A tandem of SH3-like domains participates in RNA binding in KIN17, a human protein activated in response to genotoxics

The human KIN17 protein is an essential nuclear protein conserved from yeast to human and expressed ubiquitously in mammals. Suppression of Rts2, the yeast equivalent of gene KIN17, renders the cells unviable, and silencing the human KIN17 gene slows cell growth dramatically. Moreover, the human gene KIN17 is up-regulated following exposure to ionizing radiations and UV light, depending on the integrity of the human global genome repair machinery. Its ectopic over-expression blocks S-phase progression by inhibiting DNA synthesis. The C-terminal region of human KIN17 is crucial for this anti-proliferation effect. Its high-resolution structure, presented here, reveals a tandem of SH3-like subdomains. This domain binds to ribonucleotide homopolymers with the same preferences as the whole protein. Analysis of its structure complexed with tungstate shows structural variability within the domain. The interaction with tungstate is mediated by several lysine residues located within a positively charged groove at the interface between the two subdomains. This groove could be the site of interaction with RNA, since mutagenesis of two of these highly conserved lysine residue weakens RNA binding.     

Identification of cyclin D3 as a new interaction partner of lamin A/C

Lamin A/C is a major component of the nuclear lamina. An intact nuclear lamina has been proposed to be necessary for muscle differentiation. Cyclin D3 is known to be upregulated in differentiated muscle cells and to form insoluble complexes with cell-cycle regulatory factors in these cells. We have examined the possibility of direct binding interactions between lamin A/C and cyclin D3 by in vitro binding assays and co-immunoprecipitation studies with muscle cells. Our results indicate that cyclin D3 binds specifically to amino acid residues 383-474 of lamin A/C and associates with lamin A/C in muscle cells. The identification of cyclin D3 as a novel binding partner of lamin A/C has important implications for a role for lamin A/C in muscle differentiation.     

The Ribosomal Grip of the Peptidyl-tRNA is Critical for Reading Frame Maintenance

If a ribosome shifts to an alternative reading frame during translation, the information in the message is usually lost. We have selected mutants of Salmonella typhimurium with alterations in tRNA_cmo5UGG^Pro that cause increased frameshifting when present in the ribosomal P-site. In 108 such mutants, two parts of the tRNA molecule are altered: the anticodon stem and the D-arm, including its tertiary interactions with the variable arm. Some of these alterations in tRNA_cmo5UGG^Pro are in close proximity to ribosomal components in the P-site. The crystal structure of the 30S subunit suggests that the C-terminal end of ribosomal protein S9 contacts nucleotides 32-34 of peptidyl-tRNA. We have isolated mutants with defects in the C-terminus of S9 that induce + 1 frameshifting. Combinations of changes in tRNA_cmo5UGG^Pro and S9 suggest that an interaction occurs between position 32 of the peptidyl-tRNA and the C-terminal end of S9. Together, our results suggest that the cause of frameshifting is an aberrant interaction between the peptidyl-tRNA and the P-site environment. We suggest that the “ribosomal grip” of the peptidyl-tRNA is pivotal for maintaining the reading frame.     

Vimentin binding to phosphorylated Erk sterically hinders enzymatic dephosphorylation of the kinase

Cleavage fragments of de novo synthesized vimentin were recently reported to interact with phosphorylated Erk1 and Erk2 MAP kinases (pErk) in injured sciatic nerve, thus linking pErk to a signaling complex retrogradely transported on importins and dynein. Here we clarify the structural basis for this interaction, which explains how pErk is protected from dephosphorylation while bound to vimentin. Pull-down and ELISA experiments revealed robust calcium-dependent binding of pErk to the second coiled-coil domain of vimentin, with observed affinities of binding increasing from 180 nM at 0.1 microM calcium to 15 nM at 10 microM calcium. In contrast there was little or no binding of non-phosphorylated Erk to vimentin under these conditions. Geometric and electrostatic complementarity docking generated a number of solutions wherein vimentin binding to pErk occludes the lip containing the phosphorylated residues in the kinase. Binding competition experiments with Erk peptides confirmed a solution in which vimentin covers the phosphorylation lip in pErk, interacting with residues above and below the lip. The same peptides inhibited pErk binding to the dynein complex in sciatic nerve axoplasm, and interfered with protection from phosphatases by vimentin. Thus, a soluble intermediate filament fragment interacts with a signaling kinase and protects it from dephosphorylation by calcium-dependent steric hindrance.     

