matter fiber tracts of the peripheral (PNS) and central (CNS) nervous systems during myelination and remyelination in a cuprizone-induced demyelination model. The dynamic localization of HPR3 between axons and nuclei during myelination was consistent with its axonal localization during neuritogenesis. To study this phenomenon, we identified two splice variants encoded by the HRP3 gene: the canonical isoform HRP3-I and a newly recognized isoform, HRP3-II. HRP3-I remained solely in the nucleus, whereas HRP3-II displayed distinct axonal localization both before and during myelination. Interestingly, HRP3-II remained in the nuclei of unmyelinated neurons and glial cells, suggesting the existence of a molecular machinery that transfers it to and retains it in the axons of neurons fated for myelination. Overexpression of HRP3-II, but not of HRP3-I, increased Schwann cell numbers and myelination in PNS neuron-glia co-cultures. However, HRP3-II overexpression in CNS co-cultures did not alter myelination., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

The RNA exosome is a multisubunit protein complex involved in RNA surveillance of all classes of RNA, and is essential for pre-rRNA processing. The exosome is conserved throughout evolution, present in archaea and eukaryotes from yeast to humans, where it localizes to the nucleus and cytoplasm. The catalytically active subunit Rrp44/Dis3 of the exosome in budding yeast ( Saccharomyces cerevisiae ) is considered a protein present in these two subcellular compartments, and here we report that it not only localizes mainly to the nucleus, but is concentrated in the nucleolus, where the early pre-rRNA processing reactions take place. Moreover, we show by confocal microscopy analysis that the core exosome subunits Rrp41 and Rrp43 also localize largely to the nucleus and strongly accumulate in the nucleolus. These results shown here shed additional light on the localization of the yeast exosome and have implications regarding the main function of this RNase complex, which seems to be primarily in early pre-rRNA processing and surveillance., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Okuda et al.)

The transcriptional coactivator YAP1 (yes-associated protein 1) regulates cell proliferation, cell-cell interactions, organ size, and tumorigenesis. Post-transcriptional modifications and nuclear translocation of YAP1 are crucial for its nuclear activity. The objective of this study was to elucidate the mechanism by which the steroid hormone androgen regulates YAP1 nuclear entry and functions in several human prostate cancer cell lines. We demonstrate that androgen exposure suppresses the inactivating post-translational modification phospho-Ser-127 in YAP1, coinciding with increased YAP1 nuclear accumulation and activity. Pharmacological and genetic experiments revealed that intact androgen receptor signaling is necessary for androgen's inactivating effect on phospho-Ser-127 levels and increased YAP1 nuclear entry. We also found that androgen exposure antagonizes Ser/Thr kinase 4 (STK4/MST1) signaling, stimulates the activity of protein phosphatase 2A, and thereby attenuates the phospho-Ser-127 modification and promotes YAP1 nuclear localization. Results from quantitative RT-PCR and CRISPR/Cas9-aided gene knockout experiments indicated that androgen differentially regulates YAP1-dependent gene expression. Furthermore, an unbiased computational analysis of the prostate cancer data from The Cancer Genome Atlas revealed that YAP1 and androgen receptor transcript levels correlate with each other in prostate cancer tissues. These findings indicate that androgen regulates YAP1 nuclear localization and its transcriptional activity through the androgen receptor-STK4/MST1-protein phosphatase 2A axis, which may have important implications for human diseases such as prostate cancer., Competing Interests: Conflict of interest—The authors declare no competing conflicts of interest with the content of this article., (© 2020 Cinar et al.)

Nuclear accumulation of the small phosphoprotein integrin cytoplasmic domain-associated protein-1 (ICAP1) results in recruitment of its binding partner, Krev/Rap1 interaction trapped-1 (KRIT1), to the nucleus. KRIT1 loss is the most common cause of cerebral cavernous malformation, a neurovascular dysplasia resulting in dilated, thin-walled vessels that tend to rupture, increasing the risk for hemorrhagic stroke. KRIT1's nuclear roles are unknown, but it is known to function as a scaffolding or adaptor protein at cell-cell junctions and in the cytosol, supporting normal blood vessel integrity and development. As ICAP1 controls KRIT1 subcellular localization, presumably influencing KRIT1 function, in this work, we investigated the signals that regulate ICAP1 and, hence, KRIT1 nuclear localization. ICAP1 contains a nuclear localization signal within an unstructured, N-terminal region that is rich in serine and threonine residues, several of which are reportedly phosphorylated. Using quantitative microscopy, we revealed that phosphorylation-mimicking substitutions at Ser-10, or to a lesser extent at Ser-25, within this N-terminal region inhibit ICAP1 nuclear accumulation. Conversely, phosphorylation-blocking substitutions at these sites enhanced ICAP1 nuclear accumulation. We further demonstrate that p21-activated kinase 4 (PAK4) can phosphorylate ICAP1 at Ser-10 both in vitro and in cultured cells and that active PAK4 inhibits ICAP1 nuclear accumulation in a Ser-10-dependent manner. Finally, we show that ICAP1 phosphorylation controls nuclear localization of the ICAP1-KRIT1 complex. We conclude that serine phosphorylation within the ICAP1 N-terminal region can prevent nuclear ICAP1 accumulation, providing a mechanism that regulates KRIT1 localization and signaling, potentially influencing vascular development., (© 2020 Su et al.)

