Regulation of Chk1 by its C-terminal domain

Chk1 is a protein kinase that is the effector molecule in the G2 DNA damage checkpoint. Chk1 homologues have an N-terminal kinase domain, and a C-terminal domain of ~200 amino acids that contains activating phosphorylation sites for the ATM/R kinases, though the mechanism of activation remains unknown. Structural studies of the human Chk1 kinase domain show an open conformation; the activity of the kinase domain alone is substantially higher in vitro than full-length Chk1, and coimmunoprecipitation studies suggest the C-terminal domain may contain an autoinhibitory activity. However, we show that truncation of the C-terminal domain inactivates Chk1 in vivo. We identify additional mutations within the C-terminal domain that activate ectopically expressed Chk1 without the need for activating phosphorylation. When expressed from the endogenous locus, activated alleles show a temperature-sensitive loss of function, suggesting these mutations confer a semiactive state to the protein. Intragenic suppressors of these activated alleles cluster to regions in the catalytic domain on the face of the protein that interacts with substrate, suggesting these are the regions that interact with the C-terminal domain. Thus, rather than being an autoinhibitory domain, the C-terminus of Chk1 also contains domains critical for adopting an active configuration.     

Regulation of IRAK-1 activation by its C-terminal domain

Macrophages are important mediators of the immune response to infection by virtue of their ability to secrete cytokines that trigger inflammation. Toll-like receptors (TLRs) are largely responsible for meditating the activation of macrophages by pathogens. IRAK-1 is a proximal protein kinase in TLR signalling pathways and hence its activation must be tightly regulated. However, the mechanisms which control the activation of IRAK-1 are poorly understood. IRAK-1 contains a death domain at its N-terminus that mediates its interaction with other death domain containing proteins, a central Ser/Thr kinase domain, and a C-terminal domain that contains binding motifs for TRAF6. We show here that deletion of the death domain or the majority of the C-terminal domain markedly enhanced the capacity of IRAK-1 to activate NF-κB in a TLR-independent manner in RAW 264.7 macrophages. Furthermore, the C-terminal truncation mutant spontaneously oligomerised and formed complexes with the negative regulator IRAK-M in the absence of TLR activation. In contrast to the binding of IRAK-M to IRAK-1, the death domain of IRAK-1 was not required for the interaction of IRAK-4 with IRAK-1. On the basis of these results we propose a model in which IRAK-1 is held in a closed, inactive conformation via an intramolecular mechanism involving its C-terminal domain and possibly the death domain. Phosphorylation of IRAK-1 by IRAK-4 in response to TLR activation may then release IRAK-1 from the inhibitory constraint exerted by its C-terminal domain.     

Functional cross-talk of HIV-1 Tat with p53 through its C-terminal domain

Human immunodeficiency virus type 1 (HIV-1) Tat repressed the p53-dependent gene expression through its C-terminal domain of Tat (amino acid residues 73-86) independent of the involvement of NF-kappaB and coactivator CBP/p300. Although Tat did not directly bind to p53, this repression required the N-terminal domain of p53. In contrast, Tat and p53 cooperated in the activation of HIV-1 gene expression. Thus, the cross-talk between Tat and p53 may be linked with cellular transformation by HIV-1 infection or activation of HIV-1 replication.     

C-terminal domain of APETALA1 is essential for its functional divergence from CAULIFLOWER in Arabidopsis

Journal of Plant Biochemistry and Biotechnology - APETALA1 (AP1) and CAULIFLOWER (CAL) are involved in floral meristem identity and suppress the inflorescence meristem program in flower meristem in...     

C-terminal domain mutations in ClpX uncouple substrate binding from an engagement step required for unfolding

ClpX mediates ATP-dependent denaturation of specific target proteins and disassembly of protein complexes. Like other AAA + family members, ClpX contains an alphabeta ATPase domain and an alpha-helical C-terminal domain. ClpX proteins with mutations in the C-terminal domain were constructed and screened for disassembly activity in vivo. Seven mutant enzymes with defective phenotypes were purified and characterized. Three of these proteins (L381K, D382K and Y385A) had low activity in disassembly or unfolding assays in vitro. In contrast to wild-type ClpX, substrate binding to these mutants inhibited ATP hydrolysis instead of increasing it. These mutants appear to be defective in a reaction step that engages bound substrate proteins and is required both for enhancement of ATP hydrolysis and for unfolding/disassembly. Some of these side chains form part of the interface between the C-terminal domain of one ClpX subunit and the ATPase domain of an adjacent subunit in the hexamer and appear to be required for communication between adjacent nucleotide binding sites.     

