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

Abstract

2+-and phospholipid-binding module; and domain IV is characterized by the presence of multiple EF-hand motifs in some members, including the classical calpains (m-and -calpains) (Croall and Ersfeld, 2007;Goll et al., 2003). Among the 14 or 15 members of the calpain family in mammals, calpain-6 (CAPN6) is unique in that it lacks the active-site catalytic cysteine residue and is therefore SummaryCrosstalk 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.

Calpain-6 confers atherogenicity to macrophages by dysregulating pre-mRNA splicing

“…Our data indicate that Capn6 deficiency restores the reduced expression of the Rac1 and GEF2 proteins evident in M-CSF/TNF-α-primed BMMs without altering the expression of the RhoA and RhoGDIα proteins, leading to the recovery of Rac1 activity in cells (Figure 2, A and B). This is consistent with an earlier investigation indicating that siRNA-based silencing of CAPN6 in NIH3T3 cells potentiates Rac1 activity (24). This Rac1 system recovery appears to abrogate pinocytotic ability in Capn6…”

The transcription factor Erg regulates expression of histone deacetylase 6 and multiple pathways involved in endothelial cell migration and angiogenesis

“…Whether microtubule deacetylation alone is sufficient to promote cell migration is still controversial; however, microtubule stability has been shown to affect actindependent cell motility through the small GTPases Rac-1 and Rho. 30 Possibly through multiple mechanisms, HDAC6 regulates cell migration: overexpression of HDAC6 in NIH-3T3 cells leads to increased motility, 6 while inhibition of HDAC6 activity using tubacin, a small molecule HDAC6 inhibitor that binds to its tubulin deacetylation catalytic domain, decreases migration and tumor cell invasiveness. 31 Based on the results of the microarray study and our own previous data, we propose that HDAC6 is one of several pathways and target genes involved in Erg's regulation of angiogenesis.…”

I’m coming to GEF you: Regulation of RhoGEFs during cell migration

“…127 Two recent studies have shown that GEF-H1 binding to microtubules may be regulated by Par1b, which plays a role in the regulation of cell polarity. 128,129 Different studies have shown that Par1b binds to and phosphorylates GEF-H1 at different sites in the C and N terminus, and that phosphorylation can affect its exchange activity and regulates its binding to microtubules. [129][130][131] Par1b and other kinases such as PAK1,4 and Aurora A/B, which also phosphorylate GEF-H1, may help regulate the localized binding and release of GEF-H1 from microtubules, as well as its catalytic activity in response to different stimuli.…”