scholarly journals Functional interactions between the proline-rich and repeat regions of tau enhance microtubule binding and assembly.

1997 ◽  
Vol 8 (2) ◽  
pp. 353-365 ◽  
Author(s):  
B L Goode ◽  
P E Denis ◽  
D Panda ◽  
M J Radeke ◽  
H P Miller ◽  
...  
Keyword(s):  
Amino Acid ◽  
Structural Complexity ◽  
Intramolecular Interactions ◽  
Site Directed Mutagenesis ◽  
Microtubule Assembly ◽  
Repeat Region ◽  
Rich Region ◽  
Microtubule Binding ◽  
Amino Terminal ◽  
Proline Rich Region

Tau is a neuronal microtubule-associated protein that promotes microtubule assembly, stability, and bundling in axons. Two distinct regions of tau are important for the tau-microtubule interaction, a relatively well-characterized "repeat region" in the carboxyl terminus (containing either three or four imperfect 18-amino acid repeats separated by 13- or 14-amino acid long inter-repeats) and a more centrally located, relatively poorly characterized proline-rich region. By using amino-terminal truncation analyses of tau, we have localized the microtubule binding activity of the proline-rich region to Lys215-Asn246 and identified a small sequence within this region, 215KKVAVVR221, that exerts a strong influence on microtubule binding and assembly in both three- and four-repeat tau isoforms. Site-directed mutagenesis experiments indicate that these capabilities are derived largely from Lys215/Lys216 and Arg221. In marked contrast to synthetic peptides corresponding to the repeat region, peptides corresponding to Lys215-Asn246 and Lys215-Thr222 alone possess little or no ability to promote microtubule assembly, and the peptide Lys215-Thr222 does not effectively suppress in vitro microtubule dynamics. However, combining the proline-rich region sequences (Lys215-Asn246) with their adjacent repeat region sequences within a single peptide (Lys215-Lys272) enhances microtubule assembly by 10-fold, suggesting intramolecular interactions between the proline-rich and repeat regions. Structural complexity in this region of tau also is suggested by sequential amino-terminal deletions through the proline-rich and repeat regions, which reveal an unusual pattern of loss and gain of function. Thus, these data lead to a model in which efficient microtubule binding and assembly activities by tau require intramolecular interactions between its repeat and proline-rich regions. This model, invoking structural complexity for the microtubule-bound conformation of tau, is fundamentally different from previous models of tau structure and function, which viewed tau as a simple linear array of independently acting tubulin-binding sites.

The projection domain of MAP2b regulates microtubule protrusion and process formation in Sf9 cells

2002 ◽  
Vol 115 (7) ◽  
pp. 1523-1539 ◽  
Author(s):  
Dave Bélanger ◽  
Carole Abi Farah ◽  
Minh Dang Nguyen ◽  
Michel Lauzon ◽  
Sylvie Cornibert ◽  
...  
Keyword(s):  
Cell Surface ◽  
Binding Domain ◽  
Intramolecular Interactions ◽  
Microtubule Assembly ◽  
Sf9 Cells ◽  
Microtubule Binding ◽  
Process Formation ◽  
Microtubule Binding Domain ◽  
Projection Domain

The expression of microtubule-associated protein 2 (MAP2), developmentally regulated by alternative splicing, coincides with neurite outgrowth. MAP2 proteins contain a microtubule-binding domain (C-terminal) that promotes microtubule assembly and a poorly characterized domain, the projection domain(N-terminal), extending at the surface of microtubules. MAP2b differs from MAP2c by an additional sequence of 1372 amino acids in the projection domain. In this study, we examined the role of the projection domain in the protrusion of microtubules from the cell surface and the subsequent process formation in Sf9 cells. In this system, MAP2b has a lower capacity to induce process formation than MAP2c. To investigate the role of the projection domain in this event, we expressed truncated forms of MAP2b and MAP2c that have partial or complete deletion of their projection domain in Sf9 cells. Our results indicate that process formation is induced by the microtubule-binding domain of these MAP2 proteins and is regulated by their projection domain. Furthermore, the microtubule-binding activity of MAP2b and MAP2c truncated forms as well as the structural properties of the microtubule bundles induced by them do not seem to be the only determinants that control the protrusion of microtubules from the cell surface in Sf9 cells. Rather, our data suggest that microtubule protrusion and process formation are regulated by intramolecular interactions between the projection domain and its microtubule-binding domain in MAP2b.