A nucleosome interaction module is required for normal function of Arabidopsis thaliana BRAHMA

The BRAHMA (BRM) gene encodes the SNF2-type ATPase of the putative Arabidopsis thaliana SWI/SNF chromatin remodelling complex. This family of ATPases is characterized by the presence of a conserved catalytic domain and an arrangement of auxiliary domains, whose functions in the remodelling activity remains unclear. Here, we characterize, at the molecular and functional level, the carboxy-terminal part of Arabidopsis BRM. We have found three DNA-binding regions that bind various free DNA and nucleosomal probes with different specificity. One of these regions contains an AT-hook motif. The carboxy terminus also contains a bromodomain able to bind histones H3 and H4. We propose that this array of domains constitute a nucleosome interaction module that helps BRM to interact with its substrate. We also characterize an Arabidopsis mutant that expresses a BRM protein lacking the last 454 amino acid residues (BRM-DeltaC), encompassing the bromodomain and two of the three DNA-binding activities identified. This mutant displays an intermediate phenotype between those of the wild-type and a null allele mutant, suggesting that the nucleosome interaction module is required for the normal function of BRM but it is not essential for the remodelling activity of BRM-containing SWI/SNF complexes.     

Sequence and length recognition of the C-terminal turnover element of LpxC, a soluble substrate of the membrane-bound FtsH protease

The membrane-anchored FtsH protease is essential in Escherichia coli as it adjusts the cellular amount of LpxC, the key enzyme in lipopolysaccharide (LPS) biosynthesis. Both accumulation and depletion of LpxC are toxic to E. coli. By continuous proteolysis of LpxC, FtsH maintains a low concentration of LpxC and, hence, the proper equilibrium between LPS and phospholipids. The C terminus of LpxC is required for turnover. By adding this tail to glutathione-S-transferase (GST) we show that it is necessary but not sufficient for FtsH-mediated degradation. A detailed mutational analysis revealed six non-polar residues in the C terminus of LpxC that are critical for degradation. Alteration of the C-terminal AVLA motif towards the SsrA-like sequence ALAA directed LpxC to other cellular proteases reinforcing the importance of the C-terminal tail for targeting to FtsH. Short C-terminal truncations stabilized LpxC. Most mutations in the C terminus of LpxC left its enzymatic activity intact as was shown by growth assays, microscopy and 2-keto-3-deoxyoctonate (KDO) determination. The critical length of the turnover element was defined by internal deletions. A C-terminal tail of about 20 amino acids length is required for proteolysis of LpxC by FtsH.     

Identification of B cell adaptor for PI3-kinase (BCAP) as an Abl interactor 1-regulated substrate of Abl kinases

In previous work we showed that Abl interactor 1 (Abi-1), by linking enzyme and substrate, promotes the phosphorylation of Mammalian Enabled (Mena) by c-Abl. To determine whether this mechanism extends to other c-Abl substrates, we used the yeast two-hybrid system to search for proteins that interact with Abi-1. By screening a human leukocyte cDNA library, we identified BCAP (B-cell adaptor for phosphoinositide 3-kinase) as another Abi-1-interacting protein. Binding experiments revealed that the SH3 domain of Abi-1 and the C-terminal polyproline structure of BCAP are involved in interactions between the two. In cultured cells, Abi-1 promoted phosphorylation of BCAP not only by c-Abl but also by v-Abl. The phosphorylation sites of BCAP by c-Abl were mapped to five tyrosine residues in the C-terminal region that are well conserved in mammals. These results show that Abi-1 promotes Abl-mediated BCAP phosphorylation and suggest that Abi-1 in general coordinates kinase-substrate interactions.     

