Calpain 6 Is Involved in Microtubule Stabilization and Cytoskeletal Organization

Abstract

The calpains are a family of Ca 2؉ -dependent cysteine proteases implicated in various biological processes. In this family, calpain 6 (Capn6) is unique in that it lacks the active-site cysteine residues requisite for protease activity. During the search for genes downstream of the endothelin 1 (ET-1) signaling in pharyngeal-arch development, we identified Capn6. After confirming that the expression of Capn6 in pharyngeal arches is downregulated in ET-1-null embryos by in situ hybridization, we investigated its function. In Capn6-transfected cells, cytokinesis was retarded and was often aborted to yield multinucleated cells. Capn6 overexpression also caused the formation of microtubule bundles rich in acetylated ␣-tubulin and resistant to the depolymerizing activity of nocodazole. Green fluorescent protein-Capn6 overexpression, immunostaining for endogenous Capn6, and biochemical analysis demonstrated interaction between Capn6 and microtubules, which appeared to be mainly mediated by domain III. Furthermore, RNA interference-mediated Capn6 inactivation caused microtubule instability with a loss of acetylated ␣-tubulin and induced actin reorganization, resulting in lamellipodium formation with membrane ruffling. Taken together, these results indicate that Capn6 is a microtubule-stabilizing protein expressed in embryonic tissues that may be involved in the regulation of microtubule dynamics and cytoskeletal organization.

Expanding Members and Roles of the Calpain Superfamily and Their Genetically Modified Animals

“…These molecules are considered not to have cysteine protease activity. Although the physiological roles of these nonproteolytic subfamily members have yet to be elucidated, some of the reports cited above have shown novel calpain functions other than those of protease [2,45,60,118,126,139,167]. The processes of generation of these calpain species are interesting from an evolutionary viewpoint and the elucidation of their physiological functions will shed light on novel aspects of the calpain superfamily.…”

“…By contrast, calpain-6 apparently has no active site residues in its amino acid sequence (active site Cys is substituted with Lys), strongly suggesting that calpain-6 has no proteolytic activity. Recently, calpain-6 was reported to be involved in the regulation of microtubule dynamics, which has opened up a new field of calpain study [167]. Calpain-10 was identified by large-scale genetic as-sociation studies of noninsulin-dependent diabetes mellitus (NIddM, type 2 diabetes) [8,17,63].…”

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

“…Recently, we have demonstrated that CAPN6 can bind to microtubules, mainly through domain III, and induce microtubule stabilization through nonproteolytic activity (Tonami et al, 2007). Furthermore, inactivation of CAPN6 not only destabilizes microtubules but also promotes formation of lamellipodia and lamellipodial membrane ruffling (Tonami et al, 2007). These findings have led us to study the possibility that CAPN6 participates in microtubule-actin crosstalk in order to contribute to cellular functions.…”

“…In CAPN6, as well as Capn5, the C-terminal structure is defined as a diverged C2-domain (also called domain T) instead of as domain IV, on the basis of similarity to Caenorhabditis elegans TRA-3, a nematode sex determination factor (Barnes and Hodgkin, 1996;Dear et al, 1997;Goll et al, 2003;Mugita et al, 1997). Recently, we have demonstrated that CAPN6 can bind to microtubules, mainly through domain III, and induce microtubule stabilization through nonproteolytic activity (Tonami et al, 2007). Furthermore, inactivation of CAPN6 not only destabilizes microtubules but also promotes formation of lamellipodia and lamellipodial membrane ruffling (Tonami et al, 2007).…”

Molecular Evolution Within Protease Family C2, or Calpains

“…Inactive homologues found to be abundant in some protease families and might have important roles as regulatory or inhibitory molecules, acting as dominant negatives by binding substrates through the inactive catalytic or exosite ancillary domains in nonproductive complexes, or by titrating inhibitors from the milieu to increase the net proteolytic activity (López-Otín & Overall, 2002;Puente et al, 2003;Pils & Schultz, 2004). A recent report describes a role for the non-catalytic calpain 6 in the stabilization of microtubules (Tonami et al, 2007). The expression of non-protease homologues is interesting with an evolutionary viewpoint as the discovery of their physiological functions may elucidate calpain functions distinct from proteolysis.…”