Akhmanova Anna

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  • Article
    Phosphorylation controls autoinhibition of cytoplasmic linker protein-170
    (American Society for Cell Biology, 2010-06-02) Lee, Ho-Sup ; Komarova, Yulia A. ; Nadezhdina, Elena S. ; Anjum, Rana ; Peloquin, John G. ; Schober, Joseph M. ; Danciu, Oana ; van Haren, Jeffrey ; Galjart, Niels ; Gygi, Steven P. ; Akhmanova, Anna ; Borisy, Gary G.
    Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150Glued (J. Cell Biol. 2004: 166, 1003–1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an "open" conformation and a higher binding affinity for growing MT ends and p150Glued as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the "folded back" conformation shows decreased MT association and does not interact with p150Glued. We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.
  • Article
    Mammalian end binding proteins control persistent microtubule growth
    (Rockefeller University Press, 2009-03-02) Komarova, Yulia A. ; De Groot, Christian O. ; Grigoriev, Ilya ; Gouveia, Susana Montenegro ; Munteanu, E. Laura ; Schober, Joseph M. ; Honnappa, Srinivas ; Buey, Ruben M. ; Hoogenraad, Casper C. ; Dogterom, Marileen ; Borisy, Gary G. ; Steinmetz, Michel O. ; Akhmanova, Anna
    End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.