Embryonic large molecule derived from yolk sac (ELYS) is a constituent protein of nuclear pores. It initiates assembly of nuclear pore complexes into functional nuclear pores toward the end of mitosis. Using cellular, molecular, and genetic tools, including fluorescence and Electron microscopy, quantitative PCR, and RNAi-mediated depletion, we report here that the ELYS ortholog ( d Elys) plays critical roles during Drosophila development. d Elys localized to the nuclear rim in interphase cells, but during mitosis it was absent from kinetochores and enveloped chromatin. We observed that RNAi-mediated dElys depletion leads to aberrant development and, at the cellular level, to defects in the nuclear pore and nuclear lamina assembly. Further genetic analyses indicated that dElys depletion re-activates the Dorsal (NF-κB) pathway during late larval stages. Re-activated Dorsal caused untimely expression of the Dorsal target genes in the post-embryonic stages. We also demonstrate that activated Dorsal triggers apoptosis during later developmental stages by up-regulating the pro-apoptotic genes reaper and hid The apoptosis induced by Reaper and Hid was probably the underlying cause for developmental abnormalities observed upon dElys depletion. Moreover, we noted that d Elys has conserved structural features, but contains a noncanonical AT-hook-like motif through which it strongly binds to DNA. Together, our results uncover a novel epistatic interaction that regulates Dorsal dynamics by d Elys during development., (© 2020 Mehta et al.)

Naïve CD4 + T cells in the periphery differentiate into regulatory T cells (Tregs) in which Foxp3 is expressed for their suppressive function. NLRP3, a pro-inflammatory molecule, is known to be involved in inflammasome activation associated with several diseases. Recently, the expression of NLRP3 in CD4 + T cells, as well as in myeloid cells, has been described; however, a role of T cell-intrinsic NLRP3 in Treg differentiation remains unknown. Here, we report that NLRP3 impeded the expression of Foxp3 independent of inflammasome activation in Tregs. NLRP3-deficient mice elevate Treg generation in various organs in the de novo pathway. NLRP3 deficiency increased the amount and suppressive activity of Treg populations, whereas NLRP3 overexpression reduced Foxp3 expression and Treg abundance. Importantly, NLRP3 interacted with Kpna2 and translocated to the nucleus from the cytoplasm under Treg-polarizing conditions. Taken together, our results identify a novel role for NLRP3 as a new negative regulator of Treg differentiation, mediated via its interaction with Kpna2 for nuclear translocation., (© 2019 Park et al.)

Multiplexed proteomics has emerged as a powerful tool to measure relative protein expression levels across multiple conditions. The relative protein abundances are inferred by comparing the signals generated by isobaric tags, which encode the samples' origins. Intuitively, the trust associated with a protein measurement depends on the similarity of ratios from the protein's peptides and the signal-strength of these measurements. However, typically the average peptide ratio is reported as the estimate of relative protein abundance, which is only the most likely ratio with a very naive model. Moreover, there is no sense on the confidence in these measurements. Here, we present a mathematically rigorous approach that integrates peptide signal strengths and peptide-measurement agreement into an estimation of the true protein ratio and the associated confidence (BACIQ). The main advantages of BACIQ are: (1) It removes the need to threshold reported peptide signal based on an arbitrary cut-off, thereby reporting more measurements from a given experiment; (2) Confidence can be assigned without replicates; (3) For repeated experiments BACIQ provides confidence intervals for the union, not the intersection, of quantified proteins; (4) For repeated experiments, BACIQ confidence intervals are more predictive than confidence intervals based on protein measurement agreement. To demonstrate the power of BACIQ we reanalyzed previously published data on subcellular protein movement on treatment with an Exportin-1 inhibiting drug. We detect ∼2× more highly significant movers, down to subcellular localization changes of ∼1%. Thus, our method drastically increases the value obtainable from quantitative proteomics experiments, helping researchers to interpret their data and prioritize resources. To make our approach easily accessible we distribute it via a Python/Stan package., (© 2019 Peshkin et al.)

Serine-arginine (SR) proteins are essential splicing factors that promote numerous steps associated with mRNA processing and whose biological function is tightly regulated through multi-site phosphorylation. In the nucleus, the cdc2-like kinases (CLKs) phosphorylate SR proteins on their intrinsically disordered Arg-Ser (RS) domains, mobilizing them from storage speckles to the splicing machinery. The CLKs have disordered N termini that bind tightly to RS domains, enhancing SR protein phosphorylation. The N termini also promote nuclear localization of CLKs, but their transport mechanism is presently unknown. To explore cytoplasmic-nuclear transitions, several classical nuclear localization sequences in the N terminus of the CLK1 isoform were identified, but their mutation had no effect on subcellular localization. Rather, we found that CLK1 amplifies its presence in the nucleus by forming a stable complex with the SR protein substrate and appropriating its NLS for transport. These findings indicate that, along with their well-established roles in mRNA splicing, SR proteins use disordered protein-protein interactions to carry their kinase regulator from the cytoplasm to the nucleus., (© 2019 George et al.)

Heartland virus (HRTV) is a pathogenic phlebovirus recently identified in the United States and related to severe fever with thrombocytopenia syndrome virus (SFTSV) emerging in Asia. We previously reported that SFTSV disrupts host antiviral responses directed by interferons (IFNs) and their downstream regulators, signal transducer and activator of transcription (STAT) proteins. However, whether HRTV infection antagonizes the IFN-STAT signaling axis remains unclear. Here, we show that, similar to SFTSV, HRTV also inhibits IFN-α- and IFN-λ-mediated antiviral responses. As expected, the nonstructural protein (NSs) of HRTV (HNSs) robustly antagonized both type I and III IFN signaling. Protein interaction analyses revealed that a common component downstream of type I and III IFN signaling, STAT2, is the target of HNSs. Of note, the DNA-binding and linker domains of STAT2 were required for an efficient HNSs-STAT2 interaction. Unlike the NSs of SFTSV (SNSs), which blocks both STAT2 and STAT1 nuclear accumulation, HNSs specifically blocked IFN-triggered nuclear translocation only of STAT2. However, upon HRTV infection, IFN-induced nuclear translocation of both STAT2 and STAT1 was suppressed, suggesting that STAT1 is an additional HRTV target for IFN antagonism. Consistently, despite HNSs inhibiting phosphorylation only of STAT2 and not STAT1, HRTV infection diminished both STAT2 and STAT1 phosphorylation. These results suggest that HRTV antagonizes IFN antiviral signaling by dampening both STAT2 and STAT1 activities. We propose that HNSs-specific targeting of STAT2 likely plays an important role but is not all of the "tactics" of HRTV in its immune evasion., (© 2019 Feng et al.)

