CARMIL family proteins as multidomain regulators of actin-based motility

Figures

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  FIGURE 1:. Conservation and domain architecture of CARMIL proteins. (A) Domain architecture of CARMIL proteins, illustrating the arrangement of the PH domain, linker (L), N-cap (N), LRR domain, C-cap (C), HD, CBR consisting of a CPI motif and a CSI motif, MBD, and a PRD. Sequence alignment of the CBR for selected CARMILs, including the three vertebrate isoforms in zebrafish, mouse, and human (encoded by three separate genes). The alignment includes sequences for invertebrates, which lack the CSI motif region or lack residues known to be required for the CSI-CP interaction (Zwolak et al., 2010b). Shaded residues are identical to the consensus sequences. (B) Unrooted phylogenetic tree showing the relationships among CARMIL proteins, revealing five groups of CARMIL genes. Vertebrate genomes have three genes that encode three conserved isoforms—CARMIL1, CARMIL2, and CARMIL3—and invertebrates have a single CARMIL gene and isoform. Invertebrate CARMILs can be further classified into those that contain a CSI motif and those that do not.

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  FIGURE 2:. Principal component analysis (PCA) of CP. Top, PCA of molecular dynamics simulations of the structural conformations of CP. The simulations were begun with CP that was either free (gray contour shading), bound to F-actin (blue dots and contour lines), or bound to CARMIL (red dots and contour lines). The results were projected onto the same conformational space; existing CP structures were overlaid as green dots. Based on this analysis, CARMIL-bound CP exists in a conformational space more like that of unbound CP (gray) than to that of F-actin–bound CP. Bottom, overlay of protein structures, depicting the conformations that correspond to the peaks of CARMIL-bound and F-actin bound CP. This analysis supports the findings that CARMIL allosterically inhibits CP by eliciting changes to the actin-binding surface, holding CP in a conformation similar to the free state