Regulation Of Subcellular Localization Research Articles

Regulation of Smoothened Trafficking and Abundance in Hedgehog Signaling.

The GPCR-family protein Smoothened (Smo) is essential for Hedgehog (Hh) signal transduction in both insects and vertebrates. The regulation of subcellular localization and abundance of Smo is a critical step in Hh signaling. Recent studies have demonstrated that Smo is subjected to ubiquitination mediated by multiple E3 ubiquitin ligases, leading to Smo endocytosis and subsequent degradation through the proteasome- and lysosome-mediated pathways in Drosophila. Ubiquitination of Smo also promotes its ciliary exit in mammalian cells. Hh inhibits Smo ubiquitination by blocking E3 ligase recruitment and promoting Smo deubiquitination through the ubiquitin-specific protease 8 (USP8) in Drosophila. Inhibition of Smo ubiquitination by Hh promotes Smo cell surface accumulation in Drosophila and ciliary accumulation in mammalian cells. Interestingly, Hh also induces sumoylation of Smo in both Drosophila and mammalian cells, which promotes Smo cell surface/ciliary accumulation. This review focuses on how ubiquitination and sumoylation regulate Smo intracellular trafficking and abundance and how these processes are regulated by Hh.

Yeast Phosphatidic Acid Phosphatase Pah1; Why is Tryptophan 637 Required for Cellular Function?

Pah1 in Saccharomyces cerevisiae is an Mg2+-dependent phosphatidic acid (PA) phosphatase that plays a major role in lipid synthesis by controlling the levels of triacylglycerol and membrane phospholipids. The enzyme contains two conserved domains (N-lip and HAD-like) required for catalytic activity, and non-conserved regions containing multiple phosphorylation sites involved in the regulation of subcellular localization, catalytic activity, and protein stability. In a previous study, we found that Trp-637 in a putative WW-binding domain and destruction box is essential for the function of Pah1 in vivo, but not for its catalytic activity in vitro. In this work, we examined the Pah1(W637A) for its localization, abundance, and phosphorylation state. Subcellular fractionation analysis showed that the membrane translocation of Pah1(W637A), like that of the WT enzyme, depends on the Nem1-Spo7 phosphatase function. The mutant enzyme showed a higher level of membrane association than the WT enzyme, but a significantly lower cytosolic level. The phosphorylation status of Pah1(W637A), as determined by electrophoretic mobility, showed that the mutant enzyme is less phosphorylated in vivo when compared with the WT enzyme. The Nem1-Spo7-dependent membrane translocation of Pah1(W637A) was also shown in vitro by fractionation of cytosolic Pah1 incubated with the membrane containing the phosphatase complex. These results suggest that Trp-637 is not required for the translocation of Pah1, but for its catalytic activity on PA in the cellular membrane.

Feline XLF accumulates at DNA damage sites in a Ku-dependent manner.

Resistance to radiotherapy and chemotherapy is a common problem in the treatment of cancer in humans and companion animals, including cats. There is thus an urgent need to develop new treatments. Molecularly targeted therapies hold the promise of high specificity and significant cancer‐killing effects. Accumulating evidence shows that DNA double‐strand break (DSB) repair proteins, which function in Ku‐dependent non‐homologous DNA‐end joining (NHEJ), are potential target molecules for next‐generation cancer therapies. Although cancer radioresistance in cats has been previously described, there are no reports on feline Ku‐dependent NHEJ. Here, we cloned and sequenced feline XLF cDNA and characterized X‐ray repair cross‐complementing protein 4‐like factor (XLF), which is one of the core NHEJ proteins. We demonstrated that feline XLF localizes to the nuclei of feline cells and that feline XLF immediately accumulates at laser‐induced DSB sites in a Ku‐dependent manner. Amino acid sequence alignment analysis showed that feline XLF has only 80.9% identity with human XLF protein, while the predicted nuclear localization signal and putative 14‐3‐3‐binding motif are perfectly conserved among human, cat, dog, chimpanzee, and mouse. These findings are consistent with the hypothesis that regulation of subcellular localization is important for the function of XLF. Furthermore, these findings may be useful in clarifying the mechanisms underlying feline Ku‐dependent DSB repair and feline cell radioresistance, and possibly facilitate the development of new molecularly targeted therapies that target common proteins in human and feline cancers.