Costameres are signaling hubs at the sarcolemma and important contact points between the extracellular matrix and cell interior, sensing and transducing biomechanical signals into a cellular response. The transmembrane proteoglycan syndecan-4 localizes to these attachment points and has been shown to be important in the initial stages of cardiac remodeling, but its mechanistic function in the heart remains insufficiently understood. Here, we sought to map the cardiac interactome of syndecan-4 to better understand its function and downstream signaling mechanisms. By combining two different affinity purification methods with MS analysis, we found that the cardiac syndecan-4 interactome consists of 21 novel and 29 previously described interaction partners. Nine of the novel partners were further validated to bind syndecan-4 in HEK293 cells ( i.e. CAVIN1/PTRF, CCT5, CDK9, EIF2S1, EIF4B, MPP7, PARVB, PFKM, and RASIP). We also found that 19 of the 50 interactome partners bind differently to syndecan-4 in the left ventricle lysate from aortic-banded heart failure (ABHF) rats compared with SHAM-operated animals. One of these partners was the well-known mechanotransducer muscle LIM protein (MLP), which showed direct and increased binding to syndecan-4 in ABHF. Nuclear translocation is important in MLP-mediated signaling, and we found less MLP in the nuclear-enriched fractions from syndecan-4 -/- mouse left ventricles but increased nuclear MLP when syndecan-4 was overexpressed in a cardiomyocyte cell line. In the presence of a cell-permeable syndecan-4-MLP disruptor peptide, the nuclear MLP level was reduced. These findings suggest that syndecan-4 mediates nuclear translocation of MLP in the heart., (© 2019 Mathiesen et al.)

Kinetic simulation is a useful approach for elucidating complex cell-signaling systems. The numerical simulations required for kinetic modeling in live cells critically require parameters such as protein concentrations and dissociation constants ( K d ). However, only a limited number of parameters have been measured experimentally in living cells. Here we describe an approach for quantifying the concentration and K d of endogenous proteins at the single-cell level with CRISPR/Cas9-mediated knock-in and fluorescence cross-correlation spectroscopy. First, the mEGFP gene was knocked in at the end of the mitogen-activated protein kinase 1 ( MAPK1 ) gene, encoding extracellular signal-regulated kinase 2 (ERK2), through homology-directed repair or microhomology-mediated end joining. Next, the HaloTag gene was knocked in at the end of the ribosomal S6 kinase 2 ( RSK2 ) gene. We then used fluorescence correlation spectroscopy to measure the protein concentrations of endogenous ERK2-mEGFP and RSK2-HaloTag fusion constructs in living cells, revealing substantial heterogeneities. Moreover, fluorescence cross-correlation spectroscopy analyses revealed temporal changes in the apparent K d values of the binding between ERK2-mEGFP and RSK2-HaloTag in response to epidermal growth factor stimulation. Our approach presented here provides a robust and efficient method for quantifying endogenous protein concentrations and dissociation constants in living cells., (© 2019 Komatsubara et al.)

Glycogen synthase kinase 3β (GSK3β) phosphorylates and thereby regulates a wide range of protein substrates involved in diverse cellular functions. Some GSK3β substrates, such as c-Myc and Snail, are nuclear transcription factors, suggesting the possibility that GSK3β function is controlled through its nuclear localization. Here, using ARPE-19 and MDA-MB-231 human cell lines, we found that inhibition of mTOR complex 1 (mTORC1) leads to partial redistribution of GSK3β from the cytosol to the nucleus and to a GSK3β-dependent reduction of the levels of both c-Myc and Snail. mTORC1 is known to be controlled by metabolic cues, such as by AMP-activated protein kinase (AMPK) or amino acid abundance, and we observed here that AMPK activation or amino acid deprivation promotes GSK3β nuclear localization in an mTORC1-dependent manner. GSK3β was detected on several distinct endomembrane compartments, including lysosomes. Consistently, disruption of late endosomes/lysosomes through a perturbation of RAS oncogene family member 7 (Rab7) resulted in loss of GSK3β from lysosomes and in enhanced GSK3β nuclear localization as well as GSK3β-dependent reduction of c-Myc levels. These findings indicate that the nuclear localization and function of GSK3β is suppressed by mTORC1 and suggest a link between metabolic conditions sensed by mTORC1 and GSK3β-dependent regulation of transcriptional networks controlling cellular biomass production., (© 2018 Bautista et al.)

Adaptations and responses to stress conditions are fundamental processes that all cells must accomplish to maintain or restore cellular homeostasis. Cells have a plethora of response pathways to mitigate the effect of different environmental stressors. The transcriptional regulators transcription factor EB (TFEB) and transcription factor binding to IGHM enhancer 3 (TFE3) play a key role in the control of these stress pathways. Therefore, understanding their regulation under different stress conditions is of great interest. Here, using a range of human and murine cells, we show that TFEB and TFE3 are activated upon induction of acute oxidative stress by sodium arsenite via an mTOR complex 1 (mTORC1)-independent process. We found that the mechanism of arsenite-stimulated TFEB and TFE3 activation instead involves protein phosphatase 2A (PP2A)-mediated dephosphorylation at Ser-211 and Ser-321, respectively. Depletion of either the catalytic (PPP2CA+B) or regulatory (PPP2R2A/B55α) subunits of PP2A, as well as PP2A inactivation with the specific inhibitor okadaic acid, abolished TFEB and TFE3 activation in response to sodium arsenite. Conversely, PP2A activation by ceramide or the sphingosine-like compound FTY720 was sufficient to induce TFE3 nuclear translocation. MS analysis revealed that PP2A dephosphorylates TFEB at several residues, including Ser-109, Ser-114, Ser-122, and Ser-211, thus facilitating TFEB activation. Overall, this work identifies a critical mechanism that activates TFEB and TFE3 without turning off mTORC1 activity. We propose that this mechanism may enable some cell types such as immune or cancer cells that require simultaneous TFEB/TFE3 and mTORC1 signaling to survive and achieve robust cell growth in stressful environments.

