Characterization of a murine gene encoding an acidic-basic dipeptide repeat that interacts with GADD34

GADD34 is one of a subset of proteins induced after DNA damage or cell growth arrest. To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with murine GADD34. We utilized as bait the partial product of GADD34 cDNA including the PEST region and the gamma(1)34.5. One cDNA clone was almost the same as MuRED, which encodes an acidic-basic dipeptide repeat; we named it G34BP. The interaction between GADD34 and G34BP was also confirmed in the NIH3T3 cells by in vivo two-hybrid analysis. For the binding of two proteins, the PEST region was important, and the C-terminal of G34BP was necessary. G34BP was detected in all the mouse tissues examined. Although GADD34 was significantly elevated with methyl methanesulfonate treatment, G34BP expression was not induced. Overexpression of G34BP in the NIH3T3 cells inhibited the cell growth analyzed by WST1 assay.     

Evidence for the interaction between Translin and GADD34 in mammalian cells.

To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with the murine GADD34 gene product. We utilized, as bait, the product of the GADD34 cDNA deletions including the PEST region and the gamma(1)34.5 domain. One of the cDNAs cloned encoded murine Translin which is known to bind to the DNA sequence detected in the DNA translocation. The interaction between GADD34 and Translin was also confirmed by an in vitro binding assay and in vivo two-hybrid analysis in NIH 3T3 cells. Although GADD34 expression was significantly elevated with methyl methanesulfonate treatment, we could not detect the induction of Translin mRNA.     

Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1

The growth arrest and DNA damage-inducible protein, GADD34, was identified by its interaction with human inhibitor 1 (I-1), a protein kinase A (PKA)-activated inhibitor of type 1 protein serine/threonine phosphatase (PP1), in a yeast two-hybrid screen of a human brain cDNA library. Recombinant GADD34 (amino acids 233 to 674) bound both PKA-phosphorylated and unphosphorylated I-1(1-171). Serial truncations mapped the C terminus of I-1 (amino acids 142 to 171) as essential for GADD34 binding. In contrast, PKA phosphorylation was required for PP1 binding and inhibition by the N-terminal I-1(1-80) fragment. Pulldowns of GADD34 proteins expressed in HEK293T cells showed that I-1 bound the central domain of GADD34 (amino acids 180 to 483). By comparison, affinity isolation of cellular GADD34/PP1 complexes showed that PP1 bound near the C terminus of GADD34 (amino acids 483 to 619), a region that shows sequence homology with the virulence factors ICP34.5 of herpes simplex virus and NL-S of avian sarcoma virus. While GADD34 inhibited PP1-catalyzed dephosphorylation of phosphorylase a, the GADD34-bound PP1 was an active eIF-2alpha phosphatase. In brain extracts from active ground squirrels, GADD34 bound both I-1 and PP1 and eIF-2alpha was largely dephosphorylated. In contrast, the I-1/GADD34 and PP1/GADD34 interactions were disrupted in brain from hibernating animals, in which eIF-2alpha was highly phosphorylated at serine-51 and protein synthesis was inhibited. These studies suggested that modification of the I-1/GADD34/PP1 signaling complex regulates the initiation of protein translation in mammalian tissues.     

The kinesin-like motor protein KIF1C occurs in intact cells as a dimer and associates with proteins of the 14-3-3 family