Regulation of different human NFAT isoforms by neuronal activity.

Nuclear factor of activated T-cells (NFAT) is a family of transcription factors comprising four calcium-regulated members: NFATc1, NFATc2, NFATc3, and NFATc4. Upon activation by the calcium-dependent phosphatase calcineurin (CaN), NFATs translocate from cytosol to the nucleus and regulate their target genes, which in the nervous system are involved in axon growth, synaptic plasticity, and neuronal survival. We have shown previously that there are a number of different splice variants of NFAT genes expressed in the brain. Here, we studied the subcellular localizations and transactivation capacities of alternative human NFAT isoforms in rat primary cortical or hippocampal neurons in response to membrane depolarization and compared the induced transactivation levels in neurons to those obtained from HEK293 cells in response to calcium signaling. We confirm that in neurons the translocation to the nucleus of all NFAT isoforms is reliant on the activity of CaN. However, our results suggest that both the regulation of subcellular localization and transcriptional activity of NFAT proteins in neurons is isoform specific. We show that in primary hippocampal neurons NFATc2 isoforms have very fast translocation kinetics, whereas NFATc4 isoforms translocate relatively slowly to the nucleus. Moreover, we demonstrate that the strongest transcriptional activators in HEK293 cells are NFATc1 and NFATc3, but in neurons NFATc3 and NFATc4 lead to the highest induction, and NFATc2 and NFATc1 display isoform-specific transcription activation capacities. Altogether, our results indicate that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and can differ between cell types. We show that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and differ between cell types. Although nuclear localization of all NFAT isoforms in neurons requires calcineurin, the subcellular distributions, neuronal activity-induced nuclear translocation extent and kinetics, and transcription activation capacities of alternative NFAT proteins vary.

Abstract A42: H-Ras regulates the dynamics of TRAIL death receptors

Abstract Multiple clinical trials are ongoing to evaluate the potential antitumor activity of recombinant human TNF-related apoptosis inducing ligand (rhTRAIL) and its agonistic antibodies. These protein therapies act through death receptors (DRs) 4 and/or 5 on the cell surface, followed by activation of a caspase cascade and apoptotic cell death. We have previously shown that DR4 and/or DR5 are mislocalized in nucleus and/or cytoplasm in some cancer cells thereby rendering cellular resistance to the targeted therapies. In this study, we have found a correlation between H-Ras expression and TRAIL resistance in the NCI60 panel of human cancer cell lines. The elevated expression of H-Ras is also generally associated with a deficiency of TRAIL death receptors on cell surface. Blockade of H-Ras activity in TRAIL-resistant cells upregulated the surface expression of both DR4 and DR5 without changing their total protein expressions. Subsequently, the cells were sensitized to apoptosis induction by the targeted therapies. To our knowledge, this is the first evidence that oncogenic H-Ras is involved in the regulation of subcellular localization of TRAIL death receptors. H-Ras, along with other Ras GTPases, is known to stimulate outgrowth of cancer cells. Our data suggest that H-Ras may elicit its oncogenic activity at least partly by blocking cytokine-induced apoptosis of cancer cells. Additional studies are warranted to evaluate the therapeutic potential of combinational therapies to simultaneously target H-Ras and TRAIL receptors. Citation Format: Jun-jie Chen, Xu Di, Yaqin Zhang, William Bozza, Baolin Zhang. H-Ras regulates the dynamics of TRAIL death receptors. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A42. doi: 10.1158/1557-3125.RASONC14-A42

Characterization of the zebrafish homolog of zipper interacting protein kinase.