Importin 13 is a member of the importin β family of transport receptors. Unlike most family members, importin 13 mediates both, nuclear protein import and export. To search for novel importin 13 cargoes, we used stable isotope labeling of amino acids in cell culture (SILAC) and mass spectrometry. Using stringent criteria, we identified 255 importin 13 substrates, including the known cargoes Ubc9, Mago and eIF1A, and validate many of them as transport cargoes by extensive biochemical and cell biological characterization. Several novel cargoes can also be transported by the export receptor CRM1, demonstrating a clear redundancy in receptor choice. Using importin 13 mutants, we show that many of the novel substrates contact regions on the transport receptor that are not used by Ubc9, Mago or eIF1A. Together, this study significantly expands the repertoire of importin 13 cargoes and sets the basis for a more detailed characterization of this extremely versatile transport receptor., (© 2018 Baade et al.)

The nuclear protein constitutive active/androstane receptor (CAR or NR1I3) regulates several liver functions such as drug and energy metabolism and cell growth or death, which are often involved in the development of diseases such as diabetes and hepatocellular carcinoma. CAR undergoes a conversion from inactive homodimers to active heterodimers with retinoid X receptor α (RXRα), and phosphorylation of the DNA-binding domain (DBD) at Thr-38 in CAR regulates this conversion. Here, we uncovered the molecular mechanism by which this phosphorylation regulates the intramolecular interaction between CAR's DBD and ligand-binding domain (LBD), enabling the homodimer-heterodimer conversion. Phosphomimetic substitution of Thr-38 with Asp increased co-immunoprecipitation of the CAR DBD with CAR LBD in Huh-7 cells. Isothermal titration calorimetry assays also revealed that recombinant CAR DBD-T38D, but not nonphosphorylated CAR DBD, bound the CAR LBD peptide. This DBD-LBD interaction masked CAR's dimer interface, preventing CAR homodimer formation. Of note, EGF signaling weakened the interaction of CAR DBD T38D with CAR LBD, converting CAR to the homodimer form. The DBD-T38D-LBD interaction also prevented CAR from forming a heterodimer with RXRα. However, this interaction opened up a CAR surface, allowing interaction with protein phosphatase 2A. Thr-38 dephosphorylation then dissociated the DBD-LBD interaction, allowing CAR heterodimer formation with RXRα. We conclude that the intramolecular interaction of phosphorylated DBD with the LBD enables CAR to adapt a transient monomer configuration that can be converted to either the inactive homodimer or the active heterodimer.