Proteins of the kinesin superfamily are regulated in their motor activity as well as in their ability to bind to their cargo by carboxyl-terminal associating proteins and phosphorylation. KIF1C, a recently identified member of the KIF1/Unc104 family, was shown to be involved in the retrograde vesicle transport from the Golgi-apparatus to the endoplasmic reticulum. In a yeast two-hybrid screen using the carboxyl-terminal 350 amino acids of KIF1C as a bait, we identified as binding proteins 14-3-3 beta, gamma, epsilon, and zeta. In addition, a clone encoding the carboxyl-terminal 290 amino acids of KIF1C was found, indicating a potential for KIF1C to dimerize. Subsequent transient overexpression experiments showed that KIF1C can dimerize efficiently. However, in untransfected cells, only a small portion of KIF1C was detected as a dimer. The association of 14-3-3 proteins with KIF1C could be confirmed in transient expression systems and in untransfected cells and was dependent on the phosphorylation of serine 1092 located in a consensus binding sequence for 14-3-3 ligands. Serine 1092 was a substrate for the protein kinase casein kinase II in vitro, and inhibition of casein kinase II in cells diminished the association of KIF1C with 14-3-3gamma. Our data thus suggest that KIF1C can form dimers and is associated with proteins of the 14-3-3 family.     

Growth arrest and DNA damage-inducible protein GADD34 targets protein phosphatase 1 alpha to the endoplasmic reticulum and promotes dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2

The growth arrest and DNA damage-inducible protein, GADD34, associates with protein phosphatase 1 (PP1) and promotes in vitro dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2, (eIF-2 alpha). In this report, we show that the expression of human GADD34 in cultured cells reversed eIF-2 alpha phosphorylation induced by thapsigargin and tunicamycin, agents that promote protein unfolding in the endoplasmic reticulum (ER). GADD34 expression also reversed eIF-2 alpha phosphorylation induced by okadaic acid but not that induced by another phosphatase inhibitor, calyculin A (CA), which is a result consistent with PP1 being a component of the GADD34-assembled eIF-2 alpha phosphatase. Structure-function studies identified a bipartite C-terminal domain in GADD34 that encompassed a canonical PP1-binding motif, KVRF, and a novel RARA sequence, both of which were required for PP1 binding. N-terminal deletions of GADD34 established that while PP1 binding was necessary, it was not sufficient to promote eIF-2 alpha dephosphorylation in cells. Imaging of green fluorescent protein (GFP)-GADD34 proteins showed that the N-terminal 180 residues directed the localization of GADD34 at the ER and that GADD34 targeted the alpha isoform of PP1 to the ER. These data provide new insights into the mode of action of GADD34 in assembling an ER-associated eIF-2 alpha phosphatase that regulates protein translation in mammalian cells.     

Suppression of viral replication by stress-inducible GADD34 protein via the mammalian serine/threonine protein kinase mTOR pathway

GADD34 is a protein that is induced by a variety of stressors, including DNA damage, heat shock, nutrient deprivation, energy depletion, and endoplasmic reticulum stress. Here, we demonstrated that GADD34 induced by vesicular stomatitis virus (VSV) infection suppressed viral replication in wild-type (WT) mouse embryo fibroblasts (MEFs), whereas replication was enhanced in GADD34-deficient (GADD34-KO) MEFs. Enhanced viral replication in GADD34-KO MEFs was reduced by retroviral gene rescue of GADD34. The level of VSV protein expression in GADD34-KO MEFs was significantly higher than that in WT MEFs. Neither phosphorylation of eIF2alpha nor cellular protein synthesis was correlated with viral replication in GADD34-KO MEFs. On the other hand, phosphorylation of S6 and 4EBP1, proteins downstream of mTOR, was suppressed by VSV infection in WT MEFs but not in GADD34-KO MEFs. GADD34 was able to associate with TSC1/2 and dephosphorylate TSC2 at Thr1462. VSV replication was higher in TSC2-null cells than in TSC2-expressing cells, and constitutively active Akt enhanced VSV replication. On the other hand, rapamycin, an mTOR inhibitor, significantly suppressed VSV replication in GADD34-KO MEFs. These findings demonstrate that GADD34 induced by VSV infection suppresses viral replication via mTOR pathway inhibition, indicating that cross talk between stress-inducible GADD34 and the mTOR signaling pathway plays a critical role in antiviral defense.     

