Microfilament-dependent modulation of cytoplasmic protein binding to TNFalpha mRNA AU-rich instability element in human lymphoid cells

Cytoplasmic proteins with binding capability to AU-rich instability determinant sequences (ARE) of tumour necrosis factor alpha (TNFalpha) mRNA 3' untranslated region (3'UTR) were assessed in human lymphoid cells. In vitro label transfer experiments using wild type as well as mutant sequences in which the 70 nucleotide-long AUUUA pentamer-containing portion of the 3'UTR had been deleted conferred binding specificity to five major activities of 22/25-, 38/40-, 50-, 60- and 80-kDa proteins in cytoplasmic extracts of peripheral blood mononuclear cells (PBMCs). Cytochalasin-induced disarrangement of the F-actin-based microfilament system led to a Triton X-100-insoluble to soluble redistribution of these binding activities. No such changes were observed in Jurkat tumour cells. Combination of in vivo UV-crosslinking and in vitro label transfer experiments revealed considerable differences in RNA association between proteins of the same cell type as well as between proteins of identical molecular weight (Mw) derived from either PBMCs or Jurkat cells. Our findings may explain some aspects of differential regulation of interleukin 2 (IL-2) and TNFalpha mRNA stability upon microfilament disruption in human PBMCs observed in an earlier study. These results also suggest that the physical state of cytoplasmic structural environment might contribute to important regulatory processes regarding key elements of eukaryotic mRNA metabolism, such as modulation of stability. Finally, these data highlight the possibility that the often observed disorganization of the cytoskeleton in tumour cells may partly be responsible for the maintenance of the neoplastic state, a phenomenon that potentially involves ARE-AUBP interactions.     

An inducible cytoplasmic factor (AU-B) binds selectively to AUUUA multimers in the 3'' untranslated region of lymphokine mRNA.

Considerable evidence suggests that the metabolism of lymphokine mRNAs can be selectively regulated within the cytoplasm. However, little is known about the mechanism(s) that cells use to discriminate lymphokine mRNAs from other mRNAs within the cytoplasm. In this study we report a sequence-specific cytoplasmic factor (AU-B) that binds specifically to AUUUA multimers present in the 3' untranslated region of lymphokine mRNAs. AU-B does not bind to monomeric AUUUA motifs nor to other AU-rich sequences present in the 3' untranslated region of c-myc mRNA. AU-B RNA-binding activity is not present in quiescent T cells but is rapidly induced by stimulation of the T-cell receptor/CD3 complex. Induction of AU-B RNA-binding activity requires new RNA and protein synthesis. Stabilization of lymphokine mRNA induced by costimulation with phorbol myristate acetate correlates inversely with binding by AU-B. Together, these data suggest that AU-B is a cytoplasmic regulator of lymphokine mRNA metabolism.     

The immediate-early gene product Egr-1 regulates the human interleukin-2 receptor beta-chain promoter through noncanonical Egr and Sp1 binding sites.

The interleukin-2 IL-2 receptor beta-chain (IL-2Rbeta) is an essential component of the receptors for IL-2 and IL-15. Although IL-2Rbeta is constitutively expressed by lymphocytes, its expression can be further induced by a number of stimuli, including phorbol 12-myristate 13-acetate (PMA). We have now characterized factors that bind to an enhancer region located between nucleotides -170 and -139 of the human IL-2Rbeta promoter. Both Sp1 and Sp3 bound to the 5' portion of this region, whereas a PMA-inducible factor (PIF) mainly bound to its 3' portion and bound to the Sp binding motifs as well. In Jurkat T cells, induction of PIF DNA binding activity was rapidly induced, required de novo protein synthesis, and was sustained at a high level for at least 23 h. Interestingly, PIF was constitutively activated in human T-cell leukemia virus type 1-transformed MT-2 cells. In this paper, we demonstrate that PIF is Egr-1 based on its recognition by anti-Egr-1 antisera in gel mobility shift assays, even though the IL-2Rbeta DNA binding motif differed substantially from the canonical Egr-1 binding site. In addition, Egr-1 bound to the Sp binding site. In Jurkat cells, both sites were required for maximal IL-2Rbeta promoter activity, and in HeLaS3 cells, transfection of Egr-1 could drive activity of a reporter construct containing both sites. Moreover, Sp1 and Egr-1 could form a complex with kinetics that correlated with the production of Egr-1 in Jurkat cells upon PMA stimulation. Thus, Sp1 and Egr-1 physically and functionally cooperate to mediate maximal IL-2Rbeta promoter activity.     