Human mature IL-33 is a member of the IL-1 family and a potent regulator of immunity through its pro-T helper cell 2 activity. Its precursor form, full-length interleukin-33 (FLIL33), is an intranuclear protein in many cell types, including fibroblasts, and its intracellular levels can change in response to stimuli. However, the mechanisms controlling the nuclear localization of FLIL33 or its stability in cells are not understood. Here, we identified importin-5 (IPO5), a member of the importin family of nuclear transport proteins, as an intracellular binding partner of FLIL33. By overexpressing various FLIL33 protein segments and variants in primary human lung fibroblasts and HEK293T cells, we show that FLIL33, but not mature interleukin-33, physically interacts with IPO5 and that this interaction localizes to a cluster of charged amino acids (positions 46-56) but not to an adjacent segment (positions 61-67) in the FLIL33 N-terminal region. siRNA-mediated IPO5 knockdown in cell culture did not affect nuclear localization of FLIL33. However, the IPO5 knockdown significantly decreased the intracellular levels of overexpressed FLIL33, reversed by treatment with the 20S proteasome inhibitor bortezomib. Furthermore, FLIL33 variants deficient in IPO5 binding remained intranuclear and exhibited decreased levels, which were also restored by the bortezomib treatment. These results indicate that the interaction between FLIL33 and IPO5 is localized to a specific segment of the FLIL33 protein, is not required for nuclear localization of FLIL33, and protects FLIL33 from proteasome-dependent degradation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cellular FLICE-like inhibitory protein (c-FLIP L ) is a key inhibitory protein in the extrinsic apoptotic pathway. Recent studies showed that c-FLIP L could translocate into the nucleus and might be involved in the Wnt signaling pathway. The nuclear function of c-FLIP L was still unclear. Here we found a novel c-FLIP L -associated protein TIP49, which is a nuclear protein identified as a cofactor in the transcriptional regulation of β-catenin. They had co-localization in the nucleus and the DED domain of c-FLIP L was required for the association with TIP49. By performing ChIP experiments, C-FLIP L was detected in the ITF-2 locus and facilitated TIP49 accumulation in the formation of complexes at the T-cell-specific transcription factor site of human ITF-2 promoter. When TIP49 knockdown, c-FLIP L -driven Wnt activation, and cell proliferation were inhibited, suggesting that a role of nuclear c-FLIP L involved in modulation of the Wnt pathway was in a TIP49-dependent manner. Elevated expression of c-FLIP L and TIP49 that coincided in human lung cancers were analyzed in silico using the Oncomine database. Their high expressions were reconfirmed in six lung cancer cell lines and correlated with cell growth. The association of c-FLIP L and TIP49 provided an additional mechanism involved in c-FLIP L -mediated functions, including Wnt activation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1 receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca 2+ -regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Gene-specific transcription factors (GSTFs) control gene transcription by DNA binding and specific protein complex recruitment, which regulates promoter accessibility for transcription initiation by RNA polymerase II. Mutations in the GSTFs Suppressor of Mothers Against Decapentaplegic 2 (SMAD2) and SMAD4 are frequently associated with colon and rectal carcinomas. These proteins play an important role in bone morphogenic protein (BMP) and transforming growth factor β (TGF-β) signaling pathways controlling cell fate and proliferation. To study the protein interactome of the SMAD protein family we generated a quantitative proteomics pipeline that allows for inducible expression of GFP-tagged SMAD proteins followed by affinity purification and quantitative mass spectrometry analysis. Data are available via ProteomeXchange with identifier PXD004529. The nuclear importin IPO5 was identified as a novel interacting protein of SMAD1. Overexpression of IPO5 in various cell lines specifically increases nuclear localization of BMP receptor-activated SMADs (R-SMADs) confirming a functional relationship between IPO5 and BMP but not TGF-β R-SMADs. Finally, we provide evidence that variation in length of the lysine stretch of the nuclear localization sequence is a determinant for importin specificity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Urokinase-type plasminogen activator (uPA) regulates angiogenesis and vascular permeability through proteolytic degradation of extracellular matrix and intracellular signaling initiated upon its binding to uPAR/CD87 and other cell surface receptors. Here, we describe an additional mechanism by which uPA regulates angiogenesis. Ex vivo VEGF-induced vascular sprouting from Matrigel-embedded aortic rings isolated from uPA knock-out (uPA(-/-)) mice was impaired compared with vessels emanating from wild-type mice. Endothelial cells isolated from uPA(-/-) mice show less proliferation and migration in response to VEGF than their wild type counterparts or uPA(-/-) endothelial cells in which expression of wild type uPA had been restored. We reported previously that uPA is transported from cell surface receptors to nuclei through a mechanism that requires its kringle domain. Intranuclear uPA modulates gene transcription by binding to a subset of transcription factors. Here we report that wild type single-chain uPA, but not uPA variants incapable of nuclear transport, increases the expression of cell surface VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2) by translocating to the nuclei of ECs. Intranuclear single-chain uPA binds directly to and interferes with the function of the transcription factor hematopoietically expressed homeodomain protein or proline-rich homeodomain protein (HHEX/PRH), which thereby lose their physiologic capacity to repress the activity of vehgr1 and vegfr2 gene promoters. These studies identify uPA-dependent de-repression of vegfr1 and vegfr2 gene transcription through binding to HHEX/PRH as a novel mechanism by which uPA mediates the pro-angiogenic effects of VEGF and identifies a potential new target for control of pathologic angiogenesis., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Signal transducer and activator of transcription 3 (STAT3) is involved in cell proliferation, differentiation, and cell survival during immune responses, hematopoiesis, neurogenesis, and other biological processes. STAT3 activity is regulated by a variety of mechanisms, including phosphorylation and nuclear translocation. To clarify the molecular mechanisms underlying the regulation of STAT3 activity, we performed yeast two-hybrid screening. We identified ARL3 (ADP-ribosylation factor-like 3) as a novel STAT3-binding partner. ARL3 recognizes the DNA-binding domain as well as the C-terminal region of STAT3 in vivo, and their binding was the strongest when both proteins were activated. Importantly, small interfering RNA-mediated reduction of endogenous ARL3 expression decreased IL-6-induced tyrosine phosphorylation, nuclear accumulation, and transcriptional activity of STAT3. These results indicate that ARL3 interacts with STAT3 and regulates the transcriptional activation of STAT3 by influencing its nuclear accumulation of STAT3., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The tripartite terminase complex of herpesviruses assembles in the cytoplasm of infected cells and exploits the host nuclear import machinery to gain access to the nucleus, where capsid assembly and genome-packaging occur. Here we analyzed the structure and conservation of nuclear localization signal (NLS) sequences previously identified in herpes simplex virus 1 (HSV-1) large terminase and human cytomegalovirus (HCMV) small terminase. We found a monopartite NLS at the N terminus of large terminase, flanking the ATPase domain, that is conserved only in α-herpesviruses. In contrast, small terminase exposes a classical NLS at the far C terminus of its helical structure that is conserved only in two genera of the β-subfamily and absent in α- and γ-herpesviruses. In addition, we predicted a classical NLS in the third terminase subunit that is partially conserved among herpesviruses. Bioinformatic analysis revealed that both location and potency of NLSs in terminase subunits evolved more rapidly than the rest of the amino acid sequence despite the selective pressure to keep terminase gene products active and localized in the nucleus. We propose that swapping NLSs among terminase subunits is a regulatory mechanism that allows different herpesviruses to regulate the kinetics of terminase nuclear import, reflecting a mechanism of virus:host adaptation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Hippo signaling pathway controls organ size by orchestrating cell proliferation and apoptosis. When the Hippo pathway was inactivated, the transcriptional co-activator Yorkie translocates into the nucleus and forms a complex with transcription factor Scalloped to promote the expression of Hippo pathway target genes. Therefore, the nuclear translocation of Yorkie is a critical step in Hippo signaling. Here, we provide evidence that the N-terminal 1-55 amino acids of Yorkie, especially Arg-15, were essential for its nuclear localization. By mass spectrometry and biochemical analyses, we found that Importin α1 can directly interact with the Yorkie N terminus and drive Yorkie into the nucleus. Further experiments show that the upstream component Hippo can inhibit Importin α1-mediated Yorkie nuclear import. Taken together, we identified a potential nuclear localization signal at the N-terminal end of Yorkie as well as a critical role for Importin α1 in Yorkie nuclear import., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The canonical Wnt signaling pathway, in which β-catenin nuclear localization is a crucial step, plays an important role in osteoblast differentiation. Pin1, a prolyl isomerase, is also known as a key enzyme in osteogenesis. However, the role of Pin1 in canonical Wnt signal-induced osteoblast differentiation is poorly understood. We found that Pin1 deficiency caused osteopenia and reduction of β-catenin in bone lining cells. Similarly, Pin1 knockdown or treatment with Pin1 inhibitors strongly decreased the nuclear β-catenin level, TOP flash activity, and expression of bone marker genes induced by canonical Wnt activation and vice versa in Pin1 overexpression. Pin1 interacts directly with and isomerizes β-catenin in the nucleus. The isomerized β-catenin could not bind to nuclear adenomatous polyposis coli, which drives β-catenin out of the nucleus for proteasomal degradation, which consequently increases the retention of β-catenin in the nucleus and might explain the decrease of β-catenin ubiquitination. These results indicate that Pin1 could be a critical target to modulate β-catenin-mediated osteogenesis., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Members of the receptor tyrosine kinase family (RTK) have been shown to be present in the nucleus of cells; however, the mechanisms underlying their trafficking to the nucleus, and their relevance once there are poorly understood. In the present study, we focus on the RTK ErbB3 and elucidate the mechanisms regulating its trafficking. We show that heregulin-stimulation induces trafficking of phosphorylated ErbB3 from the plasma membrane to the nucleus via a clathrin-independent mechanism. Nuclear import of ErbB3 occurs via importin β1, which drives the receptor through the nuclear pore complex. In the nucleus, ErbB3 interacts with transcription complexes, and thereby has a role in transcriptional regulation. Our results also demonstrate that ErbB3 nuclear localization is transient as it is exported out of the nucleus by the nuclear receptor protein crm-1. Analysis of normal, regenerating tissues, and tumors showed that ErbB3 nuclear translocation is a common event in proliferating tissues., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