Zipper-interacting protein kinase (ZIPK) is a conserved vertebrate-specific regulator of actomyosin contractility in smooth muscle and non-muscle cells. Murine ZIPK has undergone an unusual divergence in sequence and regulation compared to other ZIPK orthologs. In humans, subcellular localization is controlled by phosphorylation of threonines 299 and 300. In contrast, ZIPK subcellular localization in mouse and rat is controlled by interaction with PAR-4. We carried out a comparative biochemical characterization of the regulation of the zebrafish ortholog of ZIPK. Like the human orthologs zebrafish ZIPK undergoes nucleocytoplasmic-shuttling and is abundant in the cytoplasm, unlike the primarily nuclear rat ZIPK. Rat ZIPK, but not human or zebrafish ZIPK, interacts with zebrafish PAR-4. Mutation of the conserved residues required for activation of the mammalian orthologs abrogated activity of the zebrafish ZIPK. In contrast to the human ortholog, mutation of threonine 299 and 300 in the zebrafish ZIPK has no effect on the activity or subcellular localization. Thus, we found that zebrafish ZIPK functions in a manner most similar to the human ZIPK and quite distinct from murine orthologs, yet the regulation of subcellular localization is not conserved.

G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor

Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin–MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF–SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin–MRTFs interaction.

CRM1 expression in pancreatic carcinoma.

e15115 Background: Increased expression of chromosomal region maintenance (CRM1) protein, also known as exportin 1, has been described in several human cancers. It is an important regulator of subcellular localization of tumor suppressor proteins and growth regulatory proteins. Direct inhibition of CRM1 blocks cell proliferation and induces apoptosis leading to the current evaluation of CRM1 inhibitors as anticancer agents in early phase clinical trials. There is a paucity of data regarding the prevalence of CRM1 expression in pancreatic cancer. Methods: We analyzed the expression of CRM1 by immunohistochemistry (IHC) in pancreatic tissue microarray (TMA) samples (malignant and non-malignant) obtained from patients who underwent potentially curative resection for pancreatic ductal adenocarcinoma. CRM1 antibody (Santa Cruz Biotechnology) was used for immunostaining the formalin fixed paraffin embedded core sections in the TMA samples. The intensity of staining and percentage of cells stained were graded on a scale of 0 to 3, with 3 being highest. The final IHC score was obtained using the product of immunostain intensity and percentage of cells stained (Range: 0-9). Low and high CRM1 expression was considered if the IHC score was 0 to 4 and 6 to 9 respectively. Results: Seventy nonmalignant and 91 pancreatic carcinoma samples were evaluated in this study. The median IHC score was 6 (range: 0-9) and 3 (range: 1-9) in malignant and nonmalignant pancreatic tissue samples respectively (P<0.0001). High CRM1 expression was found in 11% (8/70) of normal tissue samples and 69% (63/91) of tumor samples (P<0.0001). There were 48 paired samples of pancreatic cancer tissue and normal tissue obtained from same patient. Among these patients, 33 (69%) patients had higher CRM1 expression, 7 (15%) patients had similar expression and 8 (17%) patients had lower expression in malignant tissue sample as compared to their adjacent normal tissues. Conclusions: Higher CRM1 expression occurs frequently in pancreatic cancer as compared to nonmalignant pancreatic tissue, reinforcing its putative tumor oncogenic activity, and raising the value of targeting it for pancreatic cancer therapy.

Regulated expression and neural functions of human natural killer-1 (HNK-1) carbohydrate.