Apo-calmodulin binds with its C-terminal domain to the N-methyl-D-aspartate receptor NR1 C0 region

Calmodulin (CaM) is the major Ca2+ sensor in eukaryotic cells. It consists of four EF-hand Ca2+ binding motifs, two in its N-terminal domain and two in its C-terminal domain. Through a negative feedback loop, CaM inhibits Ca2+ influx through N-methyl-D-aspartate-type glutamate receptors in neurons by binding to the C0 region in the cytosolic tail of the NR1 subunit. Ca2+ -depleted (apo)CaM is pre-associated with a variety of ion channels for fast and effective regulation of channel activities upon Ca2+ influx. Using the NR1 C0 region for fluorescence and circular dichroism spectroscopy studies we found that not only Ca2+ -saturated CaM but also apoCaM bound to NR1 C0. In vitro interaction assays showed that apoCaM also binds specifically to full-length NR1 solubilized from rat brain and to the complete C terminus of the NR1 splice form that contains the C0 plus C2' domain. The Ca2+ -independent interaction of CaM was also observed with the isolated C-but not N-terminal fragment of calmodulin in the independent spectroscopic assays. Fluorescence polarization studies indicated that apoCaM associated via its C-terminal domain with NR1 C0 in an extended conformation and collapsed to adopt a more compact conformation of faster rotational mobility in its complex with NR1 C0 upon addition of Ca2+. Our results indicate that apoCaM is associated with NR1 and that the complex of CaM bound to NR1 C0 undergoes a dramatic conformational change when Ca2+ binds to CaM.     

The role of the Brr5/Ysh1 C-terminal domain and its homolog Syc1 in mRNA 3'-end processing in Saccharomyces cerevisiae

The cleavage/polyadenylation factor (CPF) of Saccharomyces cerevisiae is thought to provide the catalytic activities of the mRNA 3'-end processing machinery, which include endonucleolytic cleavage at the poly(A) site, followed by synthesis of an adenosine polymer onto the new 3'-end by the CPF subunit Pap1. Because of similarity to other nucleases in the metallo-beta-lactamase family, the Brr5/Ysh1 subunit has been proposed to be the endonuclease. The C-terminal domain of Brr5 lies outside of beta-lactamase homology, and its function has not been elucidated. We show here that this region of Brr5 is necessary for cell viability and mRNA 3'-end processing. It is highly homologous to another CPF subunit, Syc1. Syc1 is not essential, but its removal improves the growth of other processing mutants at restrictive temperatures and restores in vitro processing activity to cleavage/ polyadenylation-defective brr5-1 extract. Our findings suggest that Syc1, by mimicking the essential Brr5 C-terminus, serves as a negative regulator of mRNA 3'-end formation.     

Enzymic product formation curves with the normal or diffusion limited reaction mechanism and in the presence of substrate receptors

1. The enzymic product formation curves for several enzymes have been studied. 2. The product formation kinetics was related to the initial velocity kinetics and to the diffusion rate limited kinetics. 3. The time curves revealed new constants characterizing structural and binding properties of the enzymic systems which are not revealed from initial velocities. 4. The influence of selected inhibitors on the time curves has been studied. 5. The time curves revealed the specific substrate-receptor binding which was not revealed from initial velocities. 6. The product formation kinetics of acid phosphatase, beta-amylase and NADPH2 cytochrome-c reductase in the absence and in the presence of inhibitors, mercuric acetate and o-iodosobenzoate is described. 7. The time curves revealed the binding of cytochrome-c to the specific natural protein receptors. 8. The activation energies of acid phosphatase and beta-amylase were determined from the time curves.     

Solution structure of the PDZ2 domain from human phosphatase hPTP1E and its interactions with C-terminal peptides from the Fas receptor

The solution structure of the second PDZ domain (PDZ2) from human phosphatase hPTP1E has been determined using 2D and 3D heteronuclear NMR experiments. The binding of peptides derived from the C-terminus of the Fas receptor to PDZ2 was studied via changes in backbone peptide and protein resonances. The structure is based on a total of 1387 nonredundant experimental NMR restraints including 1261 interproton distance restraints, 45 backbone hydrogen bonds, and 81 torsion angle restraints. Analysis of 30 lowest-energy structures resulted in rmsd values of 0.41 +/- 0.09 A for backbone atoms (N, Calpha, C') and 1.08 +/- 0.10 A for all heavy atoms, excluding the disordered N- and C-termini. The hPTP1E PDZ2 structure is similar to known PDZ domain structures but contains two unique structural features. In the peptide binding domain, the first glycine of the GLGF motif is replaced by a serine. This serine appears to replace a bound water observed in PDZ crystal structures that hydrogen bonds to the bound peptide's C-terminus. The hPTP1E PDZ2 structure also contains an unusually large loop following strand beta2 and proximal to the peptide binding site. This well-ordered loop folds back against the PDZ domain and contains several residues that undergo large amide chemical shift changes upon peptide binding. Direct observation of peptide resonances demonstrates that as many as six Fas peptide residues interact with the PDZ2 domain.     