D-Serine is a physiological co-agonist that activates N-methyl D-aspartate receptors (NMDARs) and is essential for neurotransmission, synaptic plasticity, and behavior. D-Serine may also trigger NMDAR-mediated neurotoxicity, and its dysregulation may play a role in neurodegeneration. D-Serine is synthesized by the enzyme serine racemase (SR), which directly converts L-serine to D-serine. However, many aspects concerning the regulation of D-serine production under physiological and pathological conditions remain to be elucidated. Here, we investigate possible mechanisms regulating the synthesis of D-serine by SR in paradigms relevant to neurotoxicity. We report that SR undergoes nucleocytoplasmic shuttling and that this process is dysregulated by several insults leading to neuronal death, typically by apoptotic stimuli. Cell death induction promotes nuclear accumulation of SR, in parallel with the nuclear translocation of GAPDH and Siah proteins at an early stage of the cell death process. Mutations in putative SR nuclear export signals (NESs) elicit SR nuclear accumulation and its depletion from the cytosol. Following apoptotic insult, SR associates with nuclear GAPDH along with other nuclear components, and this is accompanied by complete inactivation of the enzyme. As a result, extracellular D-serine concentration is reduced, even though extracellular glutamate concentration increases severalfold. Our observations imply that nuclear translocation of SR provides a fail-safe mechanism to prevent or limit secondary NMDAR-mediated toxicity in nearby synapses., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

SNAREs constitute the core machinery of intracellular membrane fusion, but vesicular SNAREs localize to specific compartments via largely unknown mechanisms. Here, we identified an interaction between VAMP7 and SNAP-47 using a proteomics approach. We found that SNAP-47 mainly localized to cytoplasm, the endoplasmic reticulum (ER), and ERGIC and could also shuttle between the cytoplasm and the nucleus. SNAP-47 preferentially interacted with the trans-Golgi network VAMP4 and post-Golgi VAMP7 and -8. SNAP-47 also interacted with ER and Golgi syntaxin 5 and with syntaxin 1 in the absence of Munc18a, when syntaxin 1 is retained in the ER. A C-terminally truncated SNAP-47 was impaired in interaction with VAMPs and affected their subcellular distribution. SNAP-47 silencing further shifted the subcellular localization of VAMP4 from the Golgi apparatus to the ER. WT and mutant SNAP-47 overexpression impaired VAMP7 exocytic activity. We conclude that SNAP-47 plays a role in the proper localization and function of a subset of VAMPs likely via regulation of their transport through the early secretory pathway., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

A series of in vitro and in vivo studies has shown that EAF2 can affect multiple signaling pathways involved in cellular processes. However, the molecular mechanisms underlying its effects have remained elusive. Here we report the discovery of a new functional link between EAF2 and TGF-β signaling. Promoter reporter assays indicated that EAF2 suppresses Smad3 transcriptional activity, resulting in inhibition of TGF-β signaling. Coimmunoprecipitation assays showed that EAF2 specifically interacts with Smad3 in vitro and in vivo but not with other Smad proteins. In addition, we observed that EAF2 binding does not alter Smad3 phosphorylation but causes Smad3 cytoplasmic retention, competes with Smad4 for binding to Smad3, and prevents p300-Smad3 complex formation. Furthermore, we demonstrated that EAF2 suppresses both TGF-β-induced G1 cell cycle arrest and TGF-β-induced cell migration. This study identifies and characterizes a novel repressor of TGF-β signaling., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Lipin1, an intracellular protein, plays critical roles in controlling lipid synthesis and energy metabolism through its enzymatic activity and nuclear transcriptional functions. Several mouse models of skeletal muscle wasting are associated with lipin1 mutation or altered expression. Recent human studies have suggested that children with homozygous null mutations in the LPIN1 gene suffer from rhabdomyolysis. However, the underlying pathophysiologic mechanism is still poorly understood. In the present study we examined whether lipin1 contributes to regulating muscle regeneration. We characterized the time course of skeletal muscle regeneration in lipin1-deficient fld mice after injury. We found that fld mice exhibited smaller regenerated muscle fiber cross-sectional areas compared with wild-type mice in response to injury. Our results from a series of in vitro experiments suggest that lipin1 is up-regulated and translocated to the nucleus during myoblast differentiation and plays a key role in myogenesis by regulating the cytosolic activation of ERK1/2 to form a complex and a downstream effector cyclin D3-mediated cell cycle withdrawal. Overall, our study reveals a previously unknown role of lipin1 in skeletal muscle regeneration and expands our understanding of the cellular and molecular mechanisms underlying skeletal muscle regeneration., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