Human natural killer-1 (HNK-1) carbohydrate, comprising a unique trisaccharide HSO(3)-3GlcAβ1-3Galβ1-4GlcNAc, shows well-regulated expression and unique functions in the nervous system. Recent studies have revealed sophisticated and complicated expression mechanisms for HNK-1 glycan. Activities of biosynthetic enzymes are controlled through the formation of enzyme-complexes and regulation of subcellular localization. Functional aspects of HNK-1 carbohydrate were examined by overexpression, knockdown, and knockout studies of these enzymes. HNK-1 is involved in several neural functions such as synaptic plasticity, learning and memory, and the underlying molecular mechanisms have been illustrated upon identification of the target carrier glycoproteins of HNK-1 such as the glutamate receptor subunit GluA2 or tenascin-R. In this review, we describe recent findings about HNK-1 carbohydrate that provide further insights into the mechanism of its expression and function in the nervous system.

Functional comparison of protein domains within aPKCs involved in nucleocytoplasmic shuttling

SummaryThe atypical protein kinases C (PKC) isoforms ι and ζ play crucial roles in regulation of signaling pathways related to proliferation, differentiation and cell survival. Over the years several interaction partners and phosphorylation targets have been identified. However, little is known about the regulation of atypical aPKC isoforms. To address this question, we performed a comparative analysis of atypical aPKCι/λ and ζ in MDCK cells. By using green fluorescence protein (GFP) fusion proteins containing the full-length or truncated proteins, we were able to recognize differences in subcellular localization and nucleocytoplasmic shuttling of both isoforms. We show, that an earlier described nuclear localization sequence (NLS), plays a role in the regulation of atypical aPKCζ but not in aPKCι, despite the fact that it is present in both isoforms. Leptomycin B treatment induces accumulation of GFP-fusion protein of both isoforms in the nucleus. Regardless, the loss of the NLS only decreases shuttling of aPKCζ, while aPKCι remains unaffected. In addition, we identified the hinge region as a potential regulator of localization of atypical PKCs. With a set of chimeric proteins we show that the hinge region of aPKCι mediates nuclear localization. In contrast, the hinge region of aPKCζ causes exclusion from the nucleus, indicating two different mechanisms leading to isoform specific regulation. Taken together, we show for the first time, that the atypical isoforms aPKCι and ζ underly different mechanisms regarding their regulation of subcellular localization and translocation into the nucleus in MDCK cells.

Class IIa HDACs: from important roles in differentiation to possible implications in tumourigenesis

Histone deacetylases (HDACs) are important regulators of gene expression. Specific structural features and distinct regulative mechanisms rationalize the separation of the 18 different human HDACs into four classes. The class II comprises a heterogeneous group of nuclear and cytosolic HDACs involved in the regulation of several cellular functions, not just limited to transcriptional repression. In particular, HDAC4, 5, 7 and 9 belong to the subclass IIa and share many transcriptional partners, including members of the MEF2 family. Genetic studies in mice have disclosed the fundamental contribution of class IIa HDACs to specific developmental/differentiation pathways. In this review, we discuss about the recent literature, which hints a role of class IIa HDACs in the development, growth and aggressiveness of cancer cells.

Generation of Chicken Monoclonal Antibodies Against thea1,a2, anda3 Subunit Isoforms of Vacuolar-type Proton ATPase

The vacuolar-type proton pump ATPase (V-ATPase) plays several pivotal roles in the acidification of diverse intracellular compartments and the extracellular environment. The a subunit isoforms a1, a2, and a3, constituting the membrane-embedded section, are expressed in various tissues, and they are involved in the regulation of subcellular localization and activity of the holocomplex. Therefore, the characterization of their properties is indispensable for dissection of the physiological roles of the V-ATPase in highly differentiated cells. In this study, we report the production and characterization of chicken monoclonal antibodies (MAbs) against these mouse a1, a2 and a3 subunit isoforms. These MAbs are shown to be suitable for both immunoblotting and immunofluorescence analysis. The MAbs obtained in this study are useful in understanding the pathological basis of V-ATPase dysfunction.