scholarly journals Identification of a novel microtubule binding and assembly domain in the developmentally regulated inter-repeat region of tau

1994 ◽  
Vol 124 (5) ◽  
pp. 769-782 ◽  
Author(s):  
BL Goode ◽  
SC Feinstein
Keyword(s):  
Amino Acid ◽  
Electrostatic Interactions ◽  
Weak Interactions ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Microtubule Organization ◽  
Developmentally Regulated ◽  
Microtubule Binding ◽  
Amino Acid Repeats

Tau is a developmentally regulated microtubule-associated protein that influences microtubule behavior by directly associating with tubulin. The carboxyl terminus of tau contains multiple 18-amino acid repeats that bind microtubules and are separated by 13-14-amino acid inter-repeat (IR) regions previously thought to function as "linkers." Here, we have performed a high resolution deletion analysis of tau and identified the IR region located between repeats 1 and 2 (the R1-R2 IR) as a unique microtubule binding site with more than twice the binding affinity of any individual repeat. Truncation analyses and site-directed mutagenesis reveal that the binding activity of this site is derived primarily from lys265 and lys272, with a lesser contribution from lys271. These results predict strong, discrete electrostatic interactions between the R1-R2 IR and tubulin, in contrast to the distributed array of weak interactions thought to underlie the association between 18-amino acid repeats and microtubules (Butner, K. A., and M. W. Kirschner. J. Cell Biol. 115:717-730). Moreover, competition assays suggest that the R1-R2 IR associates with microtubules at tubulin site(s) distinct from those bound by the repeats. Finally, a synthetic peptide corresponding to just 10 amino acids of the R1-R2 IR is sufficient to promote tubulin polymerization in a sequence-dependent manner. Since the R1-R2 IR is specifically expressed in adult tau, its action may underlie some of the developmental transitions observed in neuronal microtubule organization. We suggest that the R1-R2 IR may establish an adult-specific, high affinity anchor that tethers the otherwise mobile tau molecule to the tubulin lattice, thereby increasing microtubule stability. Moreover, the absence of R1-R2 IR expression during early development may allow for the cytoskeletal plasticity required of immature neurons.

scholarly journals nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain.

1991 ◽  
Vol 11 (11) ◽  
pp. 5735-5745 ◽  
Author(s):  
G F Yuan ◽  
Y H Fu ◽  
G A Marzluf
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Neurospora Crassa ◽  
Transcriptional Activation ◽  
Regulatory Gene ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Dna Binding Domain ◽  
Amino Terminal ◽  
Rich Domain

nit-4, a pathway-specific regulatory gene in the nitrogen circuit of Neurospora crassa, is required for the expression of nit-3 and nit-6, the structural genes which encode nitrate and nitrite reductase, respectively. The complete nucleotide sequence of the nit-4 gene has been determined. The predicted NIT4 protein contains 1,090 amino acids and appears to possess a single Zn(II)2Cys6 binuclear-type zinc finger, which may mediate DNA binding. Site-directed mutagenesis studies demonstrated that cysteine and other conserved amino acid residues in this possible DNA-binding domain are necessary for nit-4 function. A stretch of 27 glutamines, encoded by a CAGCAA repeating sequence, occurs in the C terminus of the NIT4 protein, and a second glutamine-rich domain occurs further upstream. A NIT4 protein deleted for the polyglutamine region was still functional in vivo. However, nit-4 function was abolished when both the polyglutamine region and the glutamine-rich domain were deleted, suggesting that the glutamine-rich domain might function in transcriptional activation. The homologous regulatory gene from Aspergillus nidulans, nirA, encodes a protein whose amino-terminal half has approximately 60% amino acid identity with NIT4 but whose carboxy terminus is completely different. A hybrid nit-4-nirA gene was constructed and found to function in N. crassa.

scholarly journals Polyphosphate initiates tau aggregation through intra- and intermolecular scaffolding

2019 ◽  
Author(s):  
S. P. Wickramasinghe ◽  
J. Lempart ◽  
H. E. Merens ◽  
J. Murphy ◽  
U. Jakob ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Cross Linking ◽  
Conformational Ensemble ◽  
Intensive Study ◽  
Rich Region ◽  
Microtubule Binding ◽  
Long Range Interactions ◽  
Tau Aggregation ◽  
Proline Rich Region

