Sequence analysis of MAP2 function in living cells

Microtubule-associated protein 2 (MAP2) is an abundant neuron-specific protein that binds to microtubules through a domain near its carboxyl terminus that contains either three or four similar repeats of a 31 amino acid motif. When expressed in non-neuronal cells by transfection MAP2 stabilises microtubules and induces their rearrangement into long bundles that are capable of supporting process outgrowth. To investigate which elements in the MAP2 sequence are involved in these functions we have constructed a series of deletion mutants of the short embryonic form of MAP2, MAP2c, and transfected them into non-neuronal cells. This showed that the strength of binding to microtubules increased with the number of repeats present in the construct. However, the repeat domain itself was insufficient for microtubule binding, which required in addition contiguous sequences either amino-terminal or carboxyl-terminal to the repeats themselves. Particularly on the amino-terminal side of the repeats, where there is a proline-rich domain, step-wise increases in the length of neighbouring sequence produced a gradual increase in microtubule binding. The apparent strength of binding to microtubules produced by mutant MAP2 forms was further correlated with the degree of bundling they induced as well as with the ability of the resulting microtubules to support process outgrowth. These results indicate that the interaction of MAP2 with microtubules is mediated by the combined action of several weak binding sites, including each of the repeat motifs and elements in the sequences on either side of them, whose additive effect produces the strong binding of the native MAP2 molecule. The results further indicate that both the bundling and stiffening of microtubules by MAP2 are correlated with the strength of its binding to them and suggest that these properties are a direct result of microtubule stabilisation.

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Demonstration and Characterization of a Specific Interaction between Gonococcal Transferrin Binding Protein A and TonB

Journal of Bacteriology ◽

10.1128/jb.184.22.6138-6145.2002 ◽

2002 ◽

Vol 184 (22) ◽

pp. 6138-6145 ◽

Cited By ~ 24

Author(s):

Christopher D. Kenney ◽

Cynthia Nau Cornelissen

Keyword(s):

Outer Membrane ◽

Iron Acquisition ◽

Specific Interaction ◽

Protein A ◽

Outer Membrane Receptor ◽

Amino Terminal ◽

Rich Domain ◽

Additional Protein ◽

Combined Action ◽

Iron Binding Proteins

ABSTRACT Iron scavenging by Neisseria gonorrhoeae is accomplished by the expression of receptors that are specific for host iron-binding proteins, such as transferrin and lactoferrin. Efficient transferrin-iron acquisition is dependent on the combined action of two proteins, designated TbpA and TbpB. TbpA is a TonB-dependent outer membrane receptor, whereas TbpB is lipid modified and serves to increase the efficiency of transferrin-iron uptake. Both proteins, together or separately, can be isolated from the gonococcal outer membrane by using affinity chromatography techniques. In the present study, we identified an additional protein in transferrin-affinity preparations, which had an apparent molecular mass of 45 kDa. The ability to copurify this protein by transferrin affinity was dependent upon the presence of TbpA and not TbpB. The amino-terminal sequence of the 45-kDa protein was identical to the amino terminus of gonococcal TonB, indicating that TbpA stably interacted with TonB, without the addition of chemical cross-linkers. Using immunoprecipitation, we could recover TbpA-TonB complexes without the addition of transferrin, suggesting that ligand binding was not a necessary prerequisite for TonB interaction. In contrast, a characterized TonB box mutant of TbpA did not facilitate interaction between these two proteins such that complexes could be isolated. We generated an in-frame deletion of gonococcal TonB, which removed 35 amino acids, including a Neisseria-specific, glycine-rich domain. This mutant protein, like the parental TonB, energized TbpA to enable growth on transferrin. Consistent with the functionality of this deletion derivative, TbpA-TonB complexes could be recovered from this strain. The results of the present study thus begin to define the requirements for a functional interaction between gonococcal TbpA and TonB.