A mitotic kinase TOPK enhances Cdk1/cyclin B1-dependent phosphorylation of PRC1 and promotes cytokinesis

A MAPKK-like mitotic kinase, TOPK, implies the formation of mitotic spindles and spindle midzone and accomplishing cytokinesis, however, its underlying mechanism remains unclear. A microtubule bundling protein, PRC1, plays a pivotal role in the formation of mitotic spindles and spindle midzone. Because of their functional resemblance, we attempted to clarify the links between these two molecules. TOPK supported mitotic advance via the cdk1/cyclin B1-dependent phosphorylation of PRC1. TOPK induced the phosphorylation of PRC1 at T481 in vivo, however, TOPK did not phosphorylate PRC1 in vitro. TOPK induced the phosphorylation of PRC1 at T481 only when the cdk1/cyclin B1 existed simultaneously in vitro. Both the enzymatic activity of TOPK and association competence of TOPK with PRC1 were mandatory for this phosphorylation. TOPK binds to cdk1/cyclin B1, microtubules and PRC1 via its unique region near the C terminus. TOPK co-localized closely with cdk1 throughout the cell cycle in vivo. Collectively, these data indicate that TOPK, which makes a kinase-substrate complex with cdk1/cyclin B1 and PRC1 on microtubules during mitosis, enhances the cdk1/cyclin B1-dependent phosphorylation of PRC1 and thereby strongly promotes cytokinesis.     

Characterising the Binding Specificities of the Subunits Associated with the KMT2/Set1 Histone Lysine Methyltransferase

KMT2/Set1 is the catalytic subunit of the complex of proteins associated with Set1 (COMPASS) that is responsible for the methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. Whereas monomethylated H3K4 (H3K4me1) is found throughout the genome, di- (H3K4me2) and tri- (H3K4me3) methylated H3K4 are enriched at specific loci, which correlates with the promoter and 5′-ends of actively transcribed genes in the case of H3K4me3. The COMPASS subunits contain a number of domains that are conserved in homologous complexes in higher eukaryotes and are reported to interact with modified histones. However, the exact organization of these subunits and their role within the complex have not been elucidated. In this study we showed that: (1) subunits Swd1 and Swd3 form a stable heterodimer that dissociates upon binding to a modified H3K4me2 tail peptide, suggesting a regulatory role in COMPASS; (2) the affinity of the subunit Spp1 for modified histone H3 substrates is much higher than that of Swd1 and Swd3; (3) Spp1 has a preference for H3K4me2/3 methylation state; and (4) Spp1 contains a high-affinity DNA-binding domain in the previously uncharacterised C-terminal region. These data allow us to suggest a mechanism for the regulation of COMPASS activity at an actively transcribed gene.     

Zebrafish Mib and Mib2 are mutual E3 ubiquitin ligases with common and specific delta substrates

It was already known that both mind bomb (mib) and mind bomb-2 (mib2) encode E3 ubiquitin ligases that target Delta in Notch activation. Here we further demonstrated that zebrafish Mib and Mib2, similar to their mouse orthologs, have a C-terminal-most RING finger-dependent E3 ubiquitin ligase activity. Mib and Mib2 are reciprocal E3 ubiquitin ligases and substrates. They function similarly in Notch signaling by using DeltaC as a common substrate. However, Mib2 behaves differently from Mib in DeltaD internalization. In addition, Mib and Mib2 bind differently to extracellular and intracellular parts of DeltaA and DeltaC. Finally, mutant Mibs, Mib(ta52b) with a missense mutation in the C-terminal-most RING finger (M1013R) and Mib(m132) with a premature stop codon that leads to a deletion of three RING fingers (C785stop), act dominant-negatively and compete with Mib2 in DeltaC ubiquitylation and internalization, suggesting a molecular basis for the antimorphic phenotypes (stronger than the null phenotypes) observed in zebrafish mib(ta52b) and mib(m132) alleles.     

Dystrophin Dp71f associates with the beta1-integrin adhesion complex to modulate PC12 cell adhesion

