Analysis of protein arginine methylation and protein arginine-methyltransferase activity.

Posttranslational modification of proteins allows cells to adapt and react quickly to their environment beyond the boundaries set forth by genetic code. Arginine methylation, a protein modification discovered almost 30 years ago, has recently experienced a renewed interest as several new arginine methyltransferases have been identified and numerous proteins were found to be regulated by methylation on arginine residues. Until recently, the detection of arginine methylation required the use of chromatography and mass-spectrometrical analysis. The following protocol provides guidelines for the straightforward identification of arginine-methylated proteins, made possible by the availability of novel, commercially available reagents.

Regulation of STAT protein synthesis by c-Cbl.

Many cytokines and growth factors induce transcription of immediate early response genes by activating members of the Signal Transducers and Activators of Transcription (STAT) family. Although significant progress has been made in understanding the events that lead to the activation of STAT proteins, less is known about the regulation of their expression. Here we report that murine embryonic fibroblasts derived from c-Cbl-deficient mice display significantly increased levels of STAT1 and STAT5 protein. In contrast, STAT2 and STAT3 expression, as well as the levels of the tyrosine kinases Jak1 and Tyk2, appear to be regulated independently of c-Cbl. Interestingly, the half-life of STAT1 was unaffected by the presence of c-Cbl, indicating that c-Cbl acts independently of STAT1 degradation. Further analysis revealed similar levels of STAT1 mRNA, however, a dramatically increased rate of STAT1 protein synthesis was observed in c-Cbl-deficient cells. Thus, our findings demonstrate an additional control mechanism over STAT1 function, and also provide a novel biological effect of the Cbl protein family.

Tumor necrosis factor receptor-associated factor (TRAF)2 represses the T helper cell type 2 response through interaction with NFAT-interacting protein (NIP45).

Recently we have identified a novel protein NIP45 (nuclear factor of activated T cells [NFAT]-interacting protein) which substantially augments interleukin (IL)-4 gene transcription. The provision of NIP45 together with NFAT and the T helper cell type 2 (Th2)-specific transcription factor c-Maf to cells normally refractory to IL-4 production, such as B cells or Th1 clones, results in substantial IL-4 secretion to levels that approximate those produced by primary Th2 cells. In studies designed to further our understanding of NIP45 activity, we have uncovered a novel facet of IL-4 gene regulation. We present evidence that members of the tumor necrosis factor receptor-associated factor (TRAF) family of proteins, generally known to function as adapter proteins that transduce signals from the tumor necrosis factor receptor superfamily, contribute to the repression of IL-4 gene transcription and that this effect is mediated through their interaction with NIP45.

Arginine methylation of STAT1 modulates IFNalpha/beta-induced transcription.

Transcriptional induction by interferons requires the tyrosine and serine phosphorylation of STAT transcription factors. The N-terminal region is highly homologous among the STAT proteins and surrounds a completely conserved arginine residue. Here we demonstrate arginine methylation of STAT1 by the protein arginine methyl-transferase PRMT1 as a novel requirement for IFNalpha/beta-induced transcription. Methyl-thioadenosine, a methyl-transferase inhibitor that accumulates in many transformed cells, inhibits STAT1-mediated IFN responses. This inhibition arises from impaired STAT1-DNA binding due to an increased association of the STAT inhibitor PIAS1 with phosphorylated STAT1 dimers in the absence of arginine methylation. Thus, arginine methylation of STAT1 is an additional posttranslational modification regulating transcription factor function, and alteration of arginine methylation might be responsible for the lack of interferon responsiveness observed in many malignancies.

Regulation of STAT1 nuclear export by Jak1.

Signal transducer and activator of transcription 1 (STAT1) mediates gene expression in response to cytokines and growth factors. Activation of STAT1 is achieved through its tyrosine phosphorylation, a process that involves Jak tyrosine kinases. Here we show that STAT1, although phosphorylated on Y701, is unable to localize in the nucleus in the absence of Jak1 or Jak1 kinase activity. In contrast, the nuclear accumulation of STAT1 in Tyk2-deficient cells remains intact. Nuclear presence of tyrosine-phosphorylated STAT1 could be restored in Jak1-deficient cells by leptomycin B, an inhibitor of nuclear export. Amino acids 197 to 205 of STAT1 were found to encode a leucine-rich nuclear export signal (NES). An L-->A mutation within the NES restored nuclear retention of STAT1 in Jak1-deficient cells. Impaired binding of the transcriptional coactivator CBP to tyrosine-phosphorylated STAT1 derived from Jak1-deficient cells offers a model for the intermolecular regulation of the nuclear export sequence.

Role of the STAT1-SH2 domain and STAT2 in the activation and nuclear translocation of STAT1.

