C-terminal Thr Research Articles

Inhibition of cell adhesion by phosphorylated Ezrin/Radixin/Moesin

Altered phosphorylation status of the C-terminal Thr residues of Ezrin/Radixin/Moesin (ERM) is often linked to cell shape change. To determine the role of phophorylated ERM, we modified phosphorylation status of ERM and investigated changes in cell adhesion and morphology. Treatment with Calyculin-A (Cal-A), a protein phosphatase inhibitor, dramatically augmented phosphorylated ERM (phospho-ERM). Cal-A-treatment or expression of phospho-mimetic Moesin mutant (Moesin-TD) induced cell rounding in adherent cells. Moreover, reattachment of detached cells to substrate was inhibited by either treatment. Phospho-ERM, Moesin-TD and actin cytoskeleton were observed at the plasma membrane of such round cells. Augmented cell surface rigidity was also observed in both cases.Meanwhile, non-adherent KG-1 cells were rather rich in phospho-ERM. Treatment with Staurosporine, a protein kinase inhibitor that dephosphorylates phospho-ERM, up-regulated the integrin-dependent adhesion of KG-1 cells to substrate.These findings strongly suggest the followings: (1) Phospho-ERM inhibit cell adhesion, and therefore, dephosphorylation of ERM proteins is essential for cell adhesion. (2) Phospho-ERM induce formation and/or maintenance of spherical cell shape. (3) ERM are constitutively both phosphorylated and dephosphorylated in cultured adherent and non-adherent cells.

Constitutively active ezrin desensitizes IL-7 signaling and impairs T cell homeostasis (LYM2P.735)

Abstract IL-7 signaling results in tethering of the IL-7 receptor to the actin cytoskeleton, which is mediated by the linker molecules ezrin and moesin. To understand the role of cytoskeleton linker molecules in cytokine signaling, we analyzed mice expressing a constitutively active ezrin transgene in T cells. The ezrin transgene was generated by introducing a point mutation at a conserved C-terminal Thr residue (567T->E) that results in a constitutively open conformation of ezrin. Interestingly, in mice expressing the constitutively active ezrin transgene (T567E-Tg), peripheral T cell numbers were significantly reduced and T cell survival was severely impaired. Moreover, T567E-Tg T cells were defect in IL-7 signaling even as they expressed high levels of surface IL-7 receptor proteins. Additionally, ezrin T567E-Tg induced downregulation of CD8 coreceptor expression, suggesting a “coreceptor tuning” effect of activated ezrin on CD8 T cells. Coreceptor tuning is a model of T cell homeostasis which posits that cytokine signals tailor CD8 coreceptor gene expression to the self-specificity of the TCR. In agreement, TCR disengagement restored IL-7 signaling in T567E-Tg CD8 T cells. Thus, constitutively active ezrin desensitizes IL-7 receptor signaling through increased TCR signaling but not through direct interference with the IL-7 receptor. The molecular mechanisms of ezrin activation on TCR signaling and T cell homeostasis will be discussed.

BLT2 phosphorylation at Thr 355 by Akt is necessary for BLT2-mediated chemotaxis

BLT2, a low-affinity leukotriene B 4 (LTB 4) receptor, is a member of the G protein-coupled receptor family and is involved in multiple cellular responses, including chemotaxis. Despite its biological significance, the mechanisms of BLT2 regulation, especially by protein kinases, are poorly characterised. In this study, we found that Akt phosphorylates BLT2 at its C-terminal Thr 355 residue and that this event is critical for BLT2-mediated chemotactic responses. In addition, we found that Rac1 stimulation and subsequent reactive oxygen species (ROS) production lie downstream of BLT2 phosphorylation, thus mediating chemotaxis. Structured summary of protein interactions BLT2 physically interacts with Akt by pull down ( View interaction) BLT2 physically interacts with Akt by pull down ( View interaction)

Structural and Functional Characterization of the Streptococcus pneumoniae RrgB Pilus Backbone D1 Domain

Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.

