Counting on mitogen-activated protein kinases--ERKs 3, 4, 5, 6, 7 and 8

Signal transduction pathways in eukaryotic cells integrate diverse extracellular signals, and regulate complex biological responses such as growth, differentiation and death. One group of proline-directed Ser/Thr protein kinases, the mitogen-activated protein kinases (MAPKs), plays a central role in these signalling pathways. Much attention has focused in recent years on three subfamilies of MAPKs, the extracellular signal regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and the p38 MAPKs. However, the ERK family is broader than the ERK1 and ERK2 proteins that have been the subject of most studies in this area. Here we overview the work on ERKs 3 to 8, emphasising where possible their biological activities as well as distinctive biochemical properties. It is clear from these studies that these additional ERKs show similarities to ERK1 and ERK2, but with some interesting differences that challenge the paradigm of the archetypical ERK1/2 MAPK pathway.     

The Effect of Cauda Equina Constriction on Nitric Oxide Synthase Activity

Nitric oxide synthase (NOS) activity was studied in the gray and white matter regions of the spinal cord 2 and 5 days after multiple cauda equina constrictions of the central processes of L7-Co5 dorsal root ganglia neurons. The results show considerable differences in enzyme activity in the thoracic, upper lumbar, lower lumbar, and sacral segments. Increased NOS activity was observed at 2 days after multiple cauda equina constrictions in the dorsal, lateral, and ventral columns of the lower lumbar segments and in the ventral column of the upper lumbar segments. The values returned to control levels within 5 postconstriction days. In the lateral columns of thoracic segments taken 2 and 5 days after surgery, NOS activity was enhanced by 54% and 55% and in the upper lumbar segments by 130% and 163%, respectively. Multiple cauda equina constrictions performed surgically for 2 and 5 days caused a significant increase in NOS activity predominantly in the gray matter regions of thoracic segments. A quite different response was found 5 days postconstriction in the upper lumbar segments, where the enzyme activity was significantly decreased in the dorsal horn, intermediate zone, and ventral horn. No such extreme differences could be seen in the lower lumbar segments, where NOS activity was significantly enhanced only in the ventral horn. The data correspond with a higher number of NOS immunoreactive somata, quantitatively evaluated in the ventral horn of the lower lumbar segments at 5 days after multiple cauda equina constrictions. While the great region-dependent heterogeneity in NOS activity seen 2 and 5 days after multiple cauda equina constrictions is quite apparent and suggestive of an active role played by nitric oxide in neuroprotective or neurotoxic processes occurring in the gray and white matter of the spinal cord, the extent of damage or the degree of neuroprotection caused by nitric oxide in compartmentalized gray and white matter in this experimental paradigm would be possible only using longer postconstriction periods.     

Loop Domain Peptides from the SOS ras-Guanine Nucleotide Exchange Protein, Identified from Molecular Dynamics Calculations, Strongly Inhibit ras Signaling

In the accompanying paper, we found, using molecular dynamics calculations, four domains of the ras-specific SOS guanine nucleotide exchange protein (residues 589-601, 654-675, 746-761, and 980-989) that differ markedly in conformation when SOS is complexed with either oncogenic (Val 12-) ras-p21 or wild-type ras-p21. Three of these domains contain three crystallographically undefined loops that we modeled in these calculations, and one is a newly identified non-loop domain containing SOS residues 980-989. We have now synthesized peptides corresponding to these four domains and find that all of them block Val 12-ras-p21-induced oocyte maturation. All of them also block insulin-induced oocyte maturation, but two of these peptides, corresponding to SOS residues 589-601 and 980-989, block oncogenic ras to a significantly greater extent. These results suggest that SOS contains domains, including the three loop domains, that are important for ras signaling and that several of these domains can activate different pathways specific to oncogenic or wild-type ras-p21.     

Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes

Heteronuclear NMR methods have been used to probe the conformation of four complexes of Escherichia coli dihydrofolate reductase (DHFR) in solution. (1)H(N), (15)N, and (13)C(alpha) resonance assignments have been made for the ternary complex with folate and oxidized NADP(+) cofactor and the ternary complex with folate and a reduced cofactor analog, 5,6-dihydroNADPH. The backbone chemical shifts have been compared with those of the binary complex of DHFR with the substrate analog folate and the binary complex with NADPH (the holoenzyme). Analysis of (1)H(N) and (15)N chemical shifts has led to the identification of marker resonances that report on the active site conformation of the enzyme. Other backbone amide resonances report on the presence of ligands in the pterin binding pocket and in the adenosine and nicotinamide-ribose binding sites of the NADPH cofactor. The chemical shift data indicate that the enzyme populates two dominant structural states in solution, with the active site loops in either the closed or occluded conformations defined by X-ray crystallography; there is no evidence that the open conformation observed in some X-ray structures of E. coli DHFR are populated in solution.     

Discord in the family Sparidae (Teleostei): divergent phylogeographical patterns across the Atlantic-Mediterranean divide

The Strait of Gibraltar has been proposed to be the divide between two marine biogeographical regions, the Mediterranean Sea and the Northeast Atlantic. Intraspecific studies have shown, for several of the examined species, a reduction of gene flow between the two basins. The present study examines genetic variation at nuclear and mitochondrial loci in five marine teleost species belonging to the family Sparidae. Four samples for each species were analysed spanning the Northeast Atlantic and the Mediterranean. For all individuals 17 allozyme loci were scored and a combined single strand conformation polymorphism-sequencing approach was used to survey approximately 190 bp of the mitochondrial DNA (mtDNA) D-loop region. All five species share similar biological features. For three species, namely Lithognathus mormyrus, Spondyliosoma cantharus, and Dentex dentex, large mtDNA divergence was observed between Atlantic and Mediterranean samples. Little or no mtDNA differentiation was found in the other two species, Pagrus pagrus and Pagellus bogaraveo. Allozyme data revealed strong differentiation when comparing Atlantic and Mediterranean samples of L. mormyrus and D. dentex, moderate for P. pagrus, and no differentiation for P. bogaraveo and S. cantharus. These results provide evidence for a sharp phylogeographical break (sensu Avise) between the Atlantic and the Mediterranean for two (or possibly three) sparid species of the five investigated. At the same time, the obtained results for the other two species raise the question on which ecological/historical factors might have caused the observed discrepancy in the geographical distribution of genetic variation among otherwise biologically similar species.     

Predicting reliable regions in protein alignments from sequence profiles

For applications such as comparative modelling one major issue is the reliability of sequence alignments. Reliable regions in alignments can be predicted using sub-optimal alignments of the same pair of sequences. Here we show that reliable regions in alignments can also be predicted from multiple sequence profile information alone.Alignments were created for a set of remotely related pairs of proteins using five different test methods. Structural alignments were used to assess the quality of the alignments and the aligned positions were scored using information from the observed frequencies of amino acid residues in sequence profiles pre-generated for each template structure. High-scoring regions of these profile-derived alignment scores were a good predictor of reliably aligned regions.These profile-derived alignment scores are easy to obtain and are applicable to any alignment method. They can be used to detect those regions of alignments that are reliably aligned and to help predict the quality of an alignment. For those residues within secondary structure elements, the regions predicted as reliably aligned agreed with the structural alignments for between 92% and 97.4% of the residues. In loop regions just under 92% of the residues predicted to be reliable agreed with the structural alignments. The percentage of residues predicted as reliable ranged from 32.1% for helix residues to 52.8% for strand residues.This information could also be used to help predict conserved binding sites from sequence alignments. Residues in the template that were identified as binding sites, that aligned to an identical amino acid residue and where the sequence alignment agreed with the structural alignment were in highly conserved, high scoring regions over 80% of the time. This suggests that many binding sites that are present in both target and template sequences are in sequence-conserved regions and that there is the possibility of translating reliability to binding site prediction.     

The solution structure of the loop E region of the 5S rRNA from spinach chloroplasts

A structure has been obtained for the loop E region of the 5S rRNA from Spinacia oleracia chloroplast ribosomes using residual dipolar coupling data as well as NOE, J coupling and chemical shift information. Even though the loop E sequence of this chloroplast 5S rRNA differs from that of Escherichia coli loop E at approximately 40% of its positions, its conformation is remarkably similar to that of E.coli loop E. Consistent with this conclusion, ribosomal protein L25 from E.coli, which binds to the loop E region of both intact E.coli 5S rRNA and to oligonucleotides containing that sequence, also binds to the chloroplast-derived oligonucleotide discussed here.     