SUMO wrestling in cell movement

SUMO wrestling in cell movement

SUMOylation of the GTPase Rac1 is required for optimal cell migration

The Rho-like GTPase Rac1 induces cytoskeletal rearrangements required for cell migration. Rac activity is regulated through a number of mechanisms, including control of nucleotide exchange and hydrolysis, regulation of subcellular localization, or modulation of protein expression levels1-3. Here, we identify the Small Ubiquitin-like MOdifier (SUMO) E3-ligase, PIAS3, as a new Rac1 interactor required for increased Rac activity and optimal cell migration in response to Hepatocyte Growth Factor (HGF) signalling. We show that Rac1 can be conjugated to SUMO-1 in response to HGF and that SUMOylation is enhanced by PIAS3. Moreover, we identify non-consensus sites within the polybasic region of Rac1 as the main locations for SUMO conjugation. We demonstrate that PIAS3-mediated SUMOylation of Rac1 controls its GTP-bound levels and its ability to stimulate lamellipodia, cell migration and invasion. This is the first time that a Ras superfamily member is found to be SUMOylated, providing a new insight into the regulation of these critical mediators of cell behaviour. Moreover, our data reveal a previously undescribed role for SUMO in the regulation of cell migration and invasion.

Regulation of subcellular localization of synapses by phosphatydylinositol signaling

Regulation of subcellular localization of synapses by phosphatydylinositol signaling

Rapid functional diversification in the structurally conserved ELAV family of neuronal RNA binding proteins

BackgroundThe Drosophila gene embryonic lethal abnormal visual system (elav) is the prototype of a gene family present in all metazoans. Its members encode structurally conserved neuronal proteins with three RNA Recognition Motifs (RRM) but they paradoxically act at diverse levels of post-transcriptional regulation. In an attempt to understand the history of this family, we searched for orthologs in eleven completely sequenced genomes, including those of humans, D. melanogaster and C. elegans, for which cDNAs are available.ResultsWe analyzed 23 orthologs/paralogs of elav, and found evidence of gain/loss of gene copy number. For one set of genes, including elav itself, the coding sequences are free of introns and their products most resemble ELAV. The remaining genes show remarkable conservation of their exon organization, and their products most resemble FNE and RBP9, proteins encoded by the two elav paralogs of Drosophila. Remarkably, three of the conserved exon junctions are both close to structural elements, involved respectively in protein-RNA interactions and in the regulation of sub-cellular localization, and in the vicinity of diverse sequence variations.ConclusionThe data indicate that the essential elav gene of Drosophila is newly emerged, restricted to dipterans and of retrotransposed origin. We propose that the conserved exon junctions constitute potential sites for sequence/function modifications, and that RRM binding proteins, whose function relies upon plastic RNA-protein interactions, may have played an important role in brain evolution.

An Essential Role for 14-3-3 Proteins in Brassinosteroid Signal Transduction in Arabidopsis

Brassinosteroids (BRs) are essential hormones for plant growth and development. BRs regulate gene expression by inducing dephosphorylation of two key transcription factors, BZR1 and BZR2/BES1, through a signal transduction pathway that involves cell-surface receptors (BRI1 and BAK1) and a GSK3 kinase (BIN2). How BR-regulated phosphorylation controls the activities of BZR1/BZR2 is not fully understood. Here, we show that BIN2-catalyzed phosphorylation of BZR1/BZR2 not only inhibits DNA binding, but also promotes binding to the 14-3-3 proteins. Mutations of a BIN2-phosphorylation site in BZR1 abolish 14-3-3 binding and lead to increased nuclear localization of BZR1 protein and enhanced BR responses in transgenic plants. Further, BR deficiency increases cytoplasmic localization, and BR treatment induces rapid nuclear localization of BZR1/BZR2. Thus, 14-3-3 binding is required for efficient inhibition of phosphorylated BR transcription factors, largely through cytoplasmic retention. This study demonstrates that multiple mechanisms are required for BR regulation of gene expression and plant growth.

