Tubulin-Dependent Transport of Connexin-36 Potentiates the Size and Strength of Electrical Synapses

Connexin-36 (Cx36) electrical synapses strengthen transmission in a calcium/calmodulin (CaM)/calmodulin-dependent kinase II (CaMKII)-dependent manner similar to a mechanism whereby the N-methyl-D-aspartate (NMDA) receptor subunit NR2B facilitates chemical transmission. Since NR2B–microtubule interactions recruit receptors to the cell membrane during plasticity, we hypothesized an analogous modality for Cx36. We determined that Cx36 binding to tubulin at the carboxy-terminal domain was distinct from Cx43 and NR2B by binding a motif overlapping with the CaM and CaMKII binding motifs. Dual patch-clamp recordings demonstrated that pharmacological interference of the cytoskeleton and deleting the binding motif at the Cx36 carboxyl-terminal (CT) reversibly abolished Cx36 plasticity. Mechanistic details of trafficking to the gap-junction plaque (GJP) were probed pharmacologically and through mutational analysis, all of which affected GJP size and formation between cell pairs. Lys279, Ile280, and Lys281 positions were particularly critical. This study demonstrates that tubulin-dependent transport of Cx36 potentiates synaptic strength by delivering channels to GJPs, reinforcing the role of protein transport at chemical and electrical synapses to fine-tune communication between neurons.

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Tubulin folding is altered by mutations in a putative GTP binding motif

Journal of Cell Science ◽

10.1242/jcs.109.6.1471 ◽

1996 ◽

Vol 109 (6) ◽

pp. 1471-1478 ◽

Cited By ~ 1

Author(s):

J.C. Zabala ◽

A. Fontalba ◽

J. Avila

Keyword(s):

Intramolecular Interaction ◽

Terminal Region ◽

Binding Motif ◽

Beta Tubulin ◽

Alpha Tubulin ◽

Proposed Model ◽

Carboxy Terminal Region ◽

Carboxy Terminal ◽

Hydrolysis Of

Tubulins contain a glycine-rich loop, that has been implicated in microtubule dynamics by means of an intramolecular interaction with the carboxy-terminal region. As a further extension of the analysis of the role of the carboxy-terminal region in tubulin folding we have mutated the glycine-rich loop of tubulin subunits. An alpha-tubulin point mutant with a T150-->G substitution (the corresponding residue present in beta-tubulin) was able to incorporate into dimers and microtubules. On the other hand, four beta-tubulin point mutants, including the G148-->T substitution, did not incorporate into dimers, did not release monomers, but were able to form C900 and C300 complexes (intermediates in the process of tubulin folding). Three other mutants within this region (which approximately encompasses residues 137–152) were incapable of forming dimers and C300 complexes but gave rise to the formation of C900 complexes. These results suggest that tubulin goes through two sequential folding states during the folding process, first in association with TCP1-complexes (C900) prior to the transfer to C300 complexes. It is this second step that implies binding/hydrolysis of GTP, reinforcing our previous proposed model for tubulin folding and assembly.

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Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin

Biochemical Journal ◽

10.1042/bj3580359 ◽

2001 ◽

Vol 358 (2) ◽

pp. 359-367 ◽

Cited By ~ 70

Author(s):

Henning B. BOLDT ◽

Michael T. OVERGAARD ◽

Lisbeth S. LAURSEN ◽

Kathrin WEYER ◽

Lars SOTTRUP-JENSEN ◽

...

Keyword(s):

Plasma Protein ◽

Active Site ◽

Mammalian Cells ◽

Mutational Analysis ◽

Protein A ◽

Residual Activity ◽

Zinc Binding ◽

Binding Motif ◽

Dependent Manner ◽

Zinc Binding Motif

The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1–6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482–His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.

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The SARS Coronavirus E Protein Interacts with PALS1 and Alters Tight Junction Formation and Epithelial Morphogenesis

Molecular Biology of the Cell ◽

10.1091/mbc.e10-04-0338 ◽

2010 ◽

Vol 21 (22) ◽

pp. 3838-3852 ◽

Cited By ~ 87

Author(s):

Kim-Tat Teoh ◽

Yu-Lam Siu ◽

Wing-Lim Chan ◽

Marc A. Schlüter ◽

Chia-Jen Liu ◽

...

