Calpain-6, a microtubule-stabilizing protein, regulates Rac1 activity and cell motility through interaction with GEF-H1

Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Journal of Cell Science (Impact Factor: 5.43). 03/2011; 124(Pt 8):1214-23. DOI: 10.1242/jcs.072561
Source: PubMed


Crosstalk between microtubules and actin filaments is crucial for various cellular functions, including cell migration, spreading and cytokinesis. The Rac1 GTPase plays a key role in such crosstalk at the leading edge of migrating cells in order to promote lamellipodial formation. However, the mechanism underlying the link between microtubules and Rac1 activation remains unclear. Here, we show that calpain-6 (CAPN6), a non-proteolytic calpain with microtubule-binding and -stabilizing activity, might participate in this crosstalk. Small interfering RNA (siRNA)-induced knockdown of Capn6 in NIH 3T3 cells resulted in Rac1 activation, which promoted cell migration, spreading and lamellipodial protrusion. This increase in Rac1 activity was abolished by knockdown of the Rho guanine nucleotide exchange factor GEF-H1 (officially known as Arhgef2). CAPN6 and GEF-H1 colocalized with microtubules and also interacted with each other through specific domains. Upon knockdown of Capn6, GEF-H1 was shown to translocate from microtubules to the lamellipodial region and to interact with Rac1. By contrast, RhoA activity was decreased upon knockdown of Capn6, although low levels of active RhoA or the presence of RhoA molecules appeared to be required for the Capn6-knockdown-induced Rac1 activation. We suggest that CAPN6 acts as a potential regulator of Rac1 activity, through a mechanism involving interaction with GEF-H1, to control lamellipodial formation and cell motility.

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    • "The Rac1-specific GEF, Arhgef6 was upregulated in Paraxis 2/2 tissue, 1.6- fold (Table 2, Fig. 2). Further, Capn6 (1.5-fold down, Table 3), coding for a microtubule-stabilizing protein, is expressed in the somites of mouse embryos (Dear and Boehm, 1999) and can promote RAC1 activity by binding ARHGEF2 (Tonami et al., 2011). The deregulation of genes encoding modifiers of RAC1 activity predicts that regulation of Rho GTPase activity by PARAXIS occurs indirectly during somitogenesis. "
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    ABSTRACT: Background: Dynamic alterations in cell shape, migration, and adhesion play a central role in tissue morphogenesis during embryonic development and congenital disease. The mesenchymal-to-epithelial transition that occurs during vertebrate somitogenesis is required for proper patterning of the axial musculoskeletal system. Somitic MET is initiated in the presomitic mesoderm by PARAXIS-dependent changes in cell adhesion, cell polarity, and the composition of the extracellular matrix. However, the target genes downstream of the transcription factor PARAXIS remain poorly described. Results: A genome-wide comparison of gene expression in the anterior presomitic mesoderm and newly formed somites of Paraxis(-/-) embryos resulted in a set of deregulated genes enriched for factors associated with extracellular matrix and cytoskeletal organization and cell-cell and cell-ECM adhesion. The greatest change in expression was seen in fibroblast activation protein alpha (Fap), encoding a dipeptidyl peptidase capable of increasing fibronectin and collagen fiber organization in extracellular matrix. Further, downstream genes in the Wnt and Notch signaling pathways were downregulated, predicting that PARAXIS participates in positive feedback loops in both pathways. Conclusions: These data demonstrate that PARAXIS initiates and stabilizes somite epithelialization by integrating signals from multiple pathways to control the reorganization of the ECM, cytoskeleton, and adhesion junctions during MET.
    Developmental Dynamics 11/2013; 242(11). DOI:10.1002/dvdy.24033 · 2.38 Impact Factor
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    • "Calpains are intracellular, non-lysosomal, calcium-dependent, cysteine proteases that are involved in proteolysis, apoptotic cell death, necrosis and other physiological events. Calpain 6 is known to bind and stabilize microtubules in the regulation of microtubule and cytoskeletal organization during embryonic development [55,56]. In consequence, CAPN6 expression should be downregulated after birth, except for placenta and for uterine cell tumors [57]. "
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    • "Although most recent studies focused on RhoA activation by GEF-H1, earlier it was also shown to exert Rac-GEF activity (Ren et al., 1998). Further, Tonami et al recently showed that knockdown of calpain-6 resulted in GEF-H1-dependent Rac activation (Tonami et al., 2011). Our study provides the first example of a signalling pathway in which GEF-H1 can act both as an activator of Rac and RhoA, depending on its phosphorylation state. "
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