Article

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: 5.33). 09/2009; 122(Pt 16):2969-79. DOI: 10.1242/jcs.046649
Source: PubMed

ABSTRACT 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.

0 Followers
 · 
178 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs, and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters is not yet understood. To examine this we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2-9XS/A) and which is resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared to myotubes that express similar levels of wild type CLASP2, or that are non-infected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2-8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends.
    Journal of Biological Chemistry 09/2014; 289(44). DOI:10.1074/jbc.M114.589457 · 4.60 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A hallmark of the neuromuscular junction (NMJ) is the high density of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane. The postsynaptic apparatus of the NMJ is organized by agrin secreted from motor neurons. The mechanisms that underlie the focal delivery of AChRs to the adult NMJ are not yet understood in detail. We previously showed that microtubule (MT) capture by the plus-end tracking protein CLASP2 regulates AChR density at agrin-induced AChR clusters in cultured myotubes via PI3 kinase acting through GSK3β. Here, we show that knock-down of the CLASP2-interaction partner LL5β by RNAi and forced expression of a CLASP2 fragment blocking the CLASP2/LL5β interaction inhibit microtubule capture. The same treatments impair focal vesicle delivery to the clusters. Consistent with these findings, knock-down of LL5β at the NMJ in vivo reduces the density and insertion of AChRs into the postsynaptic membrane. MT capturing and focal vesicle delivery to agrin-induced AChR clusters are also inhibited by microtubule and actin depolymerizing drugs, invoking both cytoskeletal systems in MT capture and in the fusion of AChR vesicles with the cluster membrane. Combined our data identify a transport system, organized by agrin through PI3 kinase, GSK3β, CLASP2 and LL5β, for precise delivery of AChR vesicles from the subsynaptic nuclei to the overlying synaptic membrane. © 2015 by The American Society for Cell Biology.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Microtubules execute diverse mitotic events that are spatially and temporally separated; the underlying regulation is poorly understood. By combining drug treatments, large-scale immunoprecipitation and mass spectrometry, we report the first comprehensive map of mitotic phase-specific protein interactions of the microtubule-end binding protein, EB1. EB1 interacts with some, but not all, of its partners throughout mitosis. We show that the interaction of EB1 with Astrin-SKAP complex, a key regulator of chromosome segregation, is enhanced during prometaphase, compared to anaphase. We find that EB1 and EB3, another EB family member, can interact directly with SKAP, in an SXIP-motif dependent manner. Using an SXIP defective mutant that cannot interact with EB, we uncover two distinct pools of SKAP at spindle microtubules and kinetochores. We demonstrate the importance of SKAP's SXIP-motif in controlling microtubule growth rates and anaphase onset, without grossly disrupting spindle function. Thus, we provide the first comprehensive map of temporal changes in EB1 interactors during mitosis and highlight the importance of EB protein interactions in ensuring normal mitosis. © 2015. Published by The Company of Biologists Ltd.
    01/2015; 4(2). DOI:10.1242/bio.201410413