title: Regulation of the Actin Binding Activity of Basic Calponin and Its Implication in Cytokinesis
Creators: name: Rozenblum, Tomàs Guido
Publisher: name: The Open University
Year: 2008

description: Calponins (CaPs) are actin-binding proteins that stabilize actin filaments. Mammals express three genetic isoforms in a tissue-specific manner. CaPs share an N-terminal calponin-homology (CH) domain followed by short tandem sequence repeats termed calponin-like (CLIK23) repeats. The extreme C-termini of CaPs contain isoform-specific tail sequences. Basic calponin (h1CaP) was the first isoform to be discovered 22 years ago in an attempt to identify troponin-like proteins in smooth muscle. H1CaP is mainly expressed in smooth muscle cells and was shown to regulate contraction by inhibiting actin-activated myosin ATPase activity. Several mechanisms have been proposed for the regulation of the actin-binding activity of h1CaP. One mechanism is based on phosphorylation. H1CaP phosphorylation inhibits its interaction with actin filaments and releases the block on the myosin ATPase activity <i>in vitro</i>. However, <i>in vivo</i> it has not been unequivocally demonstrated whether h1CaP is phosphorylated or not. The binding of extracellular regulated kinase (ERK) but not phosphorylation was proposed as another regulatory mechanism, but the physiological relevance of this interaction remains elusive. Therefore, the scope of this thesis was to reveal the mechanisms that regulate the actin-binding activity of h1CaP. First, the contribution of the major h1CaP phosphorylated sites to its actin-binding activity was re-evaluated. A mutant h1CaP mimicking its phosphorylated state behaved, however, similar to wild-type h1CaP. Thus, another regulatory mechanism was explored. It has previously been shown that the C-terminal tails regulate the actin-binding activity of all three CaP isoforms. Also, electron microscopy images of purified h1CaP show a flexible molecule that can adopt both an extended and a more compact conformation, and it was recently shown that the CH domain does not mediate the actin binding-activity of h1CaP. In light of the flexibility of h1CaP and the demonstrated regulatory role of the C-terminal tail, a possible regulatory mechanism could thus involve an intramolecular interaction between different domains. Therefore, the contribution of the C-terminal tail and the CH domain of h1CaP to its actin-binding activity was investigated in cells. The results suggest that the h1CaP CH domain blocks the inhibitory effects of its tail, which could be mediated by a CH domain-tail interaction. Therefore, the interaction between the CH domain and the tail was modelled and the plausibility of their interaction was predicted <i>in silico</i>. Furthermore, the interaction between these two domains was demonstrated <i>in vitro</i> using recombinant proteins. The proximity between the CH domain and the tail was also analyzed using FRET imaging in live cells, and the region encompassing helix A and loop 1 of the CH domain was determined to be the binding region for the tail. These results suggest that the binding of h1CaP to actin filaments is modulated by an interaction between the h1CaP N-terminal CH domain and its C-terminal tail. <br><br>One aspect of the potential <i>in-vivo</i> functions of h1CaP that has been neglected so far relates to its actin-binding activity during cell division. It has previously been reported that overexpression of CaP reduces cell proliferation and increases the number of bi-nucleated cells. This suggests that CaPs have a role during mitosis. Thus, the localization of CaPs during cell division was investigated and the data presented in this study demonstrate the specific accumulation of CaP at the ingression furrow and the contractile ring, suggesting a role in cytokinesis. Furthermore, overexpression of h1CaP mutants led to cell division defects. In summary, this study has revealed a novel regulatory mechanism that controls the actin-binding activity of h1CaP, which is mediated by the intramolecular interaction of its CH domain and its C-terminal tail.