Residues within the first subdomain of the FERM-like domain in focal adhesion kinase are important in its regulation

We have previously described regulation of focal adhesion kinase (FAK) by its amino-terminal FERM-like domain through an autoinhibitory interaction with its kinase domain (Cooper, L. A., Shen, T. L., and Guan, J. L. (2003) Mol. Cell. Biol. 23, 8030-8041). Here we show that the first two subdomains of the FERM-like domain are independently capable of inhibiting phosphorylation of FAK in trans. We characterized several point mutations within the first subdomain of the FERM-like domain and find that mutation of Lys-38 to alanine results in a FAK mutant that is strongly hyperphosphorylated when expressed in mammalian cells, and promotes increased phosphorylation of the FAK substrate paxillin. A second mutation of Lys-78 to alanine results in a FAK mutant that is underphosphorylated, but can be activated by extracellular matrix stimuli. Like deletion of the amino terminus itself the K38A mutation is phosphorylated in suspension. The Delta375 truncation mutant of FAK is strongly phosphorylated both when Tyr-397 is mutated to phenylalanine, and in the presence of the Src inhibitor, PP2, suggesting that removal of the amino terminus can render FAK Src independent. This is in contrast to the K38A mutant that is not phosphorylated in the Y397F background, and which shows decreased phosphorylation in the presence of the Src inhibitor PP2, suggesting that regulation of FAK by Src is a secondary step in its activation. The K38A mutation weakens the interaction between the amino terminus of FAK and its own kinase domain, and disrupts the ability of the amino terminus to inhibit the phosphorylation of FAK in trans. The K38A mutation of FAK also increases the ability of FAK to promote cell cycle progression and cell migration, suggesting that hyperphosphorylation of this mutant can positively affect FAK function in cells. Together, these data strongly suggest a role for the first FAK subdomain of the FERM domain in its normal regulation and function in the cell.     

Lapworthellids and other skeletonised microfossils from the Cambrian Stage 3 of the northern Montagne Noire,southern France

The Montagne Noire (southern France) possesses one of the most complete Cambrian successions in the western peri-Gondwana margin and might provide a good stratigraphic reference for both regional charts and international correlations. However, to date, the lower Cambrian succession of the northern Montagne Noire has been supposed to be devoid of biostratigraphically significant fossils. The complex tectonostratigraphic framework of the area (a range divided into Axial Zone, northern and southern Montagne Noire) exacerbated problems related to regional correlations and palaeogeographic reconstructions. As a result, the chronostratigraphic context of the lower Cambrian of northern Montagne Noire is still uncertain and stratigraphic reports have broadly relied on putative lithostratigraphic correlations with the southern Montagne Noire. The purpose of this study is to characterise, for the first time, the fossil record of carbonate beds and lenses of the northern Montagne Noire occurring at the top of the siliciclastic-dominated Marcory Formation, in order to provide regional bio- and chronostratigraphic constraints on lower Cambrian strata. Moreover, this study is aimed at improving international chronostratigraphic correlation. Carbonate beds and lenses cropping out along the Orque Cliff, in the Mélagues thrust slice, were investigated. They yielded a faunal assemblage constituted of molluscs (Igorella cf. ungulata and Igorella moncereti n. sp.), hyoliths (Conotheca brevica), chancelloriids (Archiasterella cf. pentactina and Allonnia sp.) and tommotiids (Lapworthella rete). L. rete is recorded in upper Meishucunian (Cambrian Stage 3) strata of the Yangtze Platform (South China) where it is used as index fossil of the Cambrian Stage 3 Sinosachites flabelliformis–Tannuolina zhangwentangi Assemblage Zone. Therefore, the presence of this tommotiid provides evidence that the studied carbonate beds and lenses are Cambrian Age 3 (Atdabanian according to the Siberian chart). The upper part of the Marcory Formation in the Mélagues slice, dated as Cambrian Stage 3, might represent a lateral equivalent of the mixed (carbonate-siliciclastic) Pardailhan Formation defined in the southern Montagne Noire.     