MORN motifs in plant PIPKs are involved in the regulation of subcellular localization and phospholipid binding

Multiple repeats of membrane occupation and recognition nexus (MORN) motifs were detected in plant phosphatidylinositl monophosphate kinase (PIPK), a key enzyme in PI-signaling pathway. Structural analysis indicates that all the MORN motifs (with varied numbers at ranges of 7-9), which shared high homologies to those of animal ones, were located at N-terminus and sequentially arranged, except those of OsPIPK1 and AtPIPK7, in which the last MORN motif was separated others by an approximately 100 amino-acid "island" region, revealing the presence of two kinds of MORN arrangements in plant PIPKs. Through employing a yeast-based SMET (sequence of membrane-targeting) system, the MORN motifs were shown being able to target the fusion proteins to cell plasma membrane, which were further confirmed by expression of fused MORN-GFP proteins. Further detailed analysis via deletion studies indicated the MORN motifs in OsPIPK1, together with the 104 amino-acid "island" region are involved in the regulation of differential subcellular localization, i.e. plasma membrane or nucleus, of the fused proteins. Fat Western blot analysis of the recombinant MORN polypeptide, expressed in Escherichia coli, showed that MORN motifs could strongly bind to PA and relatively slightly to PI4P and PI(4,5)P2. These results provide informative hints on mechanisms of subcellular localization, as well as regulation of substrate binding, of plant PIPKs.

ARF-BP1/Mule Is a Critical Mediator of the ARF Tumor Suppressor

Although the importance of the ARF tumor suppressor in p53 regulation is well established, numerous studies indicate that ARF also suppresses cell growth in a p53/Mdm2-independent manner. To understand the mechanism of ARF-mediated tumor suppression, we identified a ubiquitin ligase, ARF-BP1, as a key factor associated with ARF in vivo. ARF-BP1 harbors a signature HECT motif, and its ubiquitin ligase activity is inhibited by ARF. Notably, inactivation of ARF-BP1, but not Mdm2, suppresses the growth of p53 null cells in a manner reminiscent of ARF induction. Surprisingly, in p53 wild-type cells, ARF-BP1 directly binds and ubiquitinates p53, and inactivation of endogenous ARF-BP1 is crucial for ARF-mediated p53 stabilization. Thus, our study modifies the current view of ARF-mediated p53 activation and reveals that ARF-BP1 is a critical mediator of both the p53-independent and p53-dependent tumor suppressor functions of ARF. As such, ARF-BP1 may serve as a potential target for therapeutic intervention in tumors regardless of p53 status.

A Serine/Threonine-rich Motif Is One of Three Nuclear Localization Signals That Determine Unidirectional Transport of the Mineralocorticoid Receptor to the Nucleus

The mineralocorticoid receptor (MR) is a tightly regulated nuclear hormone receptor that selectively transmits corticosteroid signals. Steroid treatment transforms MR from a transcriptionally inert state, in which it is distributed equally between the nucleus and cytoplasm, to an active completely nuclear transcription factor. We report here that MR is an atypical nuclear hormone receptor that moves unidirectionally from the cytoplasm to the nucleus. We show that nuclear import of MR is controlled through three nuclear localization signals (NLSs) of distinct types. Nuclear localization of naive MR was mediated primarily through a novel serine/threonine-rich NLS (NL0) in the receptor N terminus. Specific amino acid substitutions that mimicked phosphorylation selectively enhanced or repressed NL0 activity, highlighting the potential for active regulation of this new type of NLS. The second NLS (NL2) within the ligand-binding domain also lacks a recognizable basic motif. Nuclear transfer through this signal was strictly dependent on steroid agonist, but was independent of the interaction of MR with coactivator proteins. The third MR NLS (NL1) is a bipartite basic motif localized to the C terminus of the MR DNA-binding domain with properties distinct from those of NL1 of the closely related glucocorticoid receptor. NL1 acted in concert with NL0 and NL2 to stimulate nuclear uptake of the agonist-treated receptor, but also directed the complete nuclear localization of MR in response to treatment with steroid antagonist. These results present MR as a nuclear hormone receptor whose unidirectional transfer to the nucleus may be regulated through multiple pathways.