AbstractThe aggregation and deposition of tau is a hallmark of a class of neurodegenerative diseases called tauopathies. Despite intensive study, cellular and molecular factors that trigger tau aggregation are not well understood. Here we provide evidence for two mechanisms relevant to the initiation of tau aggregation in the presence of cytoplasmic polyphosphates (polyP): changes in the conformational ensemble of monomer tau and noncovalent cross-linking of multiple tau monomers. We identified conformational changes throughout full-length tau, most notably diminishment of long-range interactions between the termini coupled with compaction of the microtubule binding and proline rich regions. We found that while the proline rich and microtubule binding regions both contain polyP binding sites, the proline rich region is a requisite for compaction of the microtubule binding region upon binding. Additionally, both the magnitude of the conformational change and the aggregation of tau are dependent on the chain length of the polyP polymer. Longer polyP chains are more effective at intermolecular, noncovalent cross-linking of tau. These observations provide an understanding of the initial steps of tau aggregation through interaction with a physiologically relevant aggregation inducer.

Interaction between the guanylate kinase domain of PSD-95 and the proline-rich region and microtubule binding repeats 2 and 3 of tau

2021 ◽  
Author(s):  
Emmanuel Prikas ◽  
Holly Ahel ◽  
Kristie Stefanoska ◽  
Prita Riana Asih ◽  
Alexander Volkerling ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Protein Complexes ◽  
Kinase Domain ◽  
Rich Region ◽  
Guanylate Kinase ◽  
Synaptic Density ◽  
Microtubule Binding ◽  
Key Factor ◽  
Post Synaptic Density ◽  
Proline Rich Region

The microtubule-associated protein tau is a key factor in neurodegenerative proteinopathies and is predominantly found in the neuronal axon. However, somatodendritic localization of tau occurs for a subset of pathological and physiologic tau. Dendritic tau can localize to post-synapses where it interacts with proteins of the post-synaptic density (PSD) protein PSD-95, a membrane-associated guanylate kinase (MAGUK) scaffold factor for organization of protein complexes within the PSD, to mediate downstream signals. The sub-molecular details of this interaction, however, remain unclear. Here, we use interaction mapping in cultured cells to demonstrate that tau interacts with the guanylate kinase (GUK) domain in the C-terminal region of PSD-95. The PSD-95 GUK domain is required and sufficient for a complex with full-length human tau. Mapping the interaction of the MAGUK core on tau revealed the microtubule binding repeats 2 and 3 and the proline-rich region contribute to this interaction, while the N- and C-terminal regions of tau inhibit interaction. These results reveal intramolecular determinants of the protein complex of tau and PSD-95 and increase our understanding of tau interactions regulating neurotoxic signaling at the molecular level.

Phosphorylation-mimicking glutamate clusters in the proline-rich region are sufficient to simulate the functional deficiencies of hyperphosphorylated tau protein

2001 ◽  
Vol 357 (3) ◽  
pp. 759-767 ◽  
Author(s):  
Jochen EIDENMÜLLER ◽  
Thomas FATH ◽  
Thorsten MAAS ◽  
Madeline POOL ◽  
Estelle SONTAG ◽  
...  
Keyword(s):  
Paired Helical Filaments ◽  
Tau Proteins ◽  
Microtubule Assembly ◽  
Tail Region ◽  
Rich Region ◽  
Hyperphosphorylated Tau Protein ◽  
Functional Consequences ◽  
Phosphatase 2A ◽  
Tau Aggregation ◽  
Proline Rich Region

The microtubule-associated tau proteins represent a family of closely related phosphoproteins that become enriched in the axons during brain development. In Alzheimer's disease (AD), tau aggregates somatodendritically in paired helical filaments in a hyperphosphorylated form. Most of the sites that are phosphorylated to a high extent in paired helical filament tau are clustered in the proline-rich region (P-region; residues 172–251) and the C-terminal tail region (C-region; residues 368–441) that flank tau's microtubule-binding repeats. This might point to a role of a region-specific phosphorylation cluster for the pathogenesis of AD. To determine the functional consequences of such modifications, mutated tau proteins were produced in which a P- or C-region-specific phosphorylation cluster was simulated by replacement of serine/threonine residues with glutamate. We show that a phosphorylation-mimicking glutamate cluster in the P-region is sufficient to block microtubule assembly and to inhibit tau's interaction with the dominant brain phosphatase protein phosphatase 2A isoform ABαC. P-region-specific mutations also decrease tau aggregation into filaments and decrease tau's process-inducing activity in a cellular transfection model. In contrast, a phosphorylation-mimicking glutamate cluster in the C-region is neutral with regard to these activities. A glutamate cluster in both the P- and C-regions induces the formation of SDS-resistant conformational domains in tau and suppresses tau's interaction with the neural membrane cortex. The results indicate that modifications in the proline-rich region are sufficient to induce the functional deficiencies of tau that have been observed in AD. They suggest that phosphorylation of the proline-rich region has a crucial role in mediating tau-related changes during disease.