Keyword(s):

Tight Junction ◽

Tight Junctions ◽

Mammalian Cells ◽

Pdz Domain ◽

Ectopic Expression ◽

Binding Motif ◽

Dependent Manner ◽

Tight Junction Formation ◽

Golgi Region ◽

Carboxy Terminal

Intercellular tight junctions define epithelial apicobasal polarity and form a physical fence which protects underlying tissues from pathogen invasions. PALS1, a tight junction-associated protein, is a member of the CRUMBS3-PALS1-PATJ polarity complex, which is crucial for the establishment and maintenance of epithelial polarity in mammals. Here we report that the carboxy-terminal domain of the SARS-CoV E small envelope protein (E) binds to human PALS1. Using coimmunoprecipitation and pull-down assays, we show that E interacts with PALS1 in mammalian cells and further demonstrate that the last four carboxy-terminal amino acids of E form a novel PDZ-binding motif that binds to PALS1 PDZ domain. PALS1 redistributes to the ERGIC/Golgi region, where E accumulates, in SARS-CoV–infected Vero E6 cells. Ectopic expression of E in MDCKII epithelial cells significantly alters cyst morphogenesis and, furthermore, delays formation of tight junctions, affects polarity, and modifies the subcellular distribution of PALS1, in a PDZ-binding motif-dependent manner. We speculate that hijacking of PALS1 by SARS-CoV E plays a determinant role in the disruption of the lung epithelium in SARS patients.

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Stereotyped Patterns of Somatic Hypermutation (SHM) in Subsets of Patients with Chronic Lymphocytic Leukemia (CLL): Implications for the Role of Antigen Selection in Leukemogenesis.

Blood ◽

10.1182/blood.v110.11.744.744 ◽

2007 ◽

Vol 110 (11) ◽

pp. 744-744

Author(s):

Fiona Murray ◽

Nikos Darzentas ◽

Anastasia Hadzidimitriou ◽

Gerard Tobin ◽

Myriam Boudjogra ◽

...

Keyword(s):

Somatic Hypermutation ◽

Protein A ◽

Specific Antigen ◽

Lymphocytic Leukemia ◽

Binding Motif ◽

Staphylococcal Protein A ◽

Binding Motifs ◽

Mutational Status ◽

Identity Groups

Abstract We examined SHM features in 1967 IGH rearrangements from 1939 patients with CLL. The sequences were divided into four “identity groups”; “truly unmutated” (100% identity to germline; 677 sequences), “minimally mutated” (99–99.9% identity; 133 sequences), “borderline mutated” (98–98.9% identity; 93 sequences) and “mutated” (<98% identity; 1064 sequences). At the cohort level, SHM patterns were typical of a canonical SHM process. However, important differences emerged on analysis of subgroups of sequences. In particular, the IGHV repertoire of the four “identity groups” differed considerably, with the IGHV1-69 and IGHV1-2 genes predominating among “truly unmutated” and “minimally mutated” sequences, respectively. In contrast, other genes were mostly used in “mutated” rearrangements (eg, IGHV4-34/3-23/3-7). Of note, IGHV3-21 and IGHV3-48 had the highest proportion of “borderline mutated” rearrangements. In selected groups of sequences, a remarkable preservation of the germline configuration was observed in superantigenic binding motifs, prompting speculation that subsets of CLL cells could also receive stimulation signals by superantigenic-like interactions. In detail, the vast majority of IGHV4-34 sequences retained germline conformation at the four FR1 positions of the IGHV4-34-specific I/i binding motif. Similarly, IGHV3-21 sequences displayed remarkably few alterations of the IGHV3-specific staphylococcal protein A binding motif. Following established criteria, we identified 530/1967 sequences (27%) as belonging to 113 different subsets with stereotyped HCDR3. The distribution of sequences among subsets differed significantly according to mutational status: 43% of “truly unmutated” sequences belonged to a subset, compared to only 16% of the “mutated” group (p<0.001). Of note, among certain IGHV genes (ie, IGHV1-2/3-21/4-34/4-4), shared “stereotyped” amino acid (AA) changes (i.e. the same AA replacement at the same position) occurred across the entire IGHV sequence significantly more frequently in cases with stereotyped vs. heterogeneous HCDR3s and therefore, could be considered as “subset-biased”. Stereotyped AA changes were also observed in subsets of minimally mutated cases, indicating that even a low level of mutations may be functionally relevant. Comparison to public-database non-CLL sequences revealed that certain stereotyped AA changes were over-represented in CLL and thus could also be considered as “CLL-biased”. The very precise targeting and distinctive features of SHM in subgroups of CLL patients provide further evidence for the important role of selection by specific antigen(s) in CLL leukemogenesis.

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Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

Journal of Bacteriology ◽

10.1128/jb.01135-09 ◽

2010 ◽

Vol 192 (8) ◽

pp. 2220-2227 ◽

Cited By ~ 41

Author(s):

Bavesh D. Kana ◽

Garth L. Abrahams ◽

Nackmoon Sung ◽

Digby F. Warner ◽

Bhavna G. Gordhan ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Alkylating Agents ◽

Spontaneous Mutation ◽

Dna Polymerases ◽

Binding Motif ◽

Dependent Manner ◽

Mouse Macrophages ◽

Y Family

ABSTRACT The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the β-clamp, consistent with its canonical C-terminal β-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N 2-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.