Kinesin superfamily protein 3 (KIF3) motor transports fodrin-associating vesicles important for neurite building

Kinesin superfamily proteins (KIFs) comprise several dozen molecular motor proteins. The KIF3 heterotrimer complex is one of the most abundantly and ubiquitously expressed KIFs in mammalian cells. To unveil the functions of KIF3, microinjection of function-blocking monovalent antibodies against KIF3 into cultured superior cervical ganglion (SCG) neurons was carried out. They significantly blocked fast axonal transport and brought about inhibition of neurite extension. A yeast two-hybrid binding assay revealed the association of fodrin with the KIF3 motor through KAP3. This was further confirmed by using vesicles collected from large bundles of axons (cauda equina), from which membranous vesicles could be prepared in pure preparations. Both immunoprecipitation and immunoelectron microscopy indicated the colocalization of fodrin and KIF3 on the same vesicles, the results reinforcing the evidence that the cargo of the KIF3 motor consists of fodrin-associating vesicles. In addition, pulse-labeling study implied partial comigration of both molecules as fast flow components. Taken together, the KIF3 motor is engaged in fast axonal transport that conveys membranous components important for neurite extension.     

Kaposi's Sarcoma-Associated Herpesvirus ORF45 Interacts with Kinesin-2 Transporting Viral Capsid-Tegument Complexes along Microtubules

Open reading frame (ORF) 45 of Kaposi''s sarcoma-associated herpesvirus (KSHV) is a tegument protein. A genetic analysis with a null mutant suggested a possible role for this protein in the events leading to viral egress. In this study, ORF45 was found to interact with KIF3A, a kinesin-2 motor protein that transports cargoes along microtubules to cell periphery in a yeast two-hybrid screen. The association was confirmed by both co-immunoprecipitation and immunoflorescence approaches in primary effusion lymphoma cells following virus reactivation. ORF45 principally mediated the docking of entire viral capsid-tegument complexes onto the cargo-binding domain of KIF3A. Microtubules served as the major highways for transportation of these complexes as evidenced by drastically reduced viral titers upon treatment of cells with a microtubule depolymerizer, nocodazole. Confocal microscopic images further revealed close association of viral particles with microtubules. Inhibition of KIF3A–ORF45 interaction either by the use of a headless dominant negative (DN) mutant of KIF3A or through shRNA-mediated silencing of endogenous KIF3A expression noticeably decreased KSHV egress reflecting as appreciable reductions in the release of extracellular virions. Both these approaches, however, failed to impact HSV-1 egress, demonstrating the specificity of KIF3A in KSHV transportation. This study thus reports on transportation of KSHV viral complexes on microtubules by KIF3A, a kinesin motor thus far not implicated in virus transportation. All these findings shed light on the understudied but significant events in the KSHV life cycle, delineating a crucial role of a KSHV tegument protein in cellular transport of viral particles.     

Binding of Murine Leukemia Virus Gag Polyproteins to KIF4, a Microtubule-Based Motor Protein

A cDNA clone encoding a cellular protein that interacts with murine leukemia virus (MuLV) Gag proteins was isolated from a T-cell lymphoma library. The sequence of the clone is identical to the C terminus of a cellular protein, KIF4, a microtubule-associated motor protein that belongs to the kinesin superfamily. KIF4-MuLV Gag associations have been detected in vitro and in vivo in mammalian cells. We suggest that KIF4 could be involved in Gag polyprotein translocation from the cytoplasm to the cell membrane.     

Mapping the GRIF-1 binding domain of the kinesin, KIF5C, substantiates a role for GRIF-1 as an adaptor protein in the anterograde trafficking of cargoes

