scholarly journals MAP7 family proteins regulate kinesin-1 recruitment and activation

2018 ◽  
Author(s):  
Peter Jan Hooikaas ◽  
Maud Martin ◽  
Tobias Mühlethaler ◽  
Gert-Jan Kuijntjes ◽  
Cathelijn A.E. Peeters ◽  
...  
Keyword(s):  
Hela Cells ◽  
Family Members ◽  
Allosteric Effect ◽  
Microtubule Binding ◽  
Lower Affinity ◽  
Linker Region ◽  
Microtubule Binding Domain ◽  
In Vitro Reconstitution ◽  
Dependent Transport

AbstractKinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family members bind to kinesin-1. In HeLa cells, MAP7, MAP7D1 and MAP7D3 act redundantly to enable kinesin-1-dependent transport and microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions. In vitro, purified MAP7 and MAP7D3 increase microtubule landing rate and processivity of kinesin-1 through transient association with the motor. MAP7 proteins promote binding of kinesin-1 to microtubules both directly, through the N-terminal microtubule-binding domain and unstructured linker region, and indirectly, through an allosteric effect exerted by the kinesin-binding C-terminal domain. Compared to MAP7, MAP7D3 has a higher affinity for kinesin-1 and a lower affinity for microtubules and, unlike MAP7, can be co-transported with the motor. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules.SummaryA combination of experiments in cells and in vitro reconstitution assays demonstrated that mammalian MAP7 family proteins act redundantly to activate kinesin-1 and promote its microtubule binding and processivity by transiently associating with the stalk region of the motor.

scholarly journals MAP7 family proteins regulate kinesin-1 recruitment and activation

2019 ◽  
Vol 218 (4) ◽  
pp. 1298-1318 ◽  
Author(s):  
Peter Jan Hooikaas ◽  
Maud Martin ◽  
Tobias Mühlethaler ◽  
Gert-Jan Kuijntjes ◽  
Cathelijn A.E. Peeters ◽  
...  
Keyword(s):  
Hela Cells ◽  
Family Members ◽  
Binding Domain ◽  
Allosteric Effect ◽  
Microtubule Binding ◽  
Lower Affinity ◽  
Linker Region ◽  
Microtubule Binding Domain ◽  
Dependent Transport

Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family members bind to kinesin-1. In HeLa cells, MAP7, MAP7D1, and MAP7D3 act redundantly to enable kinesin-1–dependent transport and microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions. In vitro, purified MAP7 and MAP7D3 increase microtubule landing rate and processivity of kinesin-1 through transient association with the motor. MAP7 proteins promote binding of kinesin-1 to microtubules both directly, through the N-terminal microtubule-binding domain and unstructured linker region, and indirectly, through an allosteric effect exerted by the kinesin-binding C-terminal domain. Compared with MAP7, MAP7D3 has a higher affinity for kinesin-1 and a lower affinity for microtubules and, unlike MAP7, can be cotransported with the motor. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules.

scholarly journals Autoregulatory control of microtubule binding in the oncogene, doublecortin-like kinase 1

2020 ◽  
Author(s):  
Melissa M. Rogers ◽  
Amrita Ramkumar ◽  
Ashlyn M. Downing ◽  
Hannah Bodin ◽  
Julia Castro ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Binding Activity ◽  
Unique Form ◽  
Microtubule Binding ◽  
Microtubule Binding Domain ◽  
In Vitro Reconstitution ◽  
Protein Map

AbstractThe microtubule-associated protein (MAP), doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for the development of kinase inhibitors. However, the physiological roles of its kinase activity and how DCLK1 kinase activity is regulated remain elusive. Here we employ in vitro reconstitution with purified proteins to analyze the role of DCLK1 kinase activity in regulating microtubule binding. We find that DCLK1 autophosphorylates a single residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation largely within the doublecortin 2 (DC2) domain, which dramatically reduces the microtubule affinity of DCLK1. Therefore, autophosphorylation at specific sites within DCLK1 have diametric effects on the molecule’s ability to associate with microtubules. Overall, our results suggest a mechanism by which DCLK1 modulates its own kinase activity to tune its microtubule binding affinity, providing molecular insights into a unique form of autoregulatory control over microtubule binding activity within the broader family of MAPs. These results provide useful molecular insights for future therapeutic efforts related to DCLK1’s role in cancer development and progression.

scholarly journals Modulation of tau phosphorylation and intracellular localization by cellular stress