The yeast nuclear cap binding complex can interact with translation factor eIF4G and mediate translation initiation

The mRNA cap structure is bound by either the nuclear (CBC) or the cytoplasmic (eIF4F) cap binding complex. Following mRNA export, CBC must be exchanged for eIF4F in the cytoplasm. It is not known how this exchange occurs or how this RNP remodeling event is integrated with mRNA function. Here we report genetic and biochemical evidence that the yeast translation initiation factor eIF4G associates with CBC, and that eIF4E, the eIF4F component that binds both the cap and eIF4G, antagonizes this interaction. Furthermore, we find that CBC can stimulate translation in extracts containing an eIF4G protein deficient for eIF4E binding. These data suggest that eIF4E binding to the eIF4G-CBC complex on newly exported mRNA displaces CBC, and that the first round of translation on mRNA may occur via a different mechanism than subsequent rounds.     

Stimulation of T cells through the CD3/T-cell receptor complex: role of cytoplasmic calcium, protein kinase C translocation, and phosphorylation of pp60c-src in the activation pathway.

Stimulation of T cells or the Jurkat T-cell line with soluble antibodies to the CD3/T-cell receptor complex causes mobilization of cytoplasmic Ca2+, which is blocked by pertussis toxin but not by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and translocation of protein kinase C activity from the cytoplasm to the membrane. Such stimulation also causes phosphorylation of pp60c-src at an amino-terminal serine residue. These activities are consistent with induction of phosphatidylinositol metabolism after antibody binding. Anti-CD3 stimulation with antibody in solution, however, does not cause Jurkat cells to release interleukin 2 and blocks rather than induces proliferation of T cells. Induction of interleukin 2 production by Jurkat cells and proliferation by normal T cells requires anti-CD3 stimulation with antibody on a solid support, such as Sepharose beads or a plastic dish. Thus, we examined phosphorylation of pp60c-src after stimulation of Jurkat cells with anti-CD3 in solution or on solid phase. Both of these caused serine phosphorylation of pp60c-src that was indistinguishable even after 4 h of stimulation. These results indicate that the mode of anti-CD3 stimulation (in solution or on solid phase) controls a cellular function that modifies the consequences of signal transduction through phosphatidylinositol turnover.     

Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells.

T-cell receptor (TCR) cross-linking increases tyrosine phosphorylation of multiple proteins, only a few of which have been identified. One of the most rapidly tyrosine-phosphorylated polypeptides is the 120-kDa product of the proto-oncogene c-cbl, a cytosolic and cytoskeletal protein containing multiple proline-rich motifs that are potential binding sites for proteins containing Src homology 3 (SH3) domains. We report here that in cultured Jurkat T cells, Cbl is coprecipitated with antibody against the adapter protein Grb2. Upon activation of Jurkat T cells via the TCR-CD3 complex, we find that high-affinity binding of Cbl requires the N-terminal SH3 domain of GST-Grb2 fusion protein but after cross-linking of the TCR-CD3 and CD4 receptors, Cbl binds equally to its SH2 domain. Grb2 antisera also precipitated p85 from serum-starved cells, while TCR activation increased p85 and tyrosine-phosphorylated Cbl but not Cbl protein in Grb2 immunocomplexes. Phosphatidylinositol (PI) 3-kinase activity was immunoprecipitated from serum-starved cells with Cbl and to a lesser extent with Grb2 antisera, and TCR cross-linking increased this activity severalfold. The PI 3-kinase activity associated with Cbl amounted to 5 to 10% of the total cellular activity that could be precipitated by p85 antisera. The Ras exchange factor Son-of-sevenless 1 (Sos-1) was not found in anti-Cbl immunoprecipitates from activated cells, and Cbl was not detectable in anti-Sos-1 precipitates, supporting the likelihood that Sos-Grb2 and Cbl-Grb2 are present as distinct complexes. Taken together, these data suggest that Cbl function in Jurkat T cells involves its constitutive association with Grb2 and its recruitment of PI 3-kinase in response to TCR activation.     

Characterization of a maize G-box binding factor that is induced by hypoxia

G-box cis-acting DNA sequence elements are present in the promoter region of a number of signal-inducible plant genes. In many cases this motif is essential for gene expression. Maize nuclear extracts contain a protein complex that binds specifically to the G-box sequence. Previously, a protein called GF14 was described that is physically associated with the G-box binding complex, but is not a DNA-binding factor in and of itself. This paper reports the isolation of a cDNA encoding a maize G-box binding factor (GBF). The deduced amino acid sequence indicates that maize GBF1 is a basic region-leucine zipper protein. GBF1 binds to the G-box element with specificity similar to that of the binding activity in nuclear extracts. Furthermore, maize GBF1 and the factor detected in nuclear extract are identical in their molecular weight and are immunologically related. GBF1 mRNA accumulates rapidly in hypoxically induced maize cells prior to the increase in Adh1 mRNA levels. Taken together with results that indicate that GBF1 binds to the hypoxia-responsive promoter of maize Adh1, these observations suggest that GBF1 may be one of the factors involved in the activation of Adh1.