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181.
Sagi SA Seasholtz TM Kobiashvili M Wilson BA Toksoz D Brown JH 《The Journal of biological chemistry》2001,276(18):15445-15452
Recent reports have shown that several heterotrimeric protein-coupled receptors that signal through Galpha(q) can induce Rho-dependent responses, but the pathways that mediate the interaction between Galpha(q) and Rho have not yet been identified. In this report we present evidence that Galpha(q) expressed in COS-7 cells coprecipitates with the Rho guanine nucleotide exchange factor (GEF) Lbc. Furthermore, Galpha(q) expression enhances Rho-dependent responses. Coexpressed Galpha(q) and Lbc have a synergistic effect on the Rho-dependent rounding of 1321N1 astrocytoma cells. In addition, serum response factor-dependent gene expression, as assessed by the SRE.L reporter gene, is synergistically activated by Galpha(q) and Rho GEFs. The synergistic effect of Galpha(q) on this response is inhibited by C3 exoenzyme and requires phospholipase C activation. Surprisingly, expression of Galpha(q), in contrast to that of Galpha(12) and Galpha(13), does not increase the amount of activated Rho. We also observe that Galpha(q) enhances SRE.L stimulation by activated Rho, indicating that the effect of Galpha(q) occurs downstream of Rho activation. Thus, Galpha(q) interacts physically and/or functionally with Rho GEFs; however this does not appear to lead to or result from increased activation of Rho. We suggest that Galpha(q)-generated signals enhance responses downstream of Rho activation. 相似文献
182.
Shackelford RE Innes CL Sieber SO Heinloth AN Leadon SA Paules RS 《The Journal of biological chemistry》2001,276(24):21951-21959
Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by neuronal degeneration accompanied by ataxia, telangiectasias, acute cancer predisposition, and sensitivity to ionizing radiation (IR). Cells from individuals with AT show unusual sensitivity to IR, severely attenuated cell cycle checkpoint functions, and poor p53 induction in response to IR compared with normal human fibroblasts (NHFs). The gene mutated in AT (ATM) has been cloned, and its product, pATM, has IR-inducible kinase activity. The AT phenotype has been suggested to be a consequence, at least in part, of an inability to respond appropriately to oxidative damage. To test this hypothesis, we examined the ability of NHFs and AT dermal fibroblasts to respond to t-butyl hydroperoxide and IR treatment. AT fibroblasts exhibit, in comparison to NHFs, increased sensitivity to the toxicity of t-butyl hydroperoxide, as measured by colony-forming efficiency assays. Unlike NHFs, AT fibroblasts fail to show G(1) and G(2) phase checkpoint functions or to induce p53 in response to t-butyl hydroperoxide. Treatment of NHFs with t-butyl hydroperoxide activates pATM-associated kinase activity. Our results indicate that pATM is involved in responding to certain aspects of oxidative damage and in signaling this information to downstream effectors of the cell cycle checkpoint functions. Our data further suggest that some of the pathologies seen in AT could arise as a consequence of an inability to respond normally to oxidative damage. 相似文献
183.
Martens JR Sakamoto N Sullivan SA Grobaski TD Tamkun MM 《The Journal of biological chemistry》2001,276(11):8409-8414
The precise subcellular localization of ion channels is often necessary to ensure rapid and efficient integration of both intracellular and extracellular signaling events. Recently, we have identified lipid raft association as a novel mechanism for the subcellular sorting of specific voltage-gated K(+) channels to regions of the membrane rich in signaling complexes. Here, we demonstrate isoform-specific targeting of voltage-gated K(+) (Kv) channels to distinct lipid raft populations with the finding that Kv1.5 specifically targets to caveolae. Multiple lines of evidence indicate that Kv1.5 and Kv2.1 exist in distinct raft domains: 1) channel/raft association shows differential sensitivity to increasing concentrations of Triton X-100; 2) unlike Kv2.1, Kv1.5 colocalizes with caveolin on the cell surface and redistributes with caveolin following microtubule disruption; and 3) immunoisolation of caveolae copurifies Kv1.5 channel. Both depletion of cellular cholesterol and inhibition of sphingolipid synthesis alter Kv1.5 channel function by inducing a hyperpolarizing shift in the voltage dependence of activation and inactivation. The differential targeting of Kv channel subtypes to caveolar and noncaveolar rafts within a single membrane represents a unique mechanism of compartmentalization, which may permit isoform-specific modulation of K(+) channel function. 相似文献
184.