2000 ◽  
Vol 345 (2) ◽  
pp. 263-270 ◽  
Author(s):  
Scott M. JENKINS ◽  
Marcus ZINNERMAN ◽  
Craig GARNER ◽  
Gail V. W. JOHNSON
Keyword(s):  
Osmotic Stress ◽  
Protein Kinases ◽  
Intracellular Localization ◽  
Tau Phosphorylation ◽  
Binding Domain ◽  
Microtubule Binding ◽  
Microtubule Binding Domain

Tau is a microtubule-associated protein that is functionally modulated by phosphorylation and hyperphosphorylated in several neurodegenerative diseases. Because phosphorylation regulates both normal and pathological tau functioning, it is of great interest to identify the signalling pathways and enzymes capable of modulating tau phosphorylation in vivo. The present study examined changes in tau phosphorylation and localization in response to osmotic stress, which activates the stress-activated protein kinases (SAPKs), a family of proline-directed protein kinases shown to phosphorylate tau in vitro and hypothesized to phosphorylate tau in Alzheimer's disease. Immunoblot analysis with phosphorylation-dependent antibodies revealed that osmotic stress increased tau phosphorylation at the non-Ser/Thr-Pro sites Ser-262/356, within the microtubule-binding domain, as well as Ser/Thr-Pro sites outside of tau's microtubule-binding domain. Although all SAPKs examined were activated by osmotic stress, none of the endogenous SAPKs mediated the increase in tau phosphorylation. However, when transfected into SH-SY5Y cells, SAPK3, but not the other SAPKs examined, phosphorylated tau in situ in response to activation by osmotic stress. Osmotic-stress-induced tau phosphorylation correlated with a decrease in the amount of tau associated with the cytoskeleton and an increase in the amount of soluble tau. This stress-induced alteration in tau localization was only partially due to phosphorylation at Ser-262/356 by a staurosporine-sensitive, non-proline-directed, protein kinase. Taken together, these results suggest that osmotic stress activates at least two tau-directed protein kinases, one proline-directed and one non-proline-directed, that SAPK3 can phosphorylate tau on Ser/Thr-Pro residues in situ, and that Ser-262/356 phosphorylation only partially regulates tau localization in the cell.

Possible Role of Each Repeat Structure of the Microtubule-Binding Domain of the Tau Protein in In Vitro Aggregation

2005 ◽  
Vol 138 (4) ◽  
pp. 413-423 ◽  
Author(s):  
Koji Tomoo ◽  
Tian-Ming Yao ◽  
Katsuhiko Minoura ◽  
Shuko Hiraoka ◽  
Miho Sumida ◽  
...  
Keyword(s):  
Tau Protein ◽  
Binding Domain ◽  
Microtubule Binding ◽  
Repeat Structure ◽  
Microtubule Binding Domain

scholarly journals Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells.

1993 ◽  
Vol 123 (2) ◽  
pp. 357-371 ◽  
Author(s):  
D Masson ◽  
T E Kreis
Keyword(s):  
Epithelial Cells ◽  
Molecular Characterization ◽  
Hela Cells ◽  
Binding Proteins ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Microtubule Binding ◽  
Terminal Domain

A novel microtubule-associated protein (MAP) of M(r) 115,000 has been identified by screening of a HeLa cell cDNA expression library with an anti-serum raised against microtubule-binding proteins from HeLa cells. Monoclonal and affinity-purified polyclonal antibodies were generated for the further characterization of this MAP. It is different from the microtubule-binding proteins of similar molecular weights, characterized so far, by its nucleotide-insensitive binding to microtubules and different sedimentation behavior. Since it is predominantly expressed in cells of epithelial origin (Caco-2, HeLa, MDCK), and rare (human skin, A72) or not detectable (Vero) in fibroblastic cells, we name it E-MAP-115 (epithelial MAP of 115 kD). In HeLa cells, E-MAP-115 is preferentially associated with subdomains or subsets of perinuclear microtubules. In Caco-2 cells, labeling for E-MAP-115 increases when they polarize and form blisters. The molecular characterization of E-MAP-115 reveals that it is a novel protein with no significant homologies to other known proteins. The secondary structure predicted from its sequence indicates two domains connected by a putative hinge region rich in proline and alanine (PAPA region). E-MAP-115 has two highly charged regions with predicted alpha-helical structure, one basic with a pI of 10.9 in the NH2-terminal domain and one neutral with a pI of 7.6 immediately following the PAPA region in the acidic COOH-terminal half of the molecule. A novel microtubule-binding site has been localized to the basic alpha-helical region in the NH2-terminal domain using in vitro microtubule-binding assays and expression of mutant polypeptides in vivo. Overexpression of this domain of E-MAP-115 by transfection of fibroblasts lacking significant levels of this protein with its cDNA renders microtubules stable to nocodazole. We conclude that E-MAP-115 is a microtubule-stabilizing protein that may play an important role during reorganization of microtubules during polarization and differentiation of epithelial cells.