Dystrophin Dp71 is the main product of the Duchenne muscular dystrophy gene in the brain; however, its function is unknown. To study the role of Dp71 in neuronal cells, we previously generated by antisense treatment PC12 neuronal cell clones with decreased Dp71 expression (antisense-Dp71 cells). PC12 cells express two different splicing isoforms of Dp71, a cytoplasmic variant called Dp71f and a nuclear isoform called Dp71d. We previously reported that antisense-Dp71 cells display deficient adhesion to substrate and reduced immunostaining of beta1-integrin in the cell area contacting the substrate. In this study, we isolated additional antisense-Dp71 clones to analyze in detail the potential involvement of Dp71f isoform with the beta1-integrin adhesion system of PC12 cells. Immunofluorescence analyses as well as immunoprecipitation assays demonstrated that the PC12 cell beta1-integrin adhesion complex is composed of beta1-integrin, talin, paxillin, alpha-actinin, FAK and actin. In addition, our results showed that Dp71f associates with most of the beta1-integrin complex components (beta1-integrin, FAK, alpha-actinin, talin and actin). In the antisense-Dp71 cells, the deficiency of Dp71 provokes a significant reduction of the beta1-integrin adhesion complex and, consequently, the deficient adhesion of these cells to laminin. In vitro binding experiments confirmed the interaction of Dp71f with FAK and beta1-integrin. Our data indicate that Dp71f is a structural component of the beta1-integrin adhesion complex of PC12 cells that modulates PC12 cell adhesion by conferring proper complex assembly and/or maintenance.     

Heat stable ssDNA/RNA-binding activity of a wheat cold shock domain protein

The cold-induced wheat WCSP1 protein belongs to the cold shock domain (CSD) protein family. In prokaryotes and eukaryotes, the CSD functions as a nucleic acid-binding domain. Here, we demonstrated that purified recombinant WCSP1 is boiling soluble and binds ss/dsDNA and mRNA. Furthermore, boiled-WCSP1 retained its characteristic nucleic acid-binding activity. A WCSP1 deletion mutant, containing only a CSD, lost ssDNA/RNA-binding activity; while a mutant containing the CSD and the first glycine-rich region (GR) displayed the activity. These data indicated that the first GR of WCSP1 is necessary for the binding activity but is not for the heat stability of the protein.     

Crystal structure at 2.8 A of the DLLRKN-containing coiled-coil domain of huntingtin-interacting protein 1 (HIP1) reveals a surface suitable for clathrin light chain binding

Huntingtin interacting protein 1 (HIP1) is a member of a family of proteins whose interaction with Huntingtin is critical to prevent cells from initiating apoptosis. HIP1, and related protein HIP12/1R, can also bind to clathrin and membrane phospholipids, and HIP12/1R links the CCV to the actin cytoskeleton. HIP1 and HIP12/1R interact with the clathrin light chain EED regulatory site and stimulate clathrin lattice assembly. Here, we report the X-ray structure of the coiled-coil domain of HIP1 (residues 482-586) that includes residues crucial for binding clathrin light chain. The dimeric HIP1 crystal structure is partially splayed open. The comparison of the HIP1 model with coiled-coil predictions revealed the heptad repeat in the dimeric trunk (S2 path) is offset relative to the register of the heptad repeat from the N-terminal portion (S1 path) of the molecule. Furthermore, surface analysis showed there is a third hydrophobic path (S3) running parallel with S1 and S2. We present structural evidence supporting a role for the S3 path as an interaction surface for clathrin light chain. Finally, comparative analysis suggests the mode of binding between sla2p and clathrin light chain may be different in yeast.     

Structures of human N-Acetylglucosamine kinase in two complexes with N-Acetylglucosamine and with ADP/glucose: insights into substrate specificity and regulation

N-Acetylglucosamine (GlcNAc), a major component of complex carbohydrates, is synthesized de novo or salvaged from lysosomally degraded glycoconjugates and from nutritional sources. The salvage pathway requires that GlcNAc kinase converts GlcNAc to GlcNAc-6-phosphate, a component utilized in UDP-GlcNAc biosynthesis or energy metabolism. GlcNAc kinase belongs to the sugar kinase/Hsp70/actin superfamily that catalyze phosphoryl transfer from ATP to their respective substrates, and in most cases catalysis is associated with a large conformational change in which the N-terminal small and C-terminal large domains enclose the substrates. Here we report two crystal structures of homodimeric human GlcNAc kinase, one in complex with GlcNAc and the other in complex with ADP and glucose. The active site of GlcNAc kinase is located in a deep cleft between the two domains of the V-shaped monomer. The enzyme adopts a "closed" configuration in the GlcNAc-bound complex and GlcNAc interacts with residues of both domains. In addition, the N-acetyl methyl group contacts residues of the other monomer in the homodimer, a unique feature compared to other members of the sugar kinase/Hsp70/actin superfamily. This contrasts an "open" configuration in the ADP/glucose-bound structure, where glucose cannot form these interactions, explaining its low binding affinity for GlcNAc kinase. Our results support functional implications derived from apo crystal structures of GlcNAc kinases from Chromobacter violaceum and Porphyromonas gingivalis and show that Tyr205, which is phosphorylated in thrombin-activated platelets, lines the GlcNAc binding pocket. This suggests that phosphorylation of Tyr205 may modulate GlcNAc kinase activity and/or specificity.     