663 Age dependence of T-cell lymphoma induction by radiation exposure in Mlh1-deficient mice

Ezrin/radixin/moesin (ERM) proteins are membrane-cytoskeleton linkers that also have roles in signal transduction. Here we show that G protein-coupled receptor kinase 2 (GRK2) regulates membrane protrusion and cell migration during wound closure in Madin-Darby canine kidney (MDCK) epithelial cell monolayers at least partly through activating phosphorylation of radixin on a conserved, regulatory C-terminal Thr residue. GRK2 phosphorylated radixin exclusively on Thr 564 in vitro. Expression of a phosphomimetic (Thr-564-to-Asp) mutant of radixin resulted in increased Rac1 activity, membrane protrusion and cell motility in MDCK cells, suggesting that radixin functions “upstream” of Rac1, presumably as a scaffolding protein. Phosphorylation of ERM proteins was highest during the most active phase of epithelial cell sheet migration over the course of wound closure. In view of these results, we explored the mode of action of quinocarmycin/quinocarcin analog DX-52-1, an inhibitor of cell migration and radixin function with considerable selectivity for radixin over the other ERM proteins, finding that its mechanism of inhibition of radixin does not appear to involve binding and antagonism at the site of regulatory phosphorylation.

G protein-coupled receptor kinase 2 activates radixin, regulating membrane protrusion and motility in epithelial cells

Ezrin/radixin/moesin (ERM) proteins are membrane-cytoskeleton linkers that also have roles in signal transduction. Here we show that G protein-coupled receptor kinase 2 (GRK2) regulates membrane protrusion and cell migration during wound closure in Madin-Darby canine kidney (MDCK) epithelial cell monolayers at least partly through activating phosphorylation of radixin on a conserved, regulatory C-terminal Thr residue. GRK2 phosphorylated radixin exclusively on Thr 564 in vitro. Expression of a phosphomimetic (Thr-564-to-Asp) mutant of radixin resulted in increased Rac1 activity, membrane protrusion and cell motility in MDCK cells, suggesting that radixin functions “upstream” of Rac1, presumably as a scaffolding protein. Phosphorylation of ERM proteins was highest during the most active phase of epithelial cell sheet migration over the course of wound closure. In view of these results, we explored the mode of action of quinocarmycin/quinocarcin analog DX-52-1, an inhibitor of cell migration and radixin function with considerable selectivity for radixin over the other ERM proteins, finding that its mechanism of inhibition of radixin does not appear to involve binding and antagonism at the site of regulatory phosphorylation.

Association of Paracellin-1 with ZO-1 Augments the Reabsorption of Divalent Cations in Renal Epithelial Cells

Paracellin-1 (PCLN-1) belongs to the claudin family of tight junction proteins and possibly plays a critical role in the reabsorption of magnesium and calcium. So far, the physiological properties of PCLN-1 have not been clarified. In the present study, we investigated whether PCLN-1 is associated with ZO-1. We also investigated whether (45)Ca(2+) transport across the paracellular barrier is affected by this association. In vitro binding analysis using glutathione S-transferase fusion protein showed that the C-terminal TRV sequence, especially Thr and Val residues, of PCLN-1 interacts with ZO-1. Next, PCLN-1 was stably expressed in Madin-Darby canine kidney cells using a FLAG tagging vector. ZO-1 was co-immunoprecipitated with the wild-type PCLN-1 and the alanine substitution (TAV) mutant. However, mutants of the deletion (Delta TRV) and the alanine substitution (ARV and TRA) inhibited the association of PCLN-1 with ZO-1. Confocal immunofluorescence demonstrated that the wild-type PCLN-1 and the TAV mutant localized in the tight junction along with ZO-1, but the Delta TRV, ARV, and TRA mutants were widely distributed in the lateral membrane including the tight junction area. Interestingly, monolayers of cells expressing the wild-type PCLN-1 and the TAV mutant showed higher activities of (45)Ca(2+) transport from apical to basal compartments, compared with those expressing the Delta TRV, ARV, and TRA mutants and the mock cells. (45)Ca(2+) transport was inhibited by increased magnesium concentration suggesting that magnesium and calcium were competitively transported by PCLN-1. It was noted that a positive electrical potential gradient enhanced (45)Ca(2+) transport from apical to basal compartments without affecting the opposite direction of transport. Thus, PCLN-1 localizes to the tight junction followed by association with ZO-1, and the PCLN-1.ZO-1 complex may play an essential role in the reabsorption of divalent cations in renal epithelial cells.