We have reported previously that dopamine D2 receptor stimulation activates calcium/calmodulin-dependent protein kinase II (CaMKII) δ3, a CaMKII nuclear isoform, increasing BDNF gene expression. However, the mechanisms underlying that activity remained unclear. Here we report that CaMKIIδ3 is dephosphorylated at Ser(332) by protein phosphatase 1 (PP1), promoting CaMKIIδ3 nuclear translocation. Neuro-2a cells transfected with CaMKIIδ3 showed cytoplasmic and nuclear staining, but the staining was predominantly nuclear when CaMKIIδ3 was coexpressed with PP1. Indeed, PP1 and CaMKIIδ3 coexpression significantly increased nuclear CaMKII activity and enhanced BDNF expression. In support of this idea, chronic administration of the dopamine D2 receptor partial agonist aripiprazole increased PP1 activity and promoted nuclear CaMKIIδ3 translocation and BDNF expression in the rat brain substantia nigra. Moreover, aripiprazole treatment enhanced neurite extension and inhibited cell death in cultured dopaminergic neurons, effects blocked by PP1γ knockdown. Taken together, nuclear translocation of CaMKIIδ3 following dephosphorylation at Ser(332) by PP1 likely accounts for BDNF expression and subsequent neurite extension and survival of dopaminergic neurons., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Activation of IκB kinase (IKK) and NF-κB by genotoxic stresses modulates apoptotic responses and production of inflammatory mediators, thereby contributing to therapy resistance and premature aging. We previously reported that genotoxic agents induce nuclear localization of NF-κB essential modulator (NEMO) via an undefined mechanism to arbitrate subsequent DNA damage-dependent IKK/NF-κB signaling. Here we show that a nonclassical nuclear import pathway via IPO3 (importin 3, transportin 2) mediates stress-induced NEMO nuclear translocation. We found putative nuclear localization signals in NEMO whose mutations disrupted stress-inducible nuclear translocation of NEMO and IKK/NF-κB activation in stably reconstituted NEMO-deficient cells. RNAi screening of both importin α and β family members, as well as co-immunoprecipitation analyses, revealed that a nonclassical importin β family member, IPO3, was the only importin that was able to associate with NEMO and whose reduced expression prevented genotoxic stress-induced NEMO nuclear translocation, IKK/NF-κB activation, and inflammatory cytokine transcription. Recombinant IPO3 interacted with recombinant NEMO but not the nuclear localization signal mutant version and induced nuclear import of NEMO in digitonin-permeabilized cells. We also provide evidence that NEMO is disengaged from IKK complex following genotoxic stress induction. Thus, the IPO3 nuclear import pathway is an early and crucial determinant of the IKK/NF-κB signaling arm of the mammalian DNA damage response., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Inflammation is widely distributed in patients with Duchenne muscular dystrophy and ultimately leads to progressive deterioration of muscle function with chronic muscle damage, oxidative stress, and reduced oxidative capacity. NF-E2-related factor 2 (Nrf2) plays a critical role in defending against inflammation in different tissues via activation of phase II enzyme heme oxygenase-1 and inhibition of the NF-κB signaling pathway. However, the role of Nrf2 in the inflammation of dystrophic muscle remains unknown. To determine whether Nrf2 may counteract inflammation in dystrophic muscle, we treated 4-week-old male mdx mice with the Nrf2 activator sulforaphane (SFN) by gavage (2 mg/kg of body weight/day) for 4 weeks. The experimental results demonstrated that SFN treatment increased the expression of muscle phase II enzyme heme oxygenase-1 in an Nrf2-dependent manner. Inflammation in mice was reduced by SFN treatment as indicated by decreased infiltration of immune cells and expression of the inflammatory cytokine CD45 and proinflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6 in the skeletal muscles of mdx mice. In addition, SFN treatment also decreased the expression of NF-κB(p65) and phosphorylated IκB kinase-α as well as increased inhibitor of κB-α expression in mdx mice in an Nrf2-dependent manner. Collectively, these results show that SFN-induced Nrf2 can alleviate muscle inflammation in mdx mice by inhibiting the NF-κB signaling pathway., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Although α-synuclein (α-syn) phosphorylation has been considered as a hallmark of sporadic and familial Parkinson disease (PD), little is known about the effect of PD-linked mutations on α-syn phosphorylation. In this study, we investigated the effects of the A30P, E46K, and A53T PD-linked mutations on α-syn phosphorylation at residues Ser-87 and Ser-129. Although the A30P and A53T mutants slightly affected Ser(P)-129 levels compared with WT α-syn, the E46K mutation significantly enhanced Ser-129 phosphorylation in yeast and mammalian cell lines. This effect was not due to the E46K mutant being a better kinase substrate nor due to alterations in endogenous kinase levels, but was mostly linked with enhanced nuclear and endoplasmic reticulum accumulation. Importantly, lentivirus-mediated overexpression in mice also showed enhanced Ser-129 phosphorylation of the E46K mutant compared to WT α-syn, thus providing in vivo validation of our findings. Altogether, our findings suggest that the different PD-linked mutations may contribute to PD pathogenesis via different mechanisms., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Herpesviruses assemble capsids in the nucleus and egress by unconventional vesicle-mediated trafficking through the nuclear envelope. Capsids bud at the inner nuclear membrane into the nuclear envelope lumen. The resulting intralumenal vesicles fuse with the outer nuclear membrane, delivering the capsids to the cytoplasm. Two viral proteins are required for vesicle formation, the tail-anchored pUL34 and its soluble interactor, pUL31. Whether cellular proteins are involved is unclear. Using giant unilamellar vesicles, we show that pUL31 and pUL34 are sufficient for membrane budding and scission. pUL34 function can be bypassed by membrane tethering of pUL31, demonstrating that pUL34 is required for pUL31 membrane recruitment but not