Phosphorylation of CLASP2 by GSK-3 beta regulates its interaction with IQGAP1, EB1 and microtubules

Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi, Japan.
Journal of Cell Science (Impact Factor: 5.43). 09/2009; 122(Pt 16):2969-79. DOI: 10.1242/jcs.046649
Source: PubMed


Polarised cell migration is required for various cell behaviours and functions. Actin and microtubules are coupled structurally and distributed asymmetrically along the front-rear axis of migrating cells. CLIP-associating proteins (CLASPs) accumulate near the ends of microtubules at the front of migrating cells to control microtubule dynamics and cytoskeletal coupling. Regional inhibition of GSK-3beta is responsible for this asymmetric distribution of CLASPs. However, it is not known how GSK-3beta regulates the activity of CLASPs for linkage between actin and microtubules. Here we identified IQGAP1, an actin-binding protein, as a novel CLASP-binding protein. GSK-3beta directly phosphorylates CLASP2 at Ser533 and Ser537 within the region responsible for the IQGAP1 binding. Phosphorylation of CLASP2 results in the dissociation of CLASP2 from IQGAP1, EB1 and microtubules. At the leading edges of migrating fibroblasts, CLASP2 near microtubule ends partially colocalises with IQGAP1. Expression of active GSK-3beta abrogates the distribution of CLASP2 on microtubules, but not that of a nonphosphorylatable CLASP2 mutant. The phosphorylated CLASP2 does not accumulate near the ends of microtubules at the leading edges. Thus, phosphorylation of CLASP2 by GSK-3beta appears to control the regional linkage of microtubules to actin filaments through IQGAP1 for cell migration.

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Available from: Kozo Kaibuchi, Jul 08, 2015
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    • "Ch-TOG might mediate the interaction of microtubules with proteins localized at the cell cortex and thereby participates to microtubule capture and stabilization. While we cannot exclude that ch-TOG associates directly with cortical components, microtubule +end cortical interactions are more likely driven by EB1, which is known to bind SxIP motif-containing cortical proteins[18,22,303132. Reconstitution experiments strongly suggest that ch-TOG allosterically enhances the binding of EB1 to the microtubule +end[22]. "
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    ABSTRACT: Non-cytotoxic concentrations of microtubule targeting agents (MTAs) interfere with the dynamics of interphase microtubules and affect cell migration, which could impair tumor angiogenesis and metastasis. The underlying mechanisms however are still ill-defined. We previously established that directed cell migration is dependent on stabilization of microtubules at the cell leading edge, which is controlled by microtubule +end interacting proteins (+TIPs). In the present study, we found that eribulin, a recently approved MTA interacting with a new class of binding site on β-tubulin, decreased microtubule growth speed, impaired their cortical stabilization and prevented directed migration of cancer cells. These effects were reminiscent of those observed when +TIP expression or cortical localization was altered. Actually, eribulin induced a dose-dependent depletion of EB1, CLIP-170 and the tubulin polymerase ch-TOG from microtubule +ends. Interestingly, eribulin doses that disturbed ch-TOG localization without significant effect on EB1 and CLIP-170 comets, had an impact on microtubule dynamics and directed migration. Moreover, knockdown of ch-TOG led to a similar inhibition of microtubule growth speed, microtubule capture and chemotaxis. Our data suggest that eribulin binding to the tip of microtubules and subsequent loss of ch-TOG is a priming event leading to alterations in microtubule dynamics and cancer cell migration.
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    • "We have also shown that the Kip2-Bim1 interaction is required for efficient localisation of Kar9 to aMT plus ends. In mammalian cells, GSK-3 phosphorylates +TIPs such as CLASP2 or APC, and weakens their interaction with MTs or other +TIPs (Rubinfeld et al., 1996; Zumbrunn et al., 2001), while local inhibition of GSK-3 stabilises microtubules and promotes their interaction with the cortical cytoskeleton during cell migration (Akhmanova et al., 2001; Etienne Manneville and Hall, 2003; Kumar et al., 2009; Watanabe et al., 2009). Regulation of Kip2 by Mck1 in budding yeast may be another example for a GSK-3-dependent mechanism utilised to couple regulation of microtubule dynamics with control of aMT-cortical interactions. "
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    • "perative to note that IQGAP1 functions as a regulator of - catenin - mediated gene transcription ( Fukata et al . , 1999 ; Wang et al . , 2008 ; Briggs et al . , 2002 ) . Direct binding of APC ( Watanabe et al . , 2004 ; Aoki and Taketo , 2007 ) or protein phosphatase 2A ( PP2A ) ( Suzuki et al . , 2005 ) and an indirect interaction with GSK - 3 ( Watanabe et al . , 2009 ) signifies the biological relevance of IQGAP1 in Wnt signaling ( Fig . 7A ) . Most importantly , a region between the IQ domains and the GRD of IQGAP1 binds the C - terminus of Disheveled ( DVL ) ( Goto et al . , 2013b ) and direct binding of - catenin itself to the RGCT domain ( Fukata et al . , 1999 ) confirms a role for IQGAP1 in th"
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