Rothbauer U Hofmann S Mühlenbein N Paschen SA Gerbitz KD Neupert W Brunner M Bauer MF 《The Journal of biological chemistry》2001,276(40):37327-37334
Tim8 and Tim13 of yeast belong to a family of evolutionary conserved zinc finger proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. Mutations in DDP1 (deafness dystonia peptide 1), the human homolog of Tim8, are associated with the Mohr-Tranebjaerg syndrome, a progressive neurodegenerative disorder. We show that DDP1 acts with human Tim13 in a complex in the intermembrane space. The DDP1.hTim13 complex is in direct contact with translocation intermediates of human Tim23 in mammalian mitochondria. The human DDP1.hTim13 complex complements the function of the TIM8.13 complex in yeast and facilitates import of yeast and human Tim23. Thus, the pathomechanism underlying the Mohr-Tranebjaerg syndrome may involve an impaired biogenesis of the human TIM23 complex causing severe pleiotropic mitochondrial dysfunction. 相似文献
185.
Haney SA Glasfeld E Hale C Keeney D He Z de Boer P 《The Journal of biological chemistry》2001,276(15):11980-11987
The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division in Escherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZ(D373G)) identified eight different changes at two residues within this sequence. In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZ(D373G) failed to interact. Two mutant proteins examined restored this interaction significantly. In vivo, the alleles tested are significantly more toxic than the wild type ftsZ and cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZ(D373G), no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently. 相似文献
186.
187.
188.
Miller BG Snider MJ Wolfenden R Short SA 《The Journal of biological chemistry》2001,276(18):15174-15176
The crystal structure of yeast orotidine-5'-phosphate decarboxylase in complex with the postulated transition state analog, 6-hydroxyuridine-5'-phosphate, reveals contacts between this inhibitor and a novel quartet of charged residues (Lys-59, Asp-91, Lys-93, and Asp-96) within the active site. The structure also suggests a possible interaction between O2 of the 6-hydroxyuridine-5'-phosphate pyrimidine ring and Gln-215. Here we report the results of mutagenesis of each of the charged active site residues and Gln-215. The activities of the Q215A and wild-type enzymes were equal indicating that any interactions between this residue and the pyrimidine ring are dispensable for efficient decarboxylation. For the D91A and K93A mutant enzymes, activity was reduced by more than 5 orders of magnitude and substrate binding could not be detected by isothermal calorimetry. For the D96A mutant enzyme, k(cat) was reduced by more than 5 orders of magnitude, and isothermal calorimetry indicated an 11-fold decrease in the affinity of this enzyme for the substrate in the ground state. For the K59A enzyme, k(cat) was reduced by a factor of 130, and K(m) had increased by a factor of 900. These results indicate that the integrity of the network of charged residues is essential for transition state stabilization. 相似文献
189.
Testing hypotheses related to the effect of gravitational orientation on neural control mechanisms is difficult for most locomotor tasks, like walking, because body orientation with respect to gravity affects both sensorimotor control and task mechanics. To examine the mechanical effect of body orientation independently from changes in workload and posture, Brown et al. (J. Biomech. 29 p. 1349, 1996) studied pedaling at altered body orientations. They found that subjects pedaling at different orientations changed needlessly their muscle excitations, putatively to preserve body-upright pedaling kinematics. We tested the feasibility of this hypothesis using simulations based on a three biomechanical-function pair organization for control of lower limb muscles (limb extension/flexion pair, extension/flexion transition pair, and foot plantarflexion/dorsiflexion pair), where each pair consists of alternating agonistic/antagonistic muscles. Adjustment of only three parameters, one to scale the muscle excitations of each pair, was sufficient to preserve pedaling kinematics to altered body orientation. Because these adjustments produced changes in muscle excitation and net joint moments similar to those observed in pedaling subjects, the hypothesis is supported. Moreover, the effectiveness of a decoupled gain adjustment procedure where each parameter was adjusted by error in only one aspect of the pedaling trajectory during each iteration (i.e., cadence adjusted the Ext/Flex parameter; peak-to-peak variation in crank velocity over the cycle adjusted the transition parameter; average ankle angle over the cycle adjusted the foot parameter) further supports the distinct function of each muscle pair. 相似文献
190.
Keilhack H Müller M Böhmer SA Frank C Weidner KM Birchmeier W Ligensa T Berndt A Kosmehl H Günther B Müller T Birchmeier C Böhmer FD 《The Journal of cell biology》2001,152(2):325-334
Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling. 相似文献