Two types of HKT transporters with different properties of Na+ and K+ transport in Oryza sativa

It is thought that Na+ and K+ homeostasis is crucial for salt-tolerance in plants. To better understand the Na+ and K+ homeostasis in important crop rice (Oryza sativa L.), a cDNA homologous to the wheat HKT1 encoding K+-Na+ symporter was isolated from japonica rice, cv Nipponbare (Ni-OsHKT1). We also isolated two cDNAs homologous to Ni-OsHKT1 from salt-tolerant indica rice, cv Pokkali (Po-OsHKT1, Po-OsHKT2). The predicted amino acid sequence of Ni-OsHKT1 shares 100% identity with Po-OsHKT1 and 91% identity with Po-OsHKT2, and they are 66-67% identical to wheat HKT1. Low-K+ conditions (less than 3 mM) induced the expression of all three OsHKT genes in roots, but mRNA accumulation was inhibited by the presence of 30 mM Na+. We further characterized the ion-transport properties of OsHKT1 and OsHKT2 using an expression system in the heterologous cells, yeast and Xenopus oocytes. OsHKT2 was capable of completely rescuing a K+-uptake deficiency mutation in yeast, whereas OsHKT1 was not under K+-limiting conditions. When OsHKTs were expressed in Na+-sensitive yeast, OsHKT1 rendered the cells more Na+-sensitive than did OsHKT2 in high NaCl conditions. The electrophysiological experiments for OsHKT1 expressed in Xenopus oocytes revealed that external Na+, but not K+, shifted the reversal potential toward depolarization. In contrast, for OsHKT2 either Na+ or K+ in the external solution shifted the reversal potential toward depolarization under the mixed Na+ and K+ containing solutions. These results suggest that two isoforms of HKT transporters, a Na+ transporter (OsHKT1) and a Na+- and K+-coupled transporter (OsHKT2), may act harmoniously in the salt tolerant indica rice.     

Stiffness of the distal loop restricts the structural heterogeneity of the transition state ensemble in SH3 domains

Protein engineering experiments and Phi(F)-value analysis of SH3 domains reveal that their transition state ensemble (TSE) is conformationally restricted, i.e. the fluctuations in the transition state (TS) structures are small. In the TS of src SH3 and alpha-spectrin SH3 the distal loop and the associated hairpin are fully structured, while the rest of the protein is relatively disordered. If native structure predominantly determines the folding mechanism, the findings for SH3 folds raise the question: What are the features of the native topology that determine the nature of the TSE? We propose that the presence of stiff loops in the native state that connect local structural elements (such as the distal hairpin in SH3 domains) conformationally restricts TSE. We validate this hypothesis using the simulations of a "control" system (16 residue beta-hairpin forming C-terminal fragment of the GBl protein) and its variants. In these fragments the role of bending rigidity in determining the nature of the TSE can be directly examined without complications arising from interactions with the rest of the protein. The TSE structures in the beta-hairpins are determined computationally using cluster analysis and limited Phi(F)-value analysis. Both techniques prove that the conformational heterogeneity decreases as the bending rigidity of the loop increases. To extend this finding to SH3 domains a measure of bending rigidity based on loop curvature, which utilizes native structures in the Protein Data Bank (PDB), is introduced. Using this measure we show that, with few exceptions, the ordering of stiffness of the distal, n-src, and RT loops in the 29 PDB structures of SH3 domains is conserved. Combining the simulation results for beta-hairpins and the analysis of PDB structures for SH3 domains, we propose that the stiff distal loop restricts the conformational fluctuations in the TSE. We also predict that constraining the distal loop to be preformed in the denatured ensemble should not alter the nature of TSE. On the other hand, if the amino and carboxy terminals are cross-linked to form a circular polypeptide chain, the pathways and TSs are altered. These contrasting scenarios are illustrated using simulations of cross-linked WT beta-hairpin fragments. Computations of bending rigidities for immunoglobulin-like domain proteins reveal no clear separation in the stiffness of their loops. In the beta-sandwich proteins, which have large fractions of non-local native contacts, the nature of the TSE cannot be apparently determined using purely local structural characteristics. Nevertheless, the measure of loop stiffness still provides qualitative predictions of the ordered regions in the TSE of Ig27 and TenFn3.