Browsing by Subject "COS Cells"

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    Critical Interaction of Actuator Domain Residues Arginine 174, Isoleucine 188, and Lysine 205 with Modulatory Nucleotide in Sarcoplasmic Reticulum Ca 2+ -ATPase
    (2008) Clausen, Johannes D; McIntosh, David B; Woolley, David G; Andersen, Jens Peter
    ATP plays dual roles in the reaction cycle of the sarcoplasmic reticulum Ca2+-ATPase by acting as the phosphorylating substrate as well as in nonphosphorylating (modulatory) modes accelerating conformational transitions of the enzyme cycle. Here we have examined the involvement of actuator domain residues Arg174, Ile188, Lys204, and Lys205 by mutagenesis. Alanine mutations to these residues had little effect on the interaction of the Ca2E1 state with nucleotide or on the HnE 2 to Ca2E1 transition of the dephosphoenzyme. The phosphoenzyme processing steps, Ca2E1P to E2P and E2P dephosphorylation, and their stimulation by MgATP/ATP were markedly affected by mutations to Arg174, Ile188, and Lys205. Replacement of Ile188 with alanine abolished nucleotide modulation of dephosphorylation but not the modulation of the Ca2E1P to E2P transition. Mutation to Arg174 interfered with nucleotide modulation of either of the phosphoenzyme processing steps, indicating a significant overlap between the modulatory nucleotide-binding sites involved. Mutation to Lys205 enhanced the rates of the phosphoenzyme processing steps in the absence of nucleotide and disrupted the nucleotide modulation of the Ca2E1P to E2P transition. Remarkably, the mutants with alterations to Lys205 showed an anomalous inhibition by ATP of the dephosphorylation, and in the alanine mutant the affinity for the inhibition by ATP was indistinguishable from that for stimulation by ATP of the wild type. Hence, the actuator domain is an important player in the function of ATP as modulator of phosphoenzyme processing, with Arg174, Ile188, and Lys205 all being critically involved, although in different ways. The data support a variable site model for the modulatory effects with the nucleotide binding somewhat differently in each of the conformational states occurring during the transport cycle.
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    Roles of Conserved P Domain Residues and Mg 2+ in ATP Binding in the Ground and Ca 2+-activated States of Sarcoplasmic Reticulum Ca 2+-ATPase
    (2004) McIntosh, David B; Clausen, Johannes D; Woolley, David G; MacLennan, David H; Vilsen, Bente; Andersen, Jens Peter
    Residues in conserved motifs (625)TGD, (676)FARXXPXXK, and (701)TGDGVND in domain P of sarcoplasmic reticulum Ca(2+)-ATPase, as well as in motifs (601)DPPR and (359)NQR(/K)MSV in the hinge segments connecting domains N and P, were examined by mutagenesis to assess their roles in nucleotide and Mg(2+) binding and stabilization of the Ca(2+)-activated transition state for phosphoryl transfer. In the absence of Mg(2+), mutations removing the charges of domain P residues Asp(627), Lys(684), Asp(703), and Asp(707) increased the affinity for ATP and 2',3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine 5'-triphosphate. These mutations, as well as Gly(626)--> Ala, were inhibitory for ATP binding in the presence of Mg(2+) and for tight binding of the beta,gamma-bidentate chromium(III) complex of ATP. The hinge mutations had pronounced, but variable, effects on ATP binding only in the presence of Mg(2+). The data demonstrate an unfavorable electrostatic environment for binding of negatively charged nucleotide in domain P and show that Mg(2+) is required to anchor the phosphoryl group of ATP at the phosphorylation site. Mutants Gly(626) --> Ala, Lys(684) --> Met, Asp(703) --> Ala/Ser/Cys, and mutants with alteration to Asp(707) exhibited very slow or negligible phosphorylation, making it possible to measure ATP binding in the pseudo-transition state attained in the presence of both Mg(2+) and Ca(2+). Under these conditions, ATP binding was almost completely blocked in Gly(626) --> Ala and occurred with 12- and 7-fold reduced affinities in Asp(703) --> Ala and Asp(707) --> Cys, respectively, relative to the situation in the presence of Mg(2+) without Ca(2+), whereas in Lys(684) --> Met and Asp(707) --> Ser/Asn the affinity was enhanced 14- and 3-5-fold, respectively. Hence, Gly(626) and Asp(703) seem particularly critical for mediating entry into the transition state for phosphoryl transfer upon Ca(2+) binding at the transport sites.
