A novel protein interacts with the Werner's syndrome gene product physically and functionally

Werner's syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging. The gene responsible for WS encodes a protein homologous to Escherichia coli RecQ. Here we describe a novel Werner helicase interacting protein (WHIP), which interacts with the N-terminal portion of Werner protein (WRN), containing the exonuclease domain. WHIP, which shows homology to replication factor C family proteins, is conserved from E. coli to human. Ectopically expressed WHIP and WRN co-localized in granular structures in the nucleus. The functional relationship between WHIP and WRN was indicated by genetic analysis of yeast cells. Disruptants of the SGS1 gene of Saccharomyces cerevisiae, which is the WRN homologue in yeast, show an accelerated aging phenotype and high sensitivity to methyl methanesulfonate as compared with wild-type cells. Disruption of the yeast WHIP (yWHIP) gene in wild-type cells and sgs1 disruptants resulted in slightly accelerated aging and enhancement of the premature aging phenotype of sgs1 disruptants, respectively. In contrast, disruption of the yWHIP gene partially alleviated the sensitivity to methyl methanesulfonate of sgs1 disruptants.     

WRN interacts physically and functionally with the recombination mediator protein RAD52

Werner syndrome (WS) is a premature aging disorder that predisposes affected individuals to cancer development. The affected gene, WRN, encodes an RecQ homologue whose precise biological function remains elusive. Altered DNA recombination is a hallmark of WS cells suggesting that WRN plays an important role in these pathways. Here we report a novel physical and functional interaction between WRN and the homologous recombination mediator protein RAD52. Fluorescence resonance energy transfer (FRET) analyses show that WRN and RAD52 form a complex in vivo that co-localizes in foci associated with arrested replication forks. Biochemical studies demonstrate that RAD52 both inhibits and enhances WRN helicase activity in a DNA structure-dependent manner, whereas WRN increases the efficiency of RAD52-mediated strand annealing between non-duplex DNA and homologous sequences contained within a double-stranded plasmid. These results suggest that coordinated WRN and RAD52 activities are involved in replication fork rescue after DNA damage.     

Xeroderma pigmentosum group C protein interacts physically and functionally with thymine DNA glycosylase

The XPC-HR23B complex recognizes various helix-distorting lesions in DNA and initiates global genome nucleotide excision repair. Here we describe a novel functional interaction between XPC-HR23B and thymine DNA glycosylase (TDG), which initiates base excision repair (BER) of G/T mismatches generated by spontaneous deamination of 5-methylcytosine. XPC-HR23B stimulated TDG activity by promoting the release of TDG from abasic sites that result from the excision of mismatched T bases. In the presence of AP endonuclease (APE), XPC-HR23B had an additive effect on the enzymatic turnover of TDG without significantly inhibiting the subsequent action of APE. Our observations suggest that XPC-HR23B may participate in BER of G/T mismatches, thereby contributing to the suppression of spontaneous mutations that may be one of the contributory factors for the promotion of carcinogenesis in xeroderma pigmentosum genetic complementation group C patients.     

ATM protein physically and functionally interacts with proliferating cell nuclear antigen to regulate DNA synthesis

Ataxia telangiectasia (A-T) is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation that is caused by biallelic mutations in A-T mutated (ATM), a gene encoding a protein kinase critical for the induction of cellular responses to DNA damage, particularly to DNA double strand breaks. A long known characteristic of A-T cells is their ability to synthesize DNA even in the presence of ionizing radiation-induced DNA damage, a phenomenon termed radioresistant DNA synthesis. We previously reported that ATM kinase inhibition, but not ATM protein disruption, blocks sister chromatid exchange following DNA damage. We now show that ATM kinase inhibition, but not ATM protein disruption, also inhibits DNA synthesis. Investigating a potential physical interaction of ATM with the DNA replication machinery, we found that ATM co-precipitates with proliferating cell nuclear antigen (PCNA) from cellular extracts. Using bacterially purified ATM truncation mutants and in vitro translated PCNA, we showed that the interaction is direct and mediated by the C terminus of ATM. Indeed, a 20-amino acid region close to the kinase domain is sufficient for strong binding to PCNA. This binding is specific to ATM, because the homologous regions of other PIKK members, including the closely related kinase A-T and Rad3-related (ATR), did not bind PCNA. ATM was found to bind two regions in PCNA. To examine the functional significance of the interaction between ATM and PCNA, we tested the ability of ATM to stimulate DNA synthesis by DNA polymerase δ, which is implicated in both DNA replication and DNA repair processes. ATM was observed to stimulate DNA polymerase activity in a PCNA-dependent manner.     

FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae.