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Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645194 ◽

1990 ◽

Vol 63 (02) ◽

pp. 193-203 ◽

Cited By ~ 9

Author(s):

John R Shainoff ◽

Deborah J Stearns ◽

Patricia M DiBello ◽

Youko Hishikawa-Itoh

Keyword(s):

Peritoneal Macrophages ◽

Affinity Binding ◽

Macrophage Cell ◽

High Affinity Binding ◽

Amino Terminal ◽

High Affinity ◽

Terminal Domain ◽

Weak Binding ◽

Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

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The E1^E4 Protein of Human Papillomavirus Interacts with the Serine-Arginine-Specific Protein Kinase SRPK1

Journal of Virology ◽

10.1128/jvi.02609-06 ◽

2007 ◽

Vol 81 (11) ◽

pp. 5437-5448 ◽

Cited By ~ 24

Author(s):

Ian Bell ◽

Ashley Martin ◽

Sally Roberts

Keyword(s):

Human Papillomavirus ◽

Protein Kinase ◽

Specific Protein ◽

Replication Cycle ◽

Differentiated Cells ◽

Rich Domain ◽

Productive Phase ◽

High Level ◽

Specific Protein Kinase

ABSTRACT Human papillomavirus (HPV) infections of the squamous epithelium are associated with high-level expression of the E1^E4 protein during the productive phase of infection. However, the precise mechanisms of how E1^E4 contributes to the replication cycle of the virus are poorly understood. Here, we show that the serine-arginine (SR)-specific protein kinase SRPK1 is a novel binding partner of HPV type 1 (HPV1) E1^E4. We map critical residues within an arginine-rich domain of HPV1 E1^E4, and in a region known to facilitate E1^E4 oligomerization, that are requisite for SRPK1 binding. In vitro kinase assays show that SRPK1 binding is associated with phosphorylation of an HPV1 E1^E4 polypeptide and modulates autophosphorylation of the kinase. We show that SRPK1 is sequestered into E4 inclusion bodies in terminally differentiated cells within HPV1 warts and that colocalization between E1^E4 and SRPK1 is not dependent on additional HPV1 factors. Moreover, we also identify SRPK1 binding of E1^E4 proteins of HPV16 and HPV18. Our findings indicate that SRPK1 binding is a conserved function of E1^E4 proteins of diverse virus types. SRPK1 influences important biochemical processes within the cell, including nuclear organization and RNA metabolism. While phosphorylation of HPV1 E4 by SRPK1 may directly influence HPV1 E4 function during the infectious cycle, the modulation and sequestration of SRPK1 by E1^E4 may affect the ability of SRPK1 to phosphorylate its cellular targets, thereby facilitating the productive phase of the HPV replication cycle.

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Influence of Ser/Pro-rich domain and kinase domain of double cortin-like protein kinase on microtubule-binding activity

The Journal of Biochemistry ◽

10.1093/jb/mvr013 ◽

2011 ◽

Vol 149 (5) ◽

pp. 619-627 ◽

Cited By ~ 5

Author(s):

T. Nagamine ◽

S. Shimomura ◽

N. Sueyoshi ◽

I. Kameshita

Keyword(s):

Protein Kinase ◽

Kinase Domain ◽

Binding Activity ◽

Microtubule Binding ◽

Rich Domain

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Mutational activation of c-raf-1 and definition of the minimal transforming sequence.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2503 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2503-2512 ◽

Cited By ~ 144

Author(s):

G Heidecker ◽

M Huleihel ◽

J L Cleveland ◽

W Kolch ◽

T W Beck ◽

...

Keyword(s):

Structural Features ◽

Kinase Domain ◽

Full Length ◽

Wild Type ◽

Amino Terminal ◽

Raf Kinase ◽

Rich Domain ◽

Binding Residue ◽

Transforming Activity ◽

Mutational Activation

A series of wild-type and mutant raf genes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine- and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and CR2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.

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A Yeast-Based Functional Assay to Study Plant N-Degron – N-Recognin Interactions

Frontiers in Plant Science ◽

10.3389/fpls.2021.806129 ◽

2022 ◽

Vol 12 ◽

Author(s):

Aida Kozlic ◽

Nikola Winter ◽

Theresia Telser ◽

Jakob Reimann ◽

Katrin Rose ◽

...