Coevolution of Specificity Determinants in Eukaryotic Glutamyl- and Glutaminyl-tRNA Synthetases

The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNA^Gln for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNA^Gln and tRNA^Glu with glutamate. This ancient GluRS also separately differentiated to exclude tRNA^Gln as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but they play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNA^Gln and tRNA^Glu recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p:GluRS complex upon the diauxic shift to respiratory conditions.     

On the benefit of bivalency in peptide ligand/pin1 interactions

The human peptidyl prolyl cis/trans isomerase (PPIase) Pin1 has a key role in developmental processes and cell proliferation. Pin1 consists of an N-terminal WW domain and a C-terminal catalytic PPIase domain both targeted specifically to Ser(PO₃H₂)/Thr(PO₃H₂)-Pro sequences. Here, we report the enhanced affinity originating from bivalent binding of ligands toward Pin1 compared to monovalent binding. We developed composite peptides where an N-terminal segment represents a catalytic site-directed motif and a C-terminal segment exhibits a predominant affinity to the WW domain of Pin1 tethered by polyproline linkers of different chain length. We used NMR shift perturbation experiments to obtain information on the specific interaction of a bivalent ligand to both targeted sites of Pin1. The bivalent ligands allowed a considerable range of thermodynamic investigations using isothermal titration calorimetry and PPIase activity assays. They expressed up to 350-fold improved affinity toward Pin1 in the nanomolar range in comparison to the monovalent peptides. The distance between the two binding motifs was highly relevant for affinity. The optimum in affinity manifested by a linker length of five prolyl residues between active site- and WW domain-directed peptide fragments suggests that the corresponding domains in Pin1 are allowed to adopt preferred spatial arrangement upon ligand binding.     

Oxidation of calmodulin alters activation and regulation of CaMKII

Increases in reactive oxygen species and mis-regulation of calcium homeostasis are associated with various physiological conditions and disease states including aging, ischemia, exposure to drugs of abuse, and neurodegenerative diseases. In aged animals, this is accompanied by a reduction in oxidative repair mechanisms resulting in increased methionine oxidation of the calcium signaling protein calmodulin in the brain. Here, we show that oxidation of calmodulin results in an inability to: (1) activate CaMKII; (2) support Thr(286) autophosphorylation of CaMKII; (3) prevent Thr(305/6) autophosphorylation of CaMKII; (4) support binding of CaMKII to the NR2B subunit of the NMDA receptor; and (5) compete with alpha-actinin for binding to CaMKII. Moreover, oxidized calmodulin does not efficiently bind calcium/calmodulin-dependent protein kinase II (CaMKII) in rat brain lysates or in vitro. These observations contrast from past experiments performed with oxidized calmodulin and the plasma membrane calcium ATPase, where oxidized calmodulin binds to, and partially activates the PMCA. When taken together, these data suggest that oxidative stress may perturb neuronal and cardiac function via a decreased ability of oxidized calmodulin to bind, activate, and regulate the interactions of CaMKII.     

Co-expression of human ABCG5 and ABCG8 in insect cells generates an androstan stimulated membrane ATPase activity

Mutations in the ATP-binding cassette (ABC) proteins ABCG5 or ABCG8 cause sitosterolemia, a condition with increased accumulation of plant sterols. Upon high level expression of the ABCG5 and ABCG8 proteins in baculovirus-Sf9 cell expression system we found a distinct, vanadate sensitive ATPase activity in isolated membrane preparations only when the two proteins were co-expressed. This ATPase activity was significantly stimulated by the addition of certain androgen hormones and analogs, and was effectively inhibited by progesterone. Our results provide a new aspect of biochemical and functional characterization of the ABCG5/ABCG8 proteins and their possible involvement in steroid hormone transport or regulation.