The Numerical Solution of the Time-Dependent Nernst-Planck Equations

Calculations are reported of the time-dependent Nernst-Planck equations for a thin permeable membrane between electrolytic solutions. Charge neutrality is assumed for the time-dependent case. The response of such a membrane system to step current input is measured in terms of the time and space changes in concentration, electrical potential, and effective conductance. The report also includes discussion of boundary effects that occur when charge neutrality does not hold in the steady-state case.     

Native mass spectrometry provides direct evidence for DNA mismatch-induced regulation of asymmetric nucleotide binding in mismatch repair protein MutS

The DNA mismatch repair protein MutS recognizes mispaired bases in DNA and initiates repair in an ATP-dependent manner. Understanding of the allosteric coupling between DNA mismatch recognition and two asymmetric nucleotide binding sites at opposing sides of the MutS dimer requires identification of the relevant MutS.mmDNA.nucleotide species. Here, we use native mass spectrometry to detect simultaneous DNA mismatch binding and asymmetric nucleotide binding to Escherichia coli MutS. To resolve the small differences between macromolecular species bound to different nucleotides, we developed a likelihood based algorithm capable to deconvolute the observed spectra into individual peaks. The obtained mass resolution resolves simultaneous binding of ADP and AMP.PNP to this ABC ATPase in the absence of DNA. Mismatched DNA regulates the asymmetry in the ATPase sites; we observe a stable DNA-bound state containing a single AMP.PNP cofactor. This is the first direct evidence for such a postulated mismatch repair intermediate, and showcases the potential of native MS analysis in detecting mechanistically relevant reaction intermediates.     

Dynamic antibody specificities and virion concentrations in circulating immune complexes in acute to chronic HIV-1 infection

Understanding the interactions between human immunodeficiency virus type 1 (HIV-1) virions and antibodies (Ab) produced during acute HIV-1 infection (AHI) is critical for defining antibody antiviral capabilities. Antibodies that bind virions may prevent transmission by neutralization of virus or mechanically prevent HIV-1 migration through mucosal layers. In this study, we quantified circulating HIV-1 virion-immune complexes (ICs), present in approximately 90% of AHI subjects, and compared the levels and antibody specificity to those in chronic infection. Circulating HIV-1 virions coated with IgG (immune complexes) were in significantly lower levels relative to the viral load in acute infection than in chronic HIV-1 infection. The specificities of the antibodies in the immune complexes differed between acute and chronic infection (anti-gp41 Ab in acute infection and anti-gp120 in chronic infection), potentially suggesting different roles in immunopathogenesis for complexes arising at different stages of infection. We also determined the ability of circulating IgG from AHI to bind infectious versus noninfectious virions. Similar to a nonneutralizing anti-gp41 monoclonal antibody (MAb), purified plasma IgG from acute HIV-1 subjects bound both infectious and noninfectious virions. This was in contrast to the neutralizing antibody 2G12 MAb that bound predominantly infectious virions. Moreover, the initial antibody response captured acute HIV-1 virions without selection for different HIV-1 envelope sequences. In total, this study demonstrates that the composition of immune complexes are dynamic over the course of HIV-1 infection and are comprised initially of antibodies that nonselectively opsonize both infectious and noninfectious virions, likely contributing to the lack of efficacy of the antibody response during acute infection.     