Keyword(s):

Ubiquitin Proteasome System ◽

Functional Assay ◽

The Novel ◽

In Planta ◽

Amino Terminal ◽

Weak Binding ◽

Ubiquitin Conjugating Enzyme ◽

Ubiquitin Proteasome ◽

The Stability

The N-degron pathway is a branch of the ubiquitin-proteasome system where amino-terminal residues serve as degradation signals. In a synthetic biology approach, we expressed ubiquitin ligase PRT6 and ubiquitin conjugating enzyme 2 (AtUBC2) from Arabidopsis thaliana in a Saccharomyces cerevisiae strain with mutation in its endogenous N-degron pathway. The two enzymes re-constitute part of the plant N-degron pathway and were probed by monitoring the stability of co-expressed GFP-linked plant proteins starting with Arginine N-degrons. The novel assay allows for straightforward analysis, whereas in vitro interaction assays often do not allow detection of the weak binding of N-degron recognizing ubiquitin ligases to their substrates, and in planta testing is usually complex and time-consuming.

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Crystal Structures of the Armadillo Repeat Domain of Adenomatous Polyposis Coli and Its Complex with the Tyrosine-Rich Domain of Sam68

Structure ◽

10.1016/j.str.2011.11.010 ◽

2011 ◽

Vol 19 (12) ◽

pp. 1896

Author(s):

Ella Czarina Morishita ◽

Kazutaka Murayama ◽

Miyuki Kato-Murayama ◽

Yoshiko Ishizuka-Katsura ◽

Yuri Tomabechi ◽

...

Keyword(s):

Crystal Structures ◽

Adenomatous Polyposis Coli ◽

Adenomatous Polyposis ◽

Polyposis Coli ◽

Rich Domain ◽

Repeat Domain ◽

Armadillo Repeat

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Residual Structure in the Repeat Domain of Tau: Echoes of Microtubule Binding and Paired Helical Filament Formation†

Biochemistry ◽

10.1021/bi048953n ◽

2005 ◽

Vol 44 (3) ◽

pp. 1026-1036 ◽

Cited By ~ 68

Author(s):

David Eliezer ◽

Patrick Barré ◽

Muris Kobaslija ◽

Dylan Chan ◽

Xiaohua Li ◽

...

Keyword(s):

Paired Helical Filament ◽

Filament Formation ◽

Microtubule Binding ◽

Residual Structure ◽

Repeat Domain

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Ama1p-activated anaphase-promoting complex regulates the destruction of Cdc20p during meiosis II

Molecular Biology of the Cell ◽

10.1091/mbc.e10-04-0360 ◽

2011 ◽

Vol 22 (3) ◽

pp. 315-326 ◽

Cited By ~ 13

Author(s):

Grace S. Tan ◽

Jennifer Magurno ◽

Katrina F. Cooper

Keyword(s):

Saccharomyces Cerevisiae ◽

Fusion Protein ◽

Spore Wall ◽

Terminal Region ◽

Anaphase Promoting Complex ◽

Wall Defect ◽

Present Evidence ◽

Domain Swap ◽

Amino Terminal ◽

Repeat Domain

The execution of meiotic divisions in Saccharomyces cerevisiae is regulated by anaphase-promoting complex/cyclosome (APC/C)–mediated protein degradation. During meiosis, the APC/C is activated by association with Cdc20p or the meiosis-specific activator Ama1p. We present evidence that, as cells exit from meiosis II, APC/CAma1 mediates Cdc20p destruction. APC/CAma1 recognizes two degrons on Cdc20p, the destruction box and destruction degron, with either domain being sufficient to mediate Cdc20p destruction. Cdc20p does not need to associate with the APC/C to bind Ama1p or be destroyed. Coimmunoprecipitation analyses showed that the diverged amino-terminal region of Ama1p recognizes both Cdc20p and Clb1p, a previously identified substrate of APC/CAma1. Domain swap experiments revealed that the C-terminal WD region of Cdh1p, when fused to the N-terminal region of Ama1p, could direct most of Ama1p functions, although at a reduced level. In addition, this fusion protein cannot complement the spore wall defect in ama1Δ strains, indicating that substrate specificity is also derived from the WD repeat domain. These findings provide a mechanism to temporally down-regulate APC/CCdc20 activity as the cells complete meiosis II and form spores.

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