The peptidyl-prolyl isomerase FKBP12 was originally identified as the intracellular receptor for the immunosuppressive drugs FK506 (tacrolimus) and rapamycin (sirolimus). Although peptidyl-prolyl isomerases have been implicated in catalyzing protein folding, the cellular functions of FKBP12 in Saccharomyces cerevisiae and other organisms are largely unknown. Using the yeast two-hybrid system, we identified aspartokinase, an enzyme that catalyzes an intermediate step in threonine and methionine biosynthesis, as an in vivo binding target of FKBP12. Aspartokinase also binds FKBP12 in vitro, and drugs that bind the FKBP12 active site, or mutations in FKBP12 surface and active site residues, disrupt the FKBP12-aspartokinase complex in vivo and in vitro.fpr1 mutants lacking FKBP12 are viable, are not threonine or methionine auxotrophs, and express wild-type levels of aspartokinase protein and activity; thus, FKBP12 is not essential for aspartokinase activity. The activity of aspartokinase is regulated by feedback inhibition by product, and genetic analyses reveal that FKBP12 is important for this feedback inhibition, possibly by catalyzing aspartokinase conformational changes in response to product binding.     

eIF5A interacts functionally with eEF2

eIF5A is highly conserved from archaea to mammals, essential for cell viability and the only protein known to contain the essential amino acid residue hypusine, generated by a unique posttranslational modification. eIF5A was originally identified as a translation initiation factor due to its ability to stimulate the formation of the first peptide bond. However, recent studies have shown that depletion of eIF5A causes a significant decrease in polysome run-off and an increase in the ribosome transit time, suggesting that eIF5A is actually involved in the elongation step of protein synthesis. We have previously shown that the depletion mutant tif51A-3 (eIF5A(C39Y/G118D)) shows a sicker phenotype when combined with the dominant negative mutant eft2 ( H699K ) of the elongation factor eEF2. In this study, we used the eIF5A(K56A) mutant to further investigate the relationship between eIF5A and eEF2. The eIF5A(K56A) mutant is temperature sensitive and has a defect in protein synthesis, but instead of causing depletion of the eIF5A protein, this mutant has a defect in hypusine modification. Like the mutant tif51A-3, the eIF5A(K56A) mutant is synthetic sick with the mutant eft2 ( H699K ) of eEF2. High-copy eEF2 not only improves cell growth of the eIF5A(K56A) mutant, but also corrects its increased cell size defect. Moreover, eEF2 suppression of the eIF5A(K56A) mutant is correlated with the improvement of total protein synthesis and with the increased resistance to the protein synthesis inhibitor hygromycin B. Finally, the polysome profile defect of the eIF5A(K56A) mutant is largely corrected by high-copy eEF2. Therefore, these results demonstrate that eIF5A is closely related to eEF2 function during translation elongation.     

The eukaryotic mRNA decapping protein Dcp1 interacts physically and functionally with the eIF4F translation initiation complex

Dcp1 plays a key role in the mRNA decay process in Saccharomyces cerevisiae, cleaving off the 5' cap to leave an end susceptible to exonucleolytic degradation. The eukaryotic initiation factor complex eIF4F, which in yeast contains the core components eIF4E and eIF4G, uses the cap as a binding site, serving as an initial point of assembly for the translation apparatus, and also binds the poly(A) binding protein Pab1. We show that Dcp1 binds to eIF4G and Pab1 as free proteins, as well as to the complex eIF4E-eIF4G-Pab1. Dcp1 interacts with the N-terminal region of eIF4G but does not compete significantly with eIF4E or Pab1 for binding to eIF4G. Most importantly, eIF4G acts as a function-enhancing recruitment factor for Dcp1. However, eIF4E blocks this effect as a component of the high affinity cap-binding complex eIF4E-eIF4G. Indeed, cooperative enhancement of the eIF4E-cap interaction stabilizes yeast mRNAs in vivo. These data on interactions at the interface between translation and mRNA decay suggest how events at the 5' cap and 3' poly(A) tail might be coupled.     

Replication protein A physically interacts with the Bloom's syndrome protein and stimulates its helicase activity

Bloom's syndrome is a rare autosomal recessive disorder characterized by genomic instability and predisposition to cancer. BLM, the gene defective in Bloom's syndrome, encodes a 159-kDa protein possessing DNA-stimulated ATPase and ATP-dependent DNA helicase activities. We have examined mechanistic aspects of the catalytic functions of purified recombinant BLM protein. Through analyzing the effects of different lengths of DNA cofactor on ATPase activity, we provide evidence to suggest that BLM translocates along single-stranded DNA in a processive manner. The helicase reaction catalyzed by BLM protein was examined as a function of duplex DNA length. We show that BLM catalyzes unwinding of short DNA duplexes (/=259-bp). The presence of the human single-stranded DNA-binding protein (human replication protein A (hRPA)) stimulates the BLM unwinding reaction on the 259-bp partial duplex DNA substrate. Heterologous single-stranded DNA-binding proteins fail to stimulate similarly the helicase activity of BLM protein. This is the first demonstration of a functional interaction between BLM and another protein. Consistent with a functional interaction between hRPA and the BLM helicase, we demonstrate a direct physical interaction between the two proteins mediated by the 70-kDa subunit of RPA. The interactions between BLM and hRPA suggest that the two proteins function together in vivo to unwind DNA duplexes during replication, recombination, or repair.     