The skeletal muscle-specific glycogen-targeted protein phosphatase 1 plays a major role in the regulation of glycogen metabolism by adrenaline in vivo

Adrenaline and insulin are the major hormones regulating glycogen metabolism in skeletal muscle. We have investigated the effects of these hormones on the rate-limiting enzymes of glycogen degradation and synthesis (phosphorylase and glycogen synthase respectively) in GM-/- mice homozygous for a null allele of the major skeletal muscle glycogen targeting subunit (GM) of protein phosphatase 1 (PP1). Hyperphosphorylation of Ser14 in phosphorylase, and Ser7, Ser640 and Ser640/644 of GS, in the skeletal muscle of GM-/- mice compared with GM+/+ mice indicates that the PP1-GM complex is the major phosphatase that dephosphorylates these sites in vivo. Adrenaline caused a 2.4-fold increase in the phosphorylase (-/+AMP) activity ratio in the skeletal muscle of control mice compared to a 1.4 fold increase in GM-/- mice. Adrenaline also elicited a 67% decrease in the GS (-/+G6P) activity ratio in control mice but only a small decrease in the skeletal muscle of GM-/- mice indicating that GM is required for the full response of phosphorylase and GS to adrenaline. PP1-GM activity and the amount of PP1 bound to GM decreased 40% and 45% respectively, in response to adrenaline in control mice. The data support a model in which adrenaline stimulates phosphorylation of phosphorylase Ser14 and GS Ser7 in GM+/+ mice by both kinase activation and PP1-GM inhibition and the phosphorylation of GS Ser640 and Ser640/644 by PP1-GM inhibition alone. Insulin decreased the phosphorylation of GS Ser640 and Ser640/644 and stimulated the GS (-/+G6P) activity ratio by approximately 2-fold in the skeletal muscle of either GM-/- and or control mice, but the low basal and insulin stimulated GS activity ratios in GM-/- mice indicate that PP1-GM is essential for maintaining normal basal and maximum insulin stimulated GS activity ratios in vivo.     

Localization of ORC1 during the cell cycle in human leukemia cells

The interaction of the origin recognition complex (ORC) with replication origins is a critical parameter in eukaryotic replication initiation. In mammals the ORC remains bound except during mitosis, thus the localization of ORC complexes allows localization of origins. A monoclonal antibody that recognizes human ORC1 was used to localize ORC complexes in populations of human MOLT-4 cells separated by cell cycle position using centrifugal elutriation. ORC1 staining in cells in early G1 is diffuse and primarily peripheral. As the cells traverse G1, ORC1 accumulates and becomes more localized towards the center of the nucleus, however around the G1/S boundary the staining pattern changes and ORC1 appears peripheral. By mid to late S phase ORC1 immunofluorescence is again concentrated at the nuclear center. During anaphase, ORC1 staining is localized mainly in the pericentriolar regions. These findings suggest that concerted movements of origin DNA sequences in addition to the previously documented assembly and disassembly of protein complexes are an important aspect of replication initiation loci in eukaryotes.     

MHC class II-bound self peptides from autoimmune MRL/lpr mice reveal potential T cell epitopes for autoantibody production in murine systemic lupus erythematosus

The systemic lupus erythematosus-like syndrome in MRL/lpr mice involves high-titered IgG autoantibodies, particularly antinuclear Abs that target histones, DNA, and RNA particles. Although T cell help is required for the generation of antinuclear Abs, the epitopes recognized by such helper T cells are unknown. To address this question, we isolated and sequenced self peptides bound by MHC class II molecules from MRL/lpr mice. We identified a number of peptides that are not seen in similar preparations from nonautoimmune C3H animals. The "abnormal" peptide donors include histone, a protein component of a small nuclear ribonucleoprotein, ribosomal proteins, and RNA processing enzymes. We postulate that the peptides from these donors are T cell epitopes required for the generation of the most frequent antinuclear Abs specificities seen in MRL/lpr mice.     

Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog

Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABA_ARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABA_AR modulation. [³H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[³H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABA_AR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([³H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1β3 GABA_ARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [³H]AziPm photoincorporation into GABA_AR subunits maximally by ∼50%. When the amino acids photolabeled by [³H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the β3-α1 subunit interface (β3Met-286 in β3M3 and α1Met-236 in α1M1), previously photolabeled by [³H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [³H]AziPm photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by R-[³H]mTFD-MPAB. The propofol-inhibitable [³H]AziPm photolabeling in the GABA_AR β3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the β3-β3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABA_AR transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.