scholarly journals Contribution of the Alanine-Rich Region of Streptococcus mutans P1 to Antigenicity, Surface Expression, and Interaction with the Proline-Rich Repeat Domain

2004 ◽  
Vol 72 (8) ◽  
pp. 4699-4706 ◽  
Author(s):  
Trevor B. Seifert ◽  
Arnold S. Bleiweis ◽  
L. Jeannine Brady
Keyword(s):  
Streptococcus Mutans ◽  
Surface Protein ◽  
Surface Expression ◽  
Etiologic Agent ◽  
Tooth Surface ◽  
Oral Streptococci ◽  
Repeat Region ◽  
Rich Region ◽  
The Stability ◽  
Proline Rich Region

ABSTRACT Streptococcus mutans is considered to be the major etiologic agent of human dental caries. Attachment of S. mutans to the tooth surface is required for the development of caries and is mediated, in part, by the 185-kDa surface protein variously known as antigen I/II, PAc, and P1. Such proteins are expressed by nearly all species of oral streptococci. Characteristics of P1 include an alanine-rich repeat region and a centrally located proline-rich repeat region. The proline-rich region of P1 has been shown to be important for the translational stability and translocation of P1 through the bacterial membrane. We show here that (i) several anti-P1 monoclonal antibodies require the simultaneous presence of the alanine-rich and proline-rich regions for binding, (ii) the proline-rich region of P1 interacts with the alanine-rich region, (iii) like the proline-rich region, the alanine-rich region is required for the stability and translocation of P1, (iv) both the proline-rich and alanine-rich regions are required for secretion of P1 in Escherichia coli, and (v) in E. coli, P1 is secreted in the absence of SecB.

scholarly journals Direct Binding of CRKL to BCR-ABL Is Not Required for BCR-ABL Transformation

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 297-306 ◽  
Author(s):  
Conor Heaney ◽  
Kathryn Kolibaba ◽  
Arun Bhat ◽  
Tsukasa Oda ◽  
Sayuri Ohno ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Direct Interaction ◽  
Myelogenous Leukemia ◽  
Indirect Interaction ◽  
Yeast Two Hybrid ◽  
Rich Region ◽  
Amino Terminal ◽  
C Terminus ◽  
Two Hybrid ◽  
Proline Rich Region

CRKL has previously been shown to be a major tyrosine phosphorylated protein in neutrophils of patients with BCR-ABL+ chronic myelogenous leukemia and in cell lines expressing BCR-ABL. CRKL and BCR-ABL form a complex as demonstrated by coimmunoprecipitation and are capable of a direct interaction in a yeast two-hybrid assay. We have mapped the site of interaction of CRKL and BCR-ABL to the amino terminal SH3 domain of CRKL with a proline rich region in the C-terminus of ABL. The proline-rich region was mutated and the effect of this deletion on BCR-ABL transforming function was assayed. Our data show that this deletion does not impair the ability of BCR-ABL to render myeloid cells factor independent for growth. In cells expressing the proline deletion mutation of BCR-ABL, CRKL is still tyrosine phosphorylated and forms a complex with BCR-ABL as demonstrated by coimmunoprecipitation. Our data suggest that the interaction between CRKL and the proline deletion mutant of BCR-ABL is an indirect interaction as CRKL does not interact directly with the proline deletion mutant of BCR-ABL in a gel overlay assay or in a yeast two-hybrid assay. Thus, a direct interaction of CRKL and BCR-ABL is not required for CRKL to become tyrosine phosphorylated by BCR-ABL and suggests that CRKL function may still be required for BCR-ABL function through an indirect interaction.

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