scholarly journals MTCL2 is a new Golgi-resident microtubule-regulating protein, essential for organizing asymmetric microtubule network

2020 ◽  
Author(s):  
Risa Matsuoka ◽  
Masateru Miki ◽  
Sonoko Mizuno ◽  
Yurina Ito ◽  
Atsushi Suzuki
Keyword(s):  
Coiled Coil ◽  
Golgi Membrane ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
Microtubule Stabilization ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Ribbon Structure ◽  
Golgi Ribbon

AbstractThe Golgi apparatus plays important roles in organizing the asymmetric microtubule network essential for polarized vesicle transport. The Golgi-associated coiled-coil protein MTCL1 is crucially involved in Golgi functioning by interconnecting and stabilizing microtubules on the Golgi membrane through its N- and C-terminal microtubule-binding domains. Here, we report the presence of a mammalian paralog of MTCL1, named MTCL2, lacking the N-terminal microtubule-binding domain. MTCL2 localizes to the Golgi membrane through the N-terminal region and directly binds microtubules through the conserved C-terminal domain without promoting microtubule stabilization. Knockdown experiments demonstrated essential roles of MTCL2 in accumulating MTs around the Golgi and regulating the Golgi ribbon structure. In vitro wound healing assays further suggested a possible intriguing activity of MTCL2 in integrating the centrosomal and Golgi-associated microtubules around the Golgi ribbon, thus supporting directional migration. Altogether, the present results demonstrate that cells utilize two members of the MTCL protein family to differentially regulate the Golgi-associated microtubules for controlling cell polarity.

C-terminal inhibition of tau assembly in vitro and in Alzheimer's disease

2000 ◽  
Vol 113 (21) ◽  
pp. 3737-3745 ◽  
Author(s):  
A. Abraha ◽  
N. Ghoshal ◽  
T.C. Gamblin ◽  
V. Cryns ◽  
R.W. Berry ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Amino Acids ◽  
Disease Process ◽  
Brain Regions ◽  
Microtubule Binding ◽  
C Terminus ◽  
Microtubule Binding Domain ◽  
Tau Polymerization

Alzheimer's disease (AD) is, in part, defined by the polymerization of tau into paired helical and straight filaments (PHF/SFs) which together comprise the fibrillar pathology in degenerating brain regions. Much of the tau in these filaments is modified by phosphorylation. Additionally, a subset also appears to be proteolytically truncated, resulting in the removal of its C terminus. Antibodies that recognize tau phosphorylated at S(396/404)or truncated at E(391) do not stain control brains but do stain brain sections very early in the disease process. We modeled these phosphorylation and truncation events by creating pseudo-phosphorylation and deletion mutants derived from a full-length recombinant human tau protein isoform (ht40) that contains N-terminal exons 2 and 3 and all four microtubule-binding repeats. In vitro assembly experiments demonstrate that both modifications greatly enhance the rates of tau filament formation and that truncation increases the mass of polymer formed, as well. Removal of as few as 12 or as many as 121 amino acids from the C terminus of tau greatly increases the rate and extent of tau polymerization. However, deletion of an additional 7 amino acids, (314)DLSKVTS(320), from the third microtubule-binding repeat results in the loss of tau's ability to form filaments in vitro. These results suggest that only part of the microtubule-binding domain (repeats 1, 2 and a small portion of 3) is crucial for tau polymerization. Moreover, the C terminus of tau clearly inhibits the assembly process; this inhibition can be partially reversed by site-specific phosphorylation and completely removed by truncation events at various sites from S(320) to the end of the molecule.

scholarly journals Identification of a novel nucleotide-sensitive microtubule-binding protein in HeLa cells.