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Significance of 14-3-3 Self-Dimerization for Phosphorylation-dependent Target Binding

Molecular Biology of the Cell ◽

10.1091/mbc.e02-12-0821 ◽

2003 ◽

Vol 14 (11) ◽

pp. 4721-4733 ◽

Cited By ~ 78

Author(s):

Ying H. Shen ◽

Jakub Godlewski ◽

Agnieszka Bronisz ◽

Jun Zhu ◽

Michael J. Comb ◽

...

Keyword(s):

Significant Role ◽

Specific Antibody ◽

Binding Motif ◽

Dependent Manner ◽

Phosphorylated Proteins ◽

Associated Proteins ◽

Target Binding ◽

Eukaryotic Organisms

14-3-3 proteins via binding serine/threonine-phosphorylated proteins regulate diverse intracellular processes in all eukaryotic organisms. Here, we examine the role of 14-3-3 self-dimerization in target binding, and in the susceptibility of 14-3-3 to undergo phosphorylation. Using a phospho-specific antibody developed against a degenerated mode-1 14-3-3 binding motif (RSxpSxP), we demonstrate that most of the 14-3-3-associated proteins in COS-7 cells are phosphorylated on sites that react with this antibody. The binding of these phosphoproteins depends on 14-3-3 dimerization, inasmuch as proteins associated in vivo with a monomeric 14-3-3 form are not recognized by the phospho-specific antibody. The role of 14-3-3 dimerization in the phosphorylation-dependent target binding is further exemplified with two well-defined 14-3-3 targets, Raf and DAF-16. Raf and DAF-16 can bind both monomeric and dimeric 14-3-3; however, whereas phosphorylation of specific Raf and DAF-16 sites is required for binding to dimeric 14-3-3, binding to monomeric 14-3-3 forms is entirely independent of Raf and DAF-16 phosphorylation. We also find that dimerization diminishes 14-3-3 susceptibility to phosphorylation. These findings establish a significant role of 14-3-3 dimerization in its ability to bind targets in a phosphorylation-dependent manner and point to a mechanism in which 14-3-3 phosphorylation and dimerization counterregulate each other.

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Mutational analysis of ribosomal S6 kinase 2 shows differential regulation of its kinase activity from that of ribosomal S6 kinase 1

Biochemical Journal ◽

10.1042/bj20021794 ◽

2003 ◽

Vol 373 (2) ◽

pp. 583-591 ◽

Cited By ~ 16

Author(s):

Sopheap PHIN ◽

Deborah KUPFERWASSER ◽

Joseph LAM ◽

Kay K. LEE-FRUMAN

Keyword(s):

Protein Kinase ◽

Cellular Localization ◽

Mutational Analysis ◽

Amino Acid Sequences ◽

Mitogen Activated Protein Kinase ◽

Dependent Manner ◽

S6 Kinase ◽

Ribosomal S6 Kinase 2 ◽

Ribosomal S6 Kinase

Ribosomal S6 kinase 2 (S6K2) is a serine/threonine kinase identified as a homologue of p70 ribosomal S6 kinase 1 (S6K1). S6K1 and S6K2 show different cellular localization as well as divergent amino acid sequences in non-catalytic domains, suggesting that their cellular functions and/or regulation may not be identical. Many of the serine/threonine residues that become phosphorylated and contribute to S6K1 activation are conserved in S6K2. In this study we carry out mutational analyses of these serine/threonine residues on S6K2 in order to elucidate the mechanism of S6K2 regulation. We find that Thr-228 and Ser-370 are crucial for S6K2 activity, and the three proline-directed serines in the autoinhibitory domain, Ser-410, Ser-417 and Ser-423, play a role in S6K2 activity regulation in a mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK)-dependent manner. However, unlike S6K1, changing Thr-388 to glutamic acid in S6K2 renders the kinase fully active. This activity was resistant to the effects of rapamycin or wortmannin, indicating that mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) regulate S6K2 activity via Thr-388. MEK-dependent phosphorylation of the autoinhibitory serines in S6K2 occurs prior to Thr-388 activation. Combining T388E and T228A mutations inhibited S6K2 activation, and a kinase-inactive phosphoinositide-dependent protein kinase (PDK1) diminished T388E activity, suggesting that the role of Thr-388 is to allow further phosphorylation of Thr-228 by PDK1. Thr-388 fails to become phosphorylated in Ser-370 mutants, suggesting that the role of Ser-370 phosphorylation may be to allow Thr-388 phosphorylation. Finally, using the rapamycin-resistant T388E mutant, we provide evidence that S6K2 can phosphorylate S6 in vivo.

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Adenosine 5'-triphosphate--a new regulator of annexin function. A hypothesis.