Gamma-aminobutyric acid, type A (GABAA) receptor interacting factor-1 (GRIF-1) and N-acetylglucosamine transferase interacting protein (OIP) 106 are both members of a newly identified coiled-coil family of proteins. They are kinesin-associated proteins proposed to function as adaptors in the anterograde trafficking of organelles to synapses. Here we have studied in more detail the interaction between the prototypic kinesin heavy chain, KIF5C, kinesin light chain, and GRIF-1. The GRIF-1 binding site of KIF5C was mapped using truncation constructs in yeast two-hybrid interaction assays, co-immunoprecipitations, and co-localization studies following expression in mammalian cells. Using these approaches, it was shown that GRIF-1 and the KIF5C binding domain of GRIF-1, GRIF-1-(124-283), associated with the KIF5C non-motor domain. Refined studies using yeast two-hybrid interactions and co-immunoprecipitations showed that GRIF-1 and GRIF-1-(124-283) associated with the cargo binding region within the KIF5C non-motor domain. Substantiation that the GRIF-1-KIF5C interaction was direct was shown by fluorescence resonance energy transfer analyses using fluorescently tagged GRIF-1 and KIF5C constructs. A significant fluorescence resonance energy transfer value was found between the C-terminal EYFP-tagged KIF5C and ECFP-GRIF-1, the C-terminal EYFP-tagged KIF5C non-motor domain and ECFP-GRIF-1, but not between the N-terminal EYFP-tagged KIF5C nor the EYFP-KIF5C motor domain and ECFP-GRIF-1, thus confirming direct association between the two proteins at the KIF5C C-terminal and GRIF-1 N-terminal regions. Co-immunoprecipitation and confocal imaging strategies further showed that GRIF-1 can bind to the tetrameric kinesin light-chain/kinesin heavy-chain complex. These findings support a role for GRIF-1 as a kinesin adaptor molecule requisite for the anterograde delivery of defined cargoes such as mitochondria and/or vesicles incorporating beta2 subunit-containing GABAA receptors, in the brain.     

Association of the testis-specific TRIM/RBCC protein RNF33/TRIM60 with the cytoplasmic motor proteins KIF3A and KIF3B

The Rnf33/Trim60 gene is temporally transcribed in the preimplantation embryo before being silenced at the blastocyst stage but Rnf33 expression is detected in adult testis of the mouse. The putative RNF33 protein is a tripartite motif (TRIM)/RBCC protein composed of a typical RING zinc finger, a B-box 2, two α-helical coiled-coil segments, and a B30.2 domain. As a first step towards the elucidation of the biologic function of RNF33, we aimed in this study to elucidate proteins that associate with RNF33. RNF33-interacting proteins were first derived by the yeast two-hybrid system followed by co-immunoprecipitation assays. Interacting domains were determined by deletion mapping in genetic and biochemical analyzes. RNF33 was shown to interact with the kinesin-2 family members 3A (KIF3A) and 3B (KIF3B) motor proteins in the heterodimeric form known to transport cargos along the microtubule. Domain mapping showed that the RB and B30.2 domains of RNF33 interacted with the respective carboxyl non-motor domains of KIF3A and KIF3B. Since RNF33 interacted with the carboxyl-terminal tail of the KIF3A-KIF3B heterodimer, the motor head section of KIF3A-KIF3B was free and available for association with designated cargo(s) and movement along the microtubule. Data also suggest that RNF33 most likely interacted with KIF3A-KIF3B independent of the adaptor kinesin-associated protein KAP3. This study is a first demonstration of a TRIM protein, namely RNF33, that interacts with the kinesin molecular motors possibly contributing to kinesin-dependent mobilization of specific cargo(s) along the microtubule in the testis of the mouse.     

The novel protein KBP regulates mitochondria localization by interaction with a kinesin-like protein

Background  

Members of the Kinesin-3 family of kinesin-like proteins mediate transport of axonal vesicles (KIF1A, KIF1Bβ), distribution of mitochondria (KIF1Bα) and anterograde Golgi to ER vesicle transport (KIF1C). Until now, little is known about the regulation of kinesin-like proteins. Several proteins interact with members of this protein family. Here we report on a novel, KIF1 binding protein (KBP) that was identified in yeast two-hybrid screens.     

