The C-terminal dimerization motif of cyclase-associated protein is essential for actin monomer regulation

Abstract

Cyclase-associated protein (CAP) is a conserved actin-regulatory protein that functions together with actin-depolymerizing factor (ADF)/cofilin to enhance actin filament dynamics. CAP has multiple functional domains, and the function to regulate actin monomers is carried out by its C-terminal half containing a Wiskott-Aldrich Syndrome protein homology 2 (WH2) domain, a CAP and X-linked retinitis pigmentosa 2 (CARP) domain, and a dimerization motif. WH2 and CARP are implicated in binding to actin monomers and important for enhancing filament turnover. However, a role of the dimerization motif is unknown. Here, we investigated function of the dimerization motif of CAS-2, a CAP isoform in the nematode Caenorhabditis elegans, in actin monomer regulation. CAS-2 promotes ATP-dependent recycling of ADF/cofilin-bound actin monomers for polymerization by enhancing exchange of actin-bound nucleotides. A C-terminal half of CAS-2 (CAS-2C) has nearly as strong activity as full-length CAS-2. Maltose-binding protein (MBP)-tagged CAS-2C is a dimer. However, MBP-CAS-2C with a truncation of either one or two C-terminal β-strands is monomeric. Truncations of the dimerization motif in MBP-CAS-2C nearly completely abolish its activity to sequester actin monomers from polymerization and enhance nucleotide exchange on actin monomers. As a result, these CAS-2C variants, also in the context of full-length CAS-2, fail to compete with ADF/cofilin to release actin monomers for polymerization. CAS-2C variants lacking the dimerization motif exhibit enhanced binding to actin filaments, which is mediated by WH2. Together, these results suggest that the evolutionarily conserved dimerization motif of CAP is essential for its C-terminal region to exert the actin-monomer-specific regulatory function.

Conserved hydrophobic residues in the CARP/β‐sheet domain of cyclase‐associated protein are involved in actin monomer regulation

“…MBP‐CAS‐2C and bound actin were pulled down, and depletion of G‐actin from the supernatants was quantified by infrared imaging. As reported previously (Iwase and Ono, ), MBP alone did not bind to either ATP‐ or ADP‐G‐actin (Figure a–d), whereas MBP‐CAS‐2C (WT) bound to both ATP‐ and ADP‐G‐actin with higher affinity to ADP‐G‐actin (Figure c,d, Table ) than ATP‐G‐actin (Figure a,b). The five examined single mutants exhibited variable affinity for binding to G‐actin (Table ).…”

“…To test whether the mutations at hydrophobic residues cause major alterations in the dimerization state of CAS‐2C, MBP‐CAS‐2C variants were analyzed by analytical gel filtration chromatography. CAS‐2C dimerizes through the C‐terminal dimerization motif, which is essential for its actin‐monomer regulatory activities (Iwase and Ono, ). MBP‐CAS‐2C (WT) was eluted faster than MBP‐CAS‐2CΔβ8β9 (a deletion of the dimerization motif; Figure a).…”

“…MBP‐CAS‐2C (WT) was eluted faster than MBP‐CAS‐2CΔβ8β9 (a deletion of the dimerization motif; Figure a). We previously used dynamic light scattering analysis to estimate that the peak fractions of MBP‐CAS‐2C (WT) and MBP‐CAS‐2CΔβ8β9 were dimeric and monomeric, respectively (Iwase and Ono, ). All examined MBP‐CAS‐2C single mutants were eluted in a similar manner to MBP‐CAS‐2C (WT) (Figure b) with no detectable peaks of large aggregates, suggesting that these single mutations did not disrupt the dimeric state of CAS‐2C.…”

“…The gels were stained with Coomassie Brilliant Blue R‐250 (National Diagnostics), scanned by an Epson Perfection V700 Photo Scanner at 300 dots per inch, and analyzed by densitometry using ImageJ. Correlation of elution profiles and dimeric/monomeric states of MBP‐CAS‐2C and MBP‐CAS‐2CΔβ8β9 was determined previously by dynamic light scattering (Iwase and Ono, ).…”

“…Side chains of surface-exposed hydrophobic residues are shown in magenta. (d, e) A homology model of the CAS-2 dimeric C-terminal region (residues 306-457; Iwase and Ono, 2016) is shown in a similar manner to human CAP1. Highlighted conserved hydrophobic residues are characterized in this study.…”