for membrane remodeling. pUL31 can inwardly deform membranes by oligomerizing on their inner surface to form buds that constrict to vesicles. Therefore, a single viral protein can mediate all events necessary for membrane budding and abscission., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids that can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol-anchored serine protease, cleaved human PCI and mouse PCI (mPCI) at their reactive sites as well as at sites close to their N terminus. This cleavage was observed not only with testisin in solution but also with cell membrane-anchored testisin on U937 cells. The cleavage close to the N terminus released peptides rich in basic amino acids. Synthetic peptides corresponding to the released peptides of human PCI (His(1)-Arg(11)) and mPCI (Arg(1)-Ala(18)) functioned as cell-penetrating peptides. Because intact mPCI but not testisin-cleaved mPCI was internalized by Jurkat T cells, a truncated mPCI mimicking testisin-cleaved mPCI was created. The truncated mPCI lacking 18 amino acids at the N terminus was not taken up by Jurkat T cells. Therefore our model suggests that testisin or other proteases could regulate the internalization of PCI by removing its N terminus. This may represent one of the mechanisms regulating the intracellular functions of PCI., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The heparan sulfate proteoglycan syndecan-1 is proteolytically shed from the surface of multiple myeloma cells and is abundant in the bone marrow microenvironment where it promotes tumor growth, angiogenesis, and metastasis. In this study, we demonstrate for the first time that shed syndecan-1 present in the medium conditioned by tumor cells is taken up by bone marrow-derived stromal cells and transported to the nucleus. Translocation of shed syndecan-1 (sSDC1) to the nucleus was blocked by addition of exogenous heparin or heparan sulfate, pretreatment of conditioned medium with heparinase III, or growth of cells in sodium chlorate, indicating that sulfated heparan sulfate chains are required for nuclear translocation. Interestingly, cargo bound to sSDC1 heparan sulfate chains (i.e. hepatocyte growth factor) was transported to the nucleus along with sSDC1, and removal of heparan sulfate-bound cargo from sSDC1 abolished its translocation to the nucleus. Once in the nucleus, sSDC1 binds to the histone acetyltransferase enzyme p300, and histone acetyltransferase activity and histone acetylation are diminished. These findings reveal a novel function for shed syndecan-1 in mediating tumor-host cross-talk by shuttling growth factors to the nucleus and by altering histone acetylation in host cells. In addition, this work has broad implications beyond myeloma because shed syndecan-1 is present in high levels in many tumor types as well as in other disease states., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays roles in both energy maintenance, and stress signaling by forming a protein complex with seven in absentia homolog 1 (Siah1). Mechanisms to coordinate its glycolytic and stress cascades are likely to be very important for survival and homeostatic control of any living organism. Here we report that apoptosis signal-regulating kinase 1 (ASK1), a representative stress kinase, interacts with both GAPDH and Siah1 and is likely able to phosphorylate Siah1 at specific amino acid residues (Thr-70/Thr-74 and Thr-235/Thr-239). Phosphorylation of Siah1 by ASK1 triggers GAPDH-Siah1 stress signaling and activates a key downstream target, p300 acetyltransferase in the nucleus. This novel mechanism, together with the established S-nitrosylation/oxidation of GAPDH at Cys-150, provides evidence of how the stress signaling involving GAPDH is finely regulated. In addition, the present results imply crosstalk between the ASK1 and GAPDH-Siah1 stress cascades., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The RTS1 gene encodes a subunit of the PP2A phosphatase that regulates cell cycle progression. Ace2 and Swi5 are cell cycle-regulated transcription factors, and we recently showed that phosphorylation of Ace2 and Swi5 is altered in an rts1 mutant. Here we examine expression of Ace2 and Swi5 target genes and find that an rts1 mutation markedly reduces expression of the HO gene. The decreased HO expression in an rts1 mutant is significantly restored by an additional ace2 mutation, a surprising result because HO is normally activated by Swi5 but not by Ace2. Ace2 normally accumulates only in daughter cells, and only activates transcription in daughters. However, in an rts1 mutant, Ace2 is present in both mother and daughter cells. One of the genes activated by Ace2 is ASH1, a protein that normally accumulates mostly in daughter cells; Ash1 is a transcriptional repressor, and it blocks HO expression in daughters. We show that in the rts1 mutant, Ace2 accumulation in mother cells results in Ash1 expression in mothers, and the Ash1 can now repress HO expression in mothers., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Class IIa histone deacetylases (HDACs) regulate the activity of many transcription factors to influence liver gluconeogenesis and the development of specialized cells, including muscle, neurons, and lymphocytes. Here, we describe a conserved role for class IIa HDACs in sustaining robust circadian behavioral rhythms in Drosophila and cellular rhythms in mammalian cells. In mouse fibroblasts, overexpression of HDAC5 severely disrupts transcriptional rhythms of core clock genes. HDAC5 overexpression decreases BMAL1 acetylation on Lys-537 and pharmacological inhibition of class IIa HDACs increases BMAL1 acetylation. Furthermore, we observe cyclical nucleocytoplasmic shuttling of HDAC5 in mouse fibroblasts that is characteristically circadian. Mutation of the Drosophila homolog HDAC4 impairs locomotor activity rhythms of flies and decreases period mRNA levels. RNAi-mediated knockdown of HDAC4 in Drosophila clock cells also dampens circadian function. Given that the localization of class IIa HDACs is signal-regulated and influenced by Ca(2+) and cAMP signals, our findings offer a mechanism by which extracellular stimuli that generate these signals can feed into the molecular clock machinery., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)