KIF2Abeta: A kinesin family member enriched in mouse male germ cells, interacts with translin associated factor-X (TRAX)

Translin associated factor X (TRAX) is a binding partner of TB-RBP/Translin. A cDNA encoding the 260 C-terminal amino acids of KIF2Abeta was isolated from mouse testis cDNAs in a yeast two-hybrid library screen for specific TRAX-interacting proteins. KIF2Abeta was expressed predominantly in the mouse testis and enriched in germ cells. The interaction of full-length KIF2Abeta or its C-terminus with TRAX was verified using in vitro synthesized fusion proteins. Deletion mapping of the TRAX-binding region of KIF2Abeta indicated that amino acids 514-659 were necessary and sufficient for the interaction in vivo. Confocal microscopy studies using GFP-fusion proteins demonstrated that KIF2Abeta colocalizes with TRAX in a perinuclear location. KIF2Abeta does not interact with TB-RBP, suggesting that either TRAX can function as an adaptor molecule for motor proteins and TB-RBP, or that this interaction reveals an undescribed role for TRAX in germ cells. The interaction with KIF2Abeta suggests a role for TRAX in microtubule-based functions during spermatogenesis.     

Inhibition of palmitate‐induced GADD34 expression augments apoptosis in mouse insulinoma cells (MIN6)

Saturated fatty acids like palmitate induce endoplasmic reticulum (ER) stress in pancreatic beta‐cells, an event linked to apoptotic loss of β‐cells in type 2 diabetes. Sustained activation of the ER stress response leads to expression of growth arrest and DNA damage‐inducible protein 34 (GADD34), a regulatory subunit of protein phosphatase 1. In the present study, we have used small interfering RNA in order to knockdown GADD34 expression in insulin‐producing MIN6 cells prior to induction of ER stress by palmitate and evaluated its consequences on RNA‐activated protein kinase‐like ER‐localized eIF2alpha kinase (PERK) signalling and apoptosis. Salubrinal, a specific inhibitor of eukaryotic initiation factor 2α (eIF2α) dephosphorylation, was used as a comparison. Salubrinal treatment augmented palmitate‐induced ER stress and increased GADD34 levels. Both GADD34 knockdown and salubrinal treatment potentiated the cytotoxic effects of palmitate as evidenced by increased DNA fragmentation and activation of caspase 3, with the fundamental difference that the former did not involve enhanced levels of GADD34. The data from this study suggest that sustained activation of PERK signalling and eIF2α phosphorylation sensitizes insulin‐producing MIN6 cells to lipoapoptosis independently of GADD34 expression levels. Copyright © 2014 John Wiley & Sons, Ltd.     

Control of cellular GADD34 levels by the 26S proteasome

GADD34, the product of a growth arrest and DNA damage-inducible gene, is expressed at low levels in unstressed cells. In response to stress, the cellular content of GADD34 protein increases and, on termination of stress, rapidly declines. We investigated the mechanisms that control GADD34 levels in human cells. GADD34 proteins containing either an internal FLAG or a C-terminal green fluorescent protein epitope were degraded at rates similar to endogenous GADD34. However, the addition of epitopes at the N terminus or deletion of N-terminal sequences stabilized GADD34. N-terminal peptides of GADD34, either alone or fused to heterologous proteins, exhibited rapid degradation similar to wild-type GADD34, thereby identifying an N-terminal degron. Deletion of internal PEST repeats had no impact on GADD34 stability but modulated the binding and activity of protein phosphatase 1. Proteasomal but not lysosomal inhibitors enhanced GADD34 stability and eukaryotic initiation factor 2α (eIF-2α) dephosphorylation, a finding consistent with GADD34's role in assembling an eIF-2α phosphatase. GADD34 was polyubiquitinated, and this modification enhanced its turnover in cells. A stabilized form of GADD34 promoted the accumulation and aggregation of the mutant cystic fibrosis transmembrane conductance regulator (CFTRΔF508), highlighting the physiological importance of GADD34 turnover in protein processing in the endoplasmic reticulum and the potential impact of prolonged GADD34 expression in human disease.