Acta Biochimica Polonica ◽

10.18388/abp.1998_4267 ◽

1998 ◽

Vol 45 (3) ◽

pp. 735-744

Author(s):

J Bandorowicz-Pikuła ◽

S Pikuła

Keyword(s):

Binding Motif ◽

Dependent Manner ◽

In Vitro Activity ◽

Consensus Sequences ◽

Calcium Dependent ◽

Gtp Binding ◽

Integral Proteins

The paradigm of annexins as phospholipid-binding proteins interacting with membranes in a calcium-dependent manner has been recently questioned in light of observations that some annexin isoforms may behave like membrane integral proteins or remain associated with their target membranes at low, resting, concentrations of Ca2+ in the cytoplasm. In addition, an evidence has been presented that some annexins (annexins I, VI and VII) bind in vitro ATP and GTP, and upon binding the nucleotide the in vitro activity of these proteins is modified. However, annexins do not contain Walker A and B consensus sequences for ATP/GTP binding. This review presents the hypothesis that a new ATP-binding motif exists within the annexin molecules and that ATP may play a role of functional ligand for annexins also in vivo.

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Mutational Analysis of the Role of HPr inListeria monocytogenes

Applied and Environmental Microbiology ◽

10.1128/aem.65.5.2112-2115.1999 ◽

1999 ◽

Vol 65 (5) ◽

pp. 2112-2115 ◽

Cited By ~ 8

Author(s):

Douglas P. Christensen ◽

Andrew K. Benson ◽

Robert W. Hutkins

Keyword(s):

Listeria Monocytogenes ◽

Phosphatase Activity ◽

Mutational Analysis ◽

Active Form ◽

Regulatory Role ◽

Dependent Manner ◽

Phosphotransferase System ◽

Gram Positive ◽

A Charge

ABSTRACT The regulatory role of HPr, a protein of the phosphotransferase system (PTS), was investigated in Listeria monocytogenes. By constructing mutations in the conserved histidine 15 and serine 46 residues of HPr, we were able to examine how HPr regulates PTS activity. The results indicated that histidine 15 was phosphorylated in a phosphoenolpyruvate (PEP)-dependent manner and was essential for PTS activity. Serine 46 was phosphorylated in an ATP-dependent manner by a membrane-associated kinase. ATP-dependent phosphorylation of serine 46 was significantly enhanced in the presence of fructose 1,6-diphosphate and resulted in a reduction of PTS activity. The presence of a charge at position 15 did not inhibit ATP-dependent phosphorylation of serine 46, a finding unique to gram-positive PEP-dependent PTSs studied to this point. Finally, HPr phosphorylated at serine 46 does not appear to possess self-phosphatase activity, suggesting a specific phosphatase protein may be essential for the recycling of HPr to its active form.

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Adhesion-dependent tyrosine phosphorylation of (beta)-dystroglycan regulates its interaction with utrophin

Journal of Cell Science ◽

10.1242/jcs.113.10.1717 ◽

2000 ◽

Vol 113 (10) ◽

pp. 1717-1726 ◽

Cited By ~ 2

Author(s):

M. James ◽

A. Nuttall ◽

J.L. Ilsley ◽

K. Ottersbach ◽

J.M. Tinsley ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Binding Motif ◽

Specific Interactions ◽

Protein Fusion ◽

Physiological Regulation ◽

Cell Extracts ◽

Maltose Binding Protein Fusion ◽

Carboxy Terminal ◽

Dependent Processes

Many cell adhesion-dependent processes are regulated by tyrosine phosphorylation. In order to investigate the role of tyrosine phosphorylation of the utrophin-dystroglycan complex we treated suspended or adherent cultures of HeLa cells with peroxyvanadate and immunoprecipitated (beta)-dystroglycan and utrophin from cell extracts. Western blotting of (β)-dystroglycan and utrophin revealed adhesion- and peroxyvanadate-dependent mobility shifts which were recognised by anti-phospho-tyrosine antibodies. Using maltose binding protein fusion constructs to the carboxy-terminal domains of utrophin we were able to demonstrate specific interactions between the WW, EF and ZZ domains of utrophin and (beta)-dystroglycan by co-immunoprecipitation with endogenous (beta)-dystroglycan. In extracts from cells treated with peroxyvanadate, where endogenous (beta)-dystroglycan was tyrosine phosphorylated, (beta)-dystroglycan was no longer co-immunoprecipitated with utrophin fusion constructs. Peptide ‘SPOTs’ assays confirmed that tyrosine phosphorylation of (beta)-dystroglycan regulated the binding of utrophin. The phosphorylated tyrosine was identified as Y(892) in the (beta)-dystroglycan WW domain binding motif PPxY thus demonstrating the physiological regulation of the (beta)-dystroglycan/utrophin interaction by adhesion-dependent tyrosine phosphorylation.

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