IL-1-induced NFκB and c-Jun N-terminal kinase (JNK) activation diverge at IL-1 receptor-associated kinase (IRAK)

Mutant I1A cells, lacking IL-1 receptor-associated kinase (IRAK) mRNA and protein, have been used to study the involvement of IRAK in NFκB and c-Jun N-terminal kinase (JNK) activation. A series of IRAK deletion constructs were expressed in I1A cells, which were then tested for their ability to respond to IL-1. Both the N-terminal death domain and the C-terminal region of IRAK are required for IL-1-induced NFκB and JNK activation, whereas the N-proximal undetermined domain is required for the activation of NFκB but not JNK. The phosphorylation and ubiquitination of IRAK deletion mutants correlate tightly with their ability to activate NFκB in response to IL-1, but IRAK can mediate IL-1-induced JNK activation without being phosphorylated. These studies reveal that the IL-1-induced signaling pathways leading to NFκB and JNK activation diverge either at IRAK or at a point nearer to the receptor.

Role of MEKK2-MEK5 in the regulation of TNF-α gene expression and MEKK2-MKK7 in the activation of c-Jun N-terminal kinase in mast cells

Cross-linking of the high-affinity IgE receptor (FcɛRI) on mast cells with IgE and multivalent antigen triggers mitogen-activated protein (MAP) kinase activation and cytokine gene expression. We report here that MAP kinase kinase 4 (MKK4) gene disruption does not affect either MAP kinase activation or cytokine gene expression in response to cross-linking of FcɛRI in embryonic stem cell-derived mast cells. MKK7 is activated in response to cross-linking of FcɛRI, and this activation is inhibited by MAP/ERK kinase (MEK) kinase 2 (MEKK2) gene disruption. In addition, expression of kinase-inactive MKK7 in the murine mast cell line MC/9 inhibits c-Jun NH2-terminal kinase (JNK) activation in response to cross-linking of FcɛRI, whereas expression of kinase-inactive MKK4 does not affect JNK activation by this stimulus. However, FcɛRI-induced activation of the tumor necrosis factor-α (TNF-α) gene promoter is not affected by expression of kinase-inactive MKK7. We describe an alternative pathway by which MEKK2 activates MEK5 and big MAP kinase1/extracellular signal-regulated kinase 5 in addition to MKK7 and JNK, and interruption of this pathway inhibits TNF-α promoter activation. These findings suggest that JNK activation by antigen cross-linking is dependent on the MEKK2-MKK7 pathway, and cytokine production in mast cells is regulated in part by the signaling complex MEKK2-MEK5-ERK5.

Ca2+-binding activity of a COOH-terminal fragment of the Drosophila BK channel involved in Ca2+-dependent activation

Mutational and biophysical analysis suggests that an intracellular COOH-terminal domain of the large conductance Ca2+-activated K+ channel (BK channel) contains Ca2+-binding site(s) that are allosterically coupled to channel opening. However the structural basis of Ca2+ binding to BK channels is unknown. To pursue this question, we overexpressed the COOH-terminal 280 residues of the Drosophila slowpoke BK channel (Dslo-C280) as a FLAG- and His6-tagged protein in Escherichia coli. We purified Dslo-C280 in soluble form and used a 45Ca2+-overlay protein blot assay to detect Ca2+ binding. Dslo-C280 exhibits specific binding of 45Ca2+ in comparison with various control proteins and known EF-hand Ca2+-binding proteins. A mutation (D5N5) of Dslo-C280, in which five consecutive Asp residues of the “Ca-bowl” motif are changed to Asn, reduces 45Ca2+-binding activity by 56%. By electrophysiological assay, the corresponding D5N5 mutant of the Drosophila BK channel expressed in HEK293 cells exhibits lower Ca2+ sensitivity for activation and a shift of ≈+80 mV in the midpoint voltage for activation. This effect is associated with a decrease in the Hill coefficient (N) for activation by Ca2+ and a reduction in apparent Ca2+ affinity, suggesting the loss of one Ca2+-binding site per monomer. These results demonstrate a functional correlation between Ca2+ binding to a specific region of the BK protein and Ca2+-dependent activation, thus providing a biochemical approach to study this process.

A Conserved Acidic Motif in the N-Terminal Domain of Nitrate Reductase Is Necessary for the Inactivation of the Enzyme in the Dark by Phosphorylation and 14-3-3 Binding1

It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611–621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II.

Solution structure of DFF40 and DFF45 N-terminal domain complex and mutual chaperone activity of DFF40 and DFF45

Apoptotic DNA fragmentation is mediated by a caspase-activated DNA fragmentation factor (DFF)40. Expression and folding of DFF40 require the presence of DFF45, which also acts as a nuclease inhibitor before DFF40 activation by execution caspases. The N-terminal domains (NTDs) of both proteins are homologous, and their interaction plays a key role in the proper functioning of this two-component system. Here we report that the NTD of DFF45 alone is unstructured in solution, and its folding is induced upon binding to DFF40 NTD. Therefore, folding of both proteins regulates the formation of the DFF40/DFF45 complex. The solution structure of the heterodimeric complex between NTDs of DFF40 and DFF45 reported here shows that the mutual chaperoning includes the formation of an extensive network of intermolecular interactions that bury a hydrophobic cluster inside the interface, surrounded by intermolecular salt bridges.