1990 ◽  
Vol 110 (5) ◽  
pp. 1623-1633 ◽  
Author(s):  
J E Rickard ◽  
T E Kreis
Keyword(s):  
Hela Cells ◽  
High Speed ◽  
Binding Proteins ◽  
Motor Proteins ◽  
Cytoplasmic Dynein ◽  
Microtubule Binding ◽  
Linear Arrays ◽  
Immunofluorescence Labeling

A protein of Mr 170,000 (170K protein) has been identified in HeLa cells, using an antiserum raised against HeLa nucleotide-sensitive microtubule-binding proteins. Affinity-purified antibodies specific for this 170K polypeptide were used for its characterization. In vitro sedimentation of the 170K protein with taxol microtubules polymerized from HeLa high-speed supernatant is enhanced in the presence of an ATP depleting system, but unaffected by the non-hydrolyzable ATP analogue AMP-PNP. In addition, it can be eluted from taxol microtubules by ATP or GTP, as well as NaCl. Thus it shows microtubule-binding characteristics distinct from those of the previously described classes of nucleotide-sensitive microtubule-binding proteins, the motor proteins kinesin and cytoplasmic dynein, homologues of which are also present in HeLa cells. The 170K protein sediments on sucrose gradients at approximately 6S, separate from kinesin (9.5S) and cytoplasmic dynein (20S), further indicating that it is not associated with these motor proteins. Immunofluorescence localization of the 170K protein shows a patchy distribution in interphase HeLa cells, often organized into linear arrays that correlate with microtubules. However, not all microtubules are labeled, and there is a significant accumulation of antigen at the peripheral ends of microtubules. In mitotic cells, 170K labeling is found in the spindle, but there is also dotty labeling in the cytoplasm. After depolymerization of microtubules by nocodazole, the staining pattern is also patchy but not organized in linear arrays, suggesting that the protein may be able to associate with other intracellular structures as well as microtubules. In vinblastine-treated cells, there is strong labeling of tubulin paracrystals, and random microtubules induced in vivo by taxol are also labeled by the antibodies. These immunofluorescence labeling patterns are stable to extraction of cells with Triton X-100 before fixation, further suggesting an association of the protein with cytoplasmic structures. In vivo, therefore, the 170K protein appears to be associated with a subset of microtubules at discrete sites. Its in vitro behavior suggests that it belongs to a novel class of nucleotide-sensitive microtubule-binding proteins.

scholarly journals Conserved Lysine Acetylation within the Microtubule-Binding Domain Regulates MAP2/Tau Family Members

PLoS ONE ◽  
2016 ◽  
Vol 11 (12) ◽  
pp. e0168913 ◽  
Author(s):  
Andrew W. Hwang ◽  
Hanna Trzeciakiewicz ◽  
Dave Friedmann ◽  
Chao-Xing Yuan ◽  
Ronen Marmorstein ◽  
...  
Keyword(s):  
Family Members ◽  
Binding Domain ◽  
Lysine Acetylation ◽  
Microtubule Binding ◽  
Microtubule Binding Domain

scholarly journals Stress-induced phosphorylation of CLIP-170 by JNK promotes microtubule rescue

2020 ◽  
Vol 219 (7) ◽  
Author(s):  
Hélène Henrie ◽  
Dalal Bakhos-Douaihy ◽  
Isabelle Cantaloube ◽  
Antoine Pilon ◽  
Maya Talantikite ◽  
...  
Keyword(s):  
Binding Domain ◽  
Cell Stress ◽  
Microtubule Dynamics ◽  
Microtubule Binding ◽  
Microtubule Binding Domain ◽  
Microtubule Growth ◽  
Jnk Activation ◽  
In Cells

The stress-induced c-Jun N-terminal kinase (JNK) controls microtubule dynamics by enhancing both microtubule growth and rescues. Here, we show that upon cell stress, JNK directly phosphorylates the microtubule rescue factor CLIP-170 in its microtubule-binding domain to increase its rescue-promoting activity. Phosphomimetic versions of CLIP-170 enhance its ability to promote rescue events in vitro and in cells. Furthermore, while phosphomimetic mutations do not alter CLIP-170’s capability to form comets at growing microtubule ends, both phosphomimetic mutations and JNK activation increase the occurrence of CLIP-170 remnants on the microtubule lattice at the rear of comets. As the CLIP-170 remnants, which are potential sites of microtubule rescue, display a shorter lifetime when CLIP-170 is phosphorylated, we propose that instead of acting at the time of rescue occurrence, CLIP-170 would rather contribute in preparing the microtubule lattice for future rescues at these predetermined sites.

Sign in / Sign up

Export Citation Format

Share Document