LIM-only protein FHL3 interacts with CDC25B2 phosphatase

LIM domain proteins are important regulators of the growth, determination, and differentiation of cells. In this report, FHL3 (human four-and-a-half LIM-only protein 3) is shown to interact with human phosphatase CDC25B, a cell cycle regulator involved in the control of G2/M. We found that this interaction was specific to the CDC25B2 isoform. Deletion and point mutation studies indicated that the second LIM domain of FHL3 was essential for this interaction. FRET experiments in C2C12 cells showed that, although both proteins were colocated in the cytoplasm and the nucleus, they interacted only in the nucleus. Finally, we showed that FHL3 binding impaired neither CDC25B2 phosphatase activity nor its localization. Further work is now needed to elucidate the consequences of this interaction on myoblast fate decision and cycle control.     

FtsY, the bacterial signal-recognition particle receptor, interacts functionally and physically with the SecYEG translocon

Co-translational membrane targeting of proteins by the bacterial signal-recognition particle (SRP) requires the specific interaction of the SRP-ribosome nascent chain complex with FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRalpha-subunit of the eukaryotic SR, which is tethered to the endoplasmic-reticulum membrane by its interaction with the integral SRbeta-subunit. In contrast to SRalpha, FtsY is partly membrane associated and partly located in the cytosol. However, the mechanisms by which FtsY associates with the membrane are unclear. No gene encoding an SRbeta homologue has been found in bacterial genomes, and the presence of an FtsY-specific membrane receptor has not been shown so far. We now provide evidence for the direct interaction between FtsY and the SecY translocon. This interaction offers an explanation of how the bacterial SRP cycle is regulated in response to available translocation channels.     

MAPK phosphatase MKP2 mediates disease responses in Arabidopsis and functionally interacts with MPK3 and MPK6

Mitogen‐activated protein kinase (MAPK) cascades have important functions in plant stress responses and development and are key players in reactive oxygen species (ROS) signalling and in innate immunity. In Arabidopsis, the transmission of ROS and pathogen signalling by MAPKs involves the coordinated activation of MPK6 and MPK3; however, the specificity of their negative regulation by phosphatases is not fully known. Here, we present genetic analyses showing that MAPK phosphatase 2 (MKP2) regulates oxidative stress and pathogen defence responses and functionally interacts with MPK3 and MPK6. We show that plants lacking a functional MKP2 gene exhibit delayed wilting symptoms in response to Ralstonia solanacearum and, by contrast, acceleration of disease progression during Botrytis cinerea infection, suggesting that this phosphatase plays differential functions in biotrophic versus necrotrophic pathogen‐induced responses. MKP2 function appears to be linked to MPK3 and MPK6 regulation, as indicated by BiFC experiments showing that MKP2 associates with MPK3 and MPK6 in vivo and that in response to fungal elicitors MKP2 exerts differential affinity versus both kinases. We also found that MKP2 interacts with MPK6 in HR‐like responses triggered by fungal elicitors, suggesting that MPK3 and MPK6 are subject to differential regulation by MKP2 in this process. We propose that MKP2 is a key regulator of MPK3 and MPK6 networks controlling both abiotic and specific pathogen responses in plants.     

The AKT3 potassium channel protein interacts with the AtPP2CA protein phosphatase 2C

The AKT3 potassium channel protein was identified as a strongly interacting partner of the Arabidopsis thaliana protein phosphatase 2C (AtPP2CA) in a yeast two-hybrid screen. A deletion analysis indicated that the catalytic domain of AtPP2CA was essential for the interaction with AKT3. Furthermore, the related PP2C phosphatase ABI1 did not interact with AKT3 in yeast.     

Pseudorabies virus UL36 tegument protein physically interacts with the UL37 protein

The UL36 open reading frame encoding the tegument protein ICP1/2 represents the largest open reading frame in the genome of herpes simplex virus type 1 (HSV-1). Polypeptides homologous to the HSV-1 UL36 protein are present in all subfamilies of HERPESVIRIDAE: We sequenced the UL36 gene of the alphaherpesvirus pseudorabies virus (PrV) and prepared a monospecific polyclonal rabbit antiserum against a bacterial glutathione S-transferase (GST)-UL36 fusion protein for identification of the protein. The antiserum detected a >300-kDa protein in PrV-infected cells and in purified virions. Interestingly, in coprecipitation analyses using radiolabeled infected-cell extracts, the anti-UL36 serum reproducibly coprecipitated the UL37 tegument protein, and antiserum directed against the UL37 protein coprecipitated the UL36 protein. This physical interaction could be verified using yeast two-hybrid analysis which demonstrated that the UL37 protein interacts with a defined region within the amino-terminal part of the UL36 protein. By use of immunogold labeling, capsids which accumulate in the cytoplasm in the absence of the UL37 protein (B. G. Klupp, H. Granzow, E. Mundt, and T. C. Mettenleiter, J. Virol. 75:8927-8936, 2001) as well as wild-type intracytoplasmic and extracellular virions were decorated by the anti-UL36 antiserum, whereas perinuclear primary enveloped virions were not. We postulate that the physical interaction of the UL36 protein, which presumably constitutes the innermost layer of the tegument (Z. Zhou, D. Chen, J. Jakana, F. J. Rixon, and W. Chiu, J. Virol. 73:3210-3218, 1999), with the UL37 protein is an important early step in tegumentation during virion morphogenesis in the cytoplasm.