The role of the C-terminal domain of protein tyrosine phosphatase-1B in phosphatase activity and substrate binding

Protein tyrosine phosphatase 1B (PTP-1B) has been implicated in the regulation of the insulin receptor. Dephosphorylation of the insulin receptor results in decreased insulin signaling and thus decreased glucose uptake. PTP-1B-/- mice have increased insulin sensitivity and are resistant to weight gain when fed a high fat diet, validating PTP-1B as a potential target for the treatment of type 2 diabetes. Many groups throughout the world have been searching for selective inhibitors for PTP-1B, and most of them target inhibitors to PTP-1B-(1-298), the N-terminal catalytic domain of the enzyme. However, the C-terminal domain is quite large and could influence the activity of the enzyme. Using two constructs of PTP-1B and a phosphopeptide as substrate, steady state assays showed that the presence of the C-terminal domain decreased both the Km and the k(cat) 2-fold. Pre-steady state kinetic experiments showed that the presence of the C-terminal domain improved the affinity of the enzyme for a phosphopeptide 2-fold, primarily because the off-rate was slower. This suggests that the C-terminal domain of PTP-1B may contact the phosphopeptide in some manner, allowing it to remain at the active site longer. This could be useful when screening libraries of compounds for inhibitors of PTP-1B. A compound that is able to make contacts with the C-terminal domain of PTP-1B would not only have a modest improvement in affinity but may also provide for specificity over other phosphatases.     

Tetramerization of the AKT1 plant potassium channel involves its C-terminal cytoplasmic domain.

All plant channels identified so far show high conservation throughout the polypeptide sequence except in the ankyrin domain which is present only in those closely related to AKT1. In this study, the architecture of the AKT1 protein has been investigated. AKT1 polypeptides expressed in the baculovirus/Sf9 cells system were found to assemble into tetramers as observed with animal Shaker-like potassium channel subunits. The AKT1 C-terminal intracytoplasmic region (downstream from the transmembrane domain) alone formed tetrameric structures when expressed in Sf9 cells, revealing a tetramerization process different from that of Shaker channels. Tests of subfragments from this sequence in the two-hybrid system detected two kinds of interaction. The first, involving two identical segments (amino acids 371-516), would form a contact between subunits, probably via their putative cyclic nucleotide-binding domains. The second interaction was found between the last 81 amino acids of the protein and a region lying between the channel hydrophobic core and the putative cyclic nucleotide-binding domain. As the interacting regions are highly conserved in all known plant potassium channels, the structural organization of AKT1 is likely to extend to these channels. The significance of this model with respect to animal cyclic nucleotide-gated channels is also discussed.     

Insights into EB1 structure and the role of its C-terminal domain for discriminating microtubule tips from the lattice

End-binding proteins (EBs) comprise a conserved family of microtubule plus end-tracking proteins. The concerted action of calponin homology (CH), linker, and C-terminal domains of EBs is important for their autonomous microtubule tip tracking, regulation of microtubule dynamics, and recruitment of numerous partners to microtubule ends. Here we report the detailed structural and biochemical analysis of mammalian EBs. Small-angle X-ray scattering, electron microscopy, and chemical cross-linking in combination with mass spectrometry indicate that EBs are elongated molecules with two interacting CH domains, an arrangement reminiscent of that seen in other microtubule- and actin-binding proteins. Removal of the negatively charged C-terminal tail did not affect the overall conformation of EBs; however, it increased the dwell times of EBs on the microtubule lattice in microtubule tip-tracking reconstitution experiments. An even more stable association with the microtubule lattice was observed when the entire negatively charged C-terminal domain of EBs was replaced by a neutral coiled-coil motif. In contrast, the interaction of EBs with growing microtubule tips was not significantly affected by these C-terminal domain mutations. Our data indicate that long-range electrostatic repulsive interactions between the C-terminus and the microtubule lattice drive the specificity of EBs for growing microtubule ends.     

A network of SMG-8, SMG-9 and SMG-1 C-terminal insertion domain regulates UPF1 substrate recruitment and phosphorylation

Mammalian nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that degrades mRNAs containing premature translation termination codons. Phosphorylation of the essential NMD effector UPF1 by the phosphoinositide-3-kinase-like kinase (PIKK) SMG-1 is a key step in NMD and occurs when SMG-1, its two regulatory factors SMG-8 and SMG-9, and UPF1 form a complex at a terminating ribosome. Electron cryo-microscopy of the SMG-1–8–9-UPF1 complex shows the head and arm architecture characteristic of PIKKs and reveals different states of UPF1 docking. UPF1 is recruited to the SMG-1 kinase domain and C-terminal insertion domain, inducing an opening of the head domain that provides access to the active site. SMG-8 and SMG-9 interact with the SMG-1 C-insertion and promote high-affinity UPF1 binding to SMG-1–8–9, as well as decelerated SMG-1 kinase activity and enhanced stringency of phosphorylation site selection. The presence of UPF2 destabilizes the SMG-1–8–9-UPF1 complex leading to substrate release. Our results suggest an intricate molecular network of SMG-8, SMG-9 and the SMG-1 C-insertion domain that governs UPF1 substrate recruitment and phosphorylation by SMG-1 kinase, an event that is central to trigger mRNA decay.     