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    The Functional Microdomain in Transmembrane Helices 2 and 7 Regulates Expression, Activation, and Coupling Pathways of the Gonadotropin-releasing Hormone Receptor
    (1999) Flanagan, Colleen A; Zhou, Wei; Chi, Ling; Yuen, Tony; Rodic, Vladimir; Robertson, Derek; Johnson, Melanie; Holland, Pamela; Millar, Robert P; Weinstein, Harel; Mitchell, Rory; Sealfon, Stuart C
    Structural microdomains of G protein-coupled receptors (GPCRs) consist of spatially related side chains that mediate discrete functions. The conserved helix 2/helix 7 microdomain was identified because the gonadotropin-releasing hormone (GnRH) receptor appears to have interchanged the Asp(2.50) and Asn(7.49) residues which are conserved in transmembrane helices 2 and 7 of rhodopsin-like GPCRs. We now demonstrate that different side chains of this microdomain contribute specifically to receptor expression, heterotrimeric G protein-, and small G protein-mediated signaling. An Asn residue is required in position 2.50(87) for expression of the GnRH receptor at the cell surface, most likely through an interaction with the conserved Asn(1.50(53)) residue, which we also find is required for receptor expression. Most GPCRs require an Asp side chain at either the helix 2 or helix 7 locus of the microdomain for coupling to heterotrimeric G proteins, but the GnRH receptor has transferred the requirement for an acidic residue from helix 2 to 7. However, the presence of Asp at the helix 7 locus precludes small G protein-dependent coupling to phospholipase D. These results implicate specific components of the helix 2/helix 7 microdomain in receptor expression and in determining the ability of the receptor to adopt distinct activated conformations that are optimal for interaction with heterotrimeric and small G proteins.
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    UV-mediated Regulation of the anti-senescence factor Tbx2
    (2008) Abrahams, Amaal; Mowla, Shaheen; PARKER, M Iqbal; Goding, Colin R; Prince, Sharon
    Several lines of evidence have implicated members of the developmentally important T-box gene family in cell cycle regulation and in cancer. Importantly, the highly related T-box factors Tbx2 and Tbx3 can suppress senescence through repressing the cyclin-dependent kinase inhibitors p19(ARF) and p21(WAF1/CIP1/SDII). Furthermore, Tbx2 is up-regulated in several cancers, including melanomas where it was shown to function as an anti-senescence factor, suggesting that this may be one of the mechanisms by which T-box proteins contribute to the oncogenic process. However, very little is known about whether Tbx2 is regulated by p21-mediated stress-induced senescence signaling pathways. In this study, using the MCF-7 breast cancer cell line known to overexpress Tbx2, we show that in response to stress induced by ultraviolet irradiation the Tbx2 protein is specifically phosphorylated by the p38 mitogen-activated protein kinase. Using site-directed mutagenesis and in vitro kinase assays, we have identified serine residues 336, 623, and 675 in the Tbx2 protein as the p38 target sites and show that these sites are phosphorylated in vivo. Importantly, we show by Western blotting, immunofluorescence, and reporter assays that this phosphorylation leads to increased Tbx2 protein levels, predominant nuclear localization of the protein, and an increase in the ability of Tbx2 to repress the p21(WAF1/CIP1/SDII) promoter. These results show for the first time that the ability of Tbx2 to repress the p21 gene is enhanced in response to a stress-induced senescence pathway, which leads to a better understanding of the regulation of the anti-senescence function of Tbx2.