Structure-function studies of the C3a-receptor: C-terminal serine and threonine residues which influence receptor internalization and signaling.

The anaphylatoxic peptide C3a is a pro-inflammatory mediator generated during complement activation, whose specific G protein coupled receptor is expressed on granulocytes, monocytes, mast cells, activated lymphocytes, and in the nervous tissue. We have generated RBL-2H3 cell clones stably expressing mutants of the human C3a-receptor (C3aR) with combined alanine (Ala) substitutions of ten C-terminal serine (Ser) or threonine (Thr) residues, which may represent putative phosphorylation sites to characterize their role in ligand-induced C3aR internalization and signaling. Ser475/479 and Thr480/481 as well as Ser449 seemed not to be involved in ligand-induced receptor internalization. Either directly or by a conformational change they even "inhibit" C3aR internalization. In contrast, mutants with Ala substitutions at Ser465/470 and Thr463/466 were poorly internalized, and Thr463 seemed to be the most important C-terminal Thr or Ser residue directly effecting receptor internalization. However, it is likely that other C3aR regions additionally participate in this negative feed-back mechanism since even mutants with multiple Ala substitutions still internalized to a limited degree. Interestingly, in a mutant with a single exchange of Ser449 to Ala, the signal transduction assessed by a Ca(2+) assay and [(35)S]GTP gamma S-binding on HEK cells transiently co-transfected with G-alpha 16 or G-alpha O, respectively, was severely impaired, indicating that this residue of C3aR is involved in G protein coupling.

Can the peptide chain of a pyoverdin be bound by an ester bond to the chromophore?--The old problem of pseudobactin 7SR1.

The structure which had been proposed for the pyoverdin named pseudobactin 7SR1 (Yang and Leong, 1984) differed from those of all other pyoverdins investigated so far: its peptide chain was supposedly linked to the chromophore not by an amide bond originating from its N-terminal amino acid, but rather by an ester bond involving one of the three Ser. It will be shown that the peptide chain of pseudobactin 7SR1 is actually bound to the chromophore amidically by its N-terminal Ser and that it comprises a cyclodepsipeptidic substructure with an ester bond between the C-terminal Thr and the OH-group of the second Ser in the chain.

Phosphorylation of protein phosphatase-1 isoforms by cdc2-cyclin B in vitro.

Protein Phosphatase-1 is phosphorylated in vitro by cdc2-cyclin B (Villa-Moruzzi, FEBS Lett 304: 211-215, 1992). In the present study we show that all the three Phosphatase-1 isoforms, alpha, gamma 1, delta, are phosphorylated by cdc2-cyclin B. Phosphorylation is specific for this kinase and involves a C-terminal Thr. This site is most likely Thr 320 in alpha (shown by others to be phosphorylated also by cdc2-cyclin A). Such Thr is conserved in gamma 1, delta and in the testis-specific gamma 2, and is the only Thr that fits the cdc2-consensus sequence in the C-terminal region. Phosphorylation of Phosphatase-1 purified from skeletal muscle, which is a mixture of the alpha, gamma 1 and delta isoforms, is up to 0.4 mol/mol and induces 30-35% enzyme inactivation. Following tryptic proteolysis each isoform yields a distinct phosphopeptide map. This is in agreement with the different sequences of the isoforms in the C-terminal regions and may be useful to distinguish the isoforms in extracts from metabolically-labelled cells. Our results suggest that all the Phosphatase-1 isoforms may be potentially regulated at M-phase.

Pseudo-prolines in peptide synthesis: Direct insertion of serine and threonine derived oxazolidines in dipeptides

Dipeptides containing serine and threonine derived oxazolidines are prepared by reacting side chain unprotected dipeptides containing C-terminal Thr or Ser with dimethoxypropane. The resulting building blocks can be coupled in Fmoc-based solid phase peptide synthesis enhancing peptide solvation by its secondary structure disrupting potential. The present procedure may be considered as a first step towards a reversible modification of structural and functional properties of bioactive peptides and proteins.