The Dictyostelium Carmil Protein Links Capping Protein and the Arp2/3 Complex to Type I Myosins through Their Sh3 Domains

Abstract

Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the α and β subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH2-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end–directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker.

V-1 regulates capping protein activity in vivo

“…One prediction of this model is that cells expressing a version of CP that cannot see the CPI motif in CARMIL and related proteins (e.g., CD2AP and CKIP) should phenocopy Arp2/3-inhibited cells. This prediction has recently received direct support by the work of Edwards et al (29).Studies using gene ablation or RNAi have demonstrated that CARMIL proteins play important roles in the formation of cortical actin structures like pseudopodia and macropinocytic crowns in Dictyostelium and lamellipodia in tissue culture cells (1,19). In contrast, relatively little is known regarding the importance of V-1 in vivo (30-32), especially with respect to effects on actin assembly and organization.…”

“…Studies using gene ablation or RNAi have demonstrated that CARMIL proteins play important roles in the formation of cortical actin structures like pseudopodia and macropinocytic crowns in Dictyostelium and lamellipodia in tissue culture cells (1,19). In contrast, relatively little is known regarding the importance of V-1 in vivo (30-32), especially with respect to effects on actin assembly and organization.…”

“…To date, two direct regulators of CP activity have been identified. The first, CARMIL, is a ∼125-kDa protein that binds CP tightly via a small, highly conserved domain known as CAH3 or CPI (1,19). CP bound to CPI binds the barbed end with an affinity of ∼50 nM, which, although still quite significant, is ∼100-fold weaker than free CP (20,21).…”

Molecular Basis for Barbed End Uncapping by CARMIL Homology Domain 3 of Mouse CARMIL-1

“…Indeed, the huge discrepancy between the half-life of CP bound to the barbed end in vitro (ϳ30 min) (18,19) and in vivo (ϳ1 s) (20) suggests that CP activity is significantly controlled by regulatory molecules in vivo. In fact, several proteins have been found to bind CP to alter its activity (1,2,(21)(22)(23)(24). One possible cellular regulator of CP is V-1 (myotrophin), which binds CP in vitro with high affinity (K d ϳ40 nM) in a 1:1 complex that has no affinity for the barbed end (i.e.…”

“…CARMIL proteins comprise a recently identified family of molecules whose two most conspicuous features are a central, leucinerich repeat domain and a C-terminal proline-rich domain (24,26). Genes encoding CARMIL proteins have been identified in Acanthamoeba, Dictyostelium, Caenorhabditis elegans, Drosophila, mice, and humans (24, 26 -30).…”