Interferon (IFN) induction of immediate-early response genes is mediated through the signal transducers and activators of transcription (STATs). Activation of STAT1 by IFNalpha or IFNgamma through its tyrosine phosphorylation involves members of the Jak tyrosine kinases. In addition, STAT2 is activated by IFNalpha, and, together with STAT1 and p48/ISGF3gamma, forms the transcription factor complex ISGF3. Previous findings suggested that the STAT1-SH2 domain, which is required for the homo- or heterodimerization of STAT1, also participates in the recruitment of STAT1 to the IFN-receptors, because mutations in the SH2-domain abolished STAT1 activation by IFNgamma. Furthermore, STAT2 was reported to be required for the activation of STAT1 by IFNalpha. We were able to induce STAT1 tyrosine phosphorylation by IFNalpha/beta in the absence of STAT2 or a functional STAT1-SH2 domain. In contrast, IFNgamma was unable to cause tyrosine phosphorylation of STAT1-(SH2:Arg --> Gln). Interestingly, although STAT1 was found in the nucleus in STAT2-deficient cells, the nuclear accumulation of the tyrosine phosphorylated SH2-mutant STAT1 was impaired. In summary, our results indicate that the SH2 domain of STAT1 is not required for its ligand-dependent activation by IFNalpha/beta. Moreover, tyrosine phosphorylation is not sufficient to target STAT1 to the nucleus; rather, dimerization appears to play a critical role in the subcellular distribution of STAT1.

Characterization of a nuclear deformed epidermal autoregulatory factor-1 (DEAF-1)-related (NUDR) transcriptional regulator protein.

A monkey kidney cDNA that encodes a nuclear regulatory factor was identified by expression and affinity binding to a synthetic retinoic acid response element (RARE) and was used to isolate human placental and rat germ cell cDNAs by hybridization. The cDNAs encode a 59-kDa protein [nuclear DEAF-1-related (NUDR)] which shows sequence similarity to the Drosophila Deformed epidermal autoregulatory factor-1 (DEAF-1), a nonhomeodomain cofactor of embryonic Deformed gene expression. Similarities to other proteins indicate five functional domains in NUDR including an alanine-rich region prevalent in developmental transcription factors, a domain found in the promyelocytic leukemia-associated SP100 proteins, and a zinc finger homology domain associated with the AML1/MTG8 oncoprotein. Although NUDR mRNA displayed a wide tissue distribution in rats, elevated levels of protein were only observed in testicular germ cells, developing fetus, and transformed cell lines. Nuclear localization of NUDR was demonstrated by immunocytochemistry and by a green fluorescent protein-NUDR fusion protein. Site-directed mutagenesis of a nuclear localization signal resulted in cytoplasmic localization of the protein and eliminated NUDR-dependent transcriptional activation. Recombinant NUDR protein showed affinity for the RARE in mobility shifts; however it was efficiently displaced by retinoic acid receptor (RAR)/retinoid X receptor (RXR) complexes. In transient transfections, NUDR produced up to 26-fold inductions of a human proenkephalin promoter-reporter plasmid, with minimal effects on the promoters for prodynorphin or thymidine kinase. Placement of a RARE on the proenkephalin promoter increased NUDR-dependent activation to 41-fold, but this RARE-dependent increase was not transferable to a thymidine kinase promoter. Recombinant NUDR protein showed minimal binding affinity for proenkephalin promoter sequences, but was able to select DNA sequences from a random oligonucleotide library that had similar core-binding motifs (TTCG) as those recognized by DEAF-1. This motif is also present between the half-sites of several endogenous RAREs. The derived consensus- binding motif recognized by NUDR (TTCGGGNNTTTCCGG) was confirmed by mobility shift and deoxyribonuclease I (DNase I) protection assays; however, the consensus sequence was also unable to confer NUDR-dependent transcriptional activation to the thymidine kinase promoter. Our data suggests that NUDR may activate transcription independently of promoter binding, perhaps through protein-protein interaction with basal transcription factors, or by activation of secondary factors. The sequence and functional similarities between NUDR and DEAF-1 suggest that NUDR may also act as a cofactor to regulate the transcription of genes during fetal development or differentiation of testicular cells.

Cytomegalovirus activates interferon immediate-early response gene expression and an interferon regulatory factor 3-containing interferon-stimulated response element-binding complex.

Interferon establishes an antiviral state in numerous cell types through the induction of a set of immediate-early response genes. Activation of these genes is mediated by phosphorylation of latent transcription factors of the STAT family. We found that infection of primary foreskin fibroblasts with human cytomegalovirus (HCMV) causes selective transcriptional activation of the alpha/beta-interferon-responsive ISG54 gene. However, no activation or nuclear translocation of STAT proteins was detected. Activation of ISG54 occurs independent of protein synthesis but is prevented by protein tyrosine kinase inhibitors. Further analysis revealed that HCMV infection induced the DNA binding of a novel complex, tentatively called cytomegalovirus-induced interferon-stimulated response element binding factor (CIF). CIF is composed, at least in part, of the recently identified interferon regulatory factor 3 (IRF3), but it does not contain the STAT1 and STAT2 proteins that participate in the formation of interferon-stimulated gene factor 3. IRF3, which has previously been shown to possess no intrinsic transcriptional activation potential, interacts with the transcriptional coactivator CREB binding protein, but not with p300, to form CIF. Activating interferon-stimulated genes without the need for prior synthesis of interferons might provide the host cell with a potential shortcut in the activation of its antiviral defense.