A Phosphothreonine Residue at the C-Terminal End of the Plasma Membrane H+-ATPase Is Protected by Fusicoccin-Induced 14–3–3 Binding1

We have isolated the plasma membrane H+−ATPase in a phosphorylated form from spinach (Spinacia oleracea L.) leaf tissue incubated with fusicoccin, a fungal toxin that induces irreversible binding of 14–3–3 protein to the C terminus of the H+-ATPase, thus activating H+ pumping. We have identified threonine-948, the second residue from the C-terminal end of the H+-ATPase, as the phosphorylated amino acid. Turnover of the phosphate group of phosphothreonine-948 was inhibited by 14–3–3 binding, suggesting that this residue may form part of a binding motif for 14–3–3. This is the first identification to our knowledge of an in vivo phosphorylation site in the plant plasma membrane H+-ATPase.

Rhamnogalacturonan α-d-Galactopyranosyluronohydrolase1 : An Enzyme That Specifically Removes the Terminal Nonreducing Galacturonosyl Residue in Rhamnogalacturonan Regions of Pectin1

A new enzyme, rhamnogalacturonan (RG) α-d-galactopyranosyluronohydrolase (RG-galacturonohydrolase), able to release a galacturonic acid residue from the nonreducing end of RG chains but not from homogalacturonan, was purified from an Aspergillus aculeatus enzyme preparation. RG-galacturonohydrolase acted with inversion of anomeric configuration, initially releasing β-d-galactopyranosyluronic acid. The enzyme cleaved smaller RG substrates with the highest catalytic efficiency. A Michaelis constant of 85 μm and a maximum reaction rate of 160 units mg−1 was found toward a linear RG fragment with a degree of polymerization of 6. RG-galacturonohydrolase had a molecular mass of 66 kD, an isoelectric point of 5.12, a pH optimum of 4.0, and a temperature optimum of 50°C. The enzyme was most stable between pH 3.0 and 6.0 (for 24 h at 40°C) and up to 60°C (for 3 h).

Substrate-specific regulation of the ribosome– translocon junction by N-terminal signal sequences

Amino-terminal signal sequences target nascent secretory and membrane proteins to the endoplasmic reticulum for translocation. Subsequent interactions between the signal sequence and components of the translocation machinery at the endoplasmic reticulum are thought to be important for the productive engagement of the translocon by the ribosome-nascent chain complex. However, it is not clear whether all signal sequences carry out these posttargeting steps identically, or if there are differences in the interactions directed by one signal sequence versus another. In this study, we find substantial differences in the ability of signal sequences from different substrates to mediate closure of the ribosome–translocon junction early in translocation. We also show that these differences in some cases necessitate functional coordination between the signal sequence and mature domain for faithful translocation. Accordingly, the translocation of some proteins is sensitive to replacement of their signal sequences. In a particularly dramatic example, the topology of the prion protein was found to depend highly on the choice of signal sequence used to direct its translocation. Taken together, our results reveal an unanticipated degree of substrate-specific functionality encoded in N-terminal signal sequences.

Binding to the naturally occurring double p53 binding site of the Mdm2 promoter alleviates the requirement for p53 C-terminal activation

Genotoxic stress activation of the tumor suppressor transcription factor p53 involves post-translational C-terminal modifications that increase both protein stability and DNA binding activity. We compared the requirement for p53 protein activation of p53 target sequences in two major p53-regulated genes, p21/WAF1 (encoding a cell cycle inhibitory protein) and Mdm2 (encoding a ubiquitin ligase that targets p53 for proteolytic degradation). The p53 binding site in the proximal p21/WAF1 promoter contains a single p53 binding consensus sequence, while the p53 binding site in the Mdm2 promoter contains two consensus sequences linked by a 17 bp spacer. Binding of recombinant p53 protein to the p21/WAF1 binding site required monoclonal antibody PAb421, which can mimic activating phosphorylation and/or acetylation events at the C-terminus. In contrast, recombinant p53 bound strongly to the Mdm2 binding site in the absence of PAb421 antibody. Separate binding to each consensus sequence of the Mdm2 binding site still required PAb421, indicating that p53 binding was not simply due to greater affinity to the Mdm2 consensus sequences. Linking two p21/WAF1 binding sites with the 17 bp spacer region from the Mdm2 gene eliminated the PAb421 requirement for p53 binding to the p21/WAF1 site. These results suggest a mechanism for regulation of Mdm2 gene transcription that differs from that other p53-induced genes by its lack of a requirement for C-terminal activation of p53 protein. A steady induction of Mdm2 protein would maintain p53 protein at low levels until post-translational modifications following DNA damage increased p53 activity towards other genes, mediating p53 growth inhibitory and apoptotic activities.