The exchange factor and diacylglycerol receptor RasGRP3 interacts with dynein light chain 1 through its C-terminal domain

RasGRP3 is an exchange factor for Ras-like small GTPases that is activated in response to the second messenger diacylglycerol. As with other diacylglycerol receptors, RasGRP3 is redistributed upon diacylglycerol or phorbol ester binding. Several factors are important in determining the pattern of translocation, including the potency of the diacylglycerol analog, the affinity of the receptor for phospholipids, and in some cases, protein-protein interactions. However, little is known about the mechanisms that play a role in RasGRP3 redistribution aside from the nature of the ligand. To discover potential protein binding partners for RasGRP3, we screened a human brain cDNA library using a yeast two-hybrid approach. We identified dynein light chain 1 as a novel RasGRP3-interacting protein. The interaction was confirmed both in vitro and in vivo and required the C-terminal domain encompassing the last 127 amino acids of RasGRP3. A truncated mutant form of RasGRP3 that lacked this C-terminal domain was unable to interact with dynein light chain 1 and displayed a dramatically altered subcellular localization, with a strong reticular distribution and perinuclear and nuclear localization. These findings suggest that dynein light chain 1 represents a novel anchoring protein for RasGRP3 that may regulate subcellular localization of the exchange factor and, as such, may participate in the signaling mediated by diacylglycerol through RasGRP3.     

Cooperative interaction of the C-terminal domain of histone H1 with DNA

We have studied the interaction of the isolated C-terminal domain of histone H1 with linear DNA using precipitation curves and electron microscopy. The C-terminal domain shows a salt-dependent transition towards cooperative binding, which reaches completion at 60 mM NaCl. At this salt concentration, the C-terminal domain binds to some of the DNA molecules, leaving the rest free. A binding site of 22 base-pairs can be calculated from the stoichiometry of the precipitated fractions. The C-terminal domain condenses the DNA in toroidal particles. The average inner radius of the particles is of the order of 195 A. Consideration of the value of the inner radius of the toroids in the light of counterion condensation theory suggests that in these complexes the isolated C-terminal domain is capable of nearly full electrostatic neutralization of the DNA phosphate charge.     

Phenazine derivatives as the mutagenic reaction product from o- or m-phenylenediamine derivatives with hydrogen peroxide

8 Kinds of o- and m-phenylenediamine (PD) derivatives, which are used as oxidative-type hair dyes, were treated with hydrogen peroxide (H2O2). Both before and after H2O2 treatment, their mutagenicity was tested by using Salmonella typhimurium TA98 in the presence or absence of a mammalian metabolic activation system (S9 mix). After H2O2 treatment, the mutagenic potencies of p-nitro-o-phenylenediamine, 3,4-diaminotoluene, p-nitro-m-phenylenediamine and 2,4-diaminophenol did not vary or slightly increased in comparison with those of the starting materials. The mutagenicity of o-PD, p-chloro-o-phenylenediamine (p-Cl-o-PD), m-PD and 2,4-diaminoanisole (p-OMe-m-PD) was enhanced remarkably by treatment with H2O2 and all the oxidation products required metabolic activation by S9 mix for their mutagenesis. In a gas chromatography/mass spectrometric study, 2,3-diaminophenazine and 2,7-diaminophenazine were identified with authentic samples in o-PD and m-PD oxidation mixture, respectively. The oxidation mixture obtained from p-Cl-o-PD and p-OMe-m-PD was separated into several fractions by repeated column chromatography. Brownish yellow crystals were isolated from oxidized p-Cl-o-PD and the structure of the compound was determined to be 2,3-diamino-7-chlorophenazine from physicochemical and chemical evidence. Two reddish yellow crystals, obtained from oxidized p-OMe-m-PD, were 2,7-diamino-3,8-dimethoxyphenazine and 2,7-diamino-3-methoxyphenazine. The number of revertants induced by 1 nmole of phenazines detected from oxidized PD derivatives was as follows; 2,3-diaminophenazine: 349 rev.; 2,3-diamino-7-chlorophenazine; 406 rev.: 2,7-diaminophenazine: 12 110 rev.; 2,7-diamino-3,8-dimethoxyphenazine: 4229 rev.; 2,7-diamino-3-methoxyphenazine: 24 640 rev. in S. typhimurium TA98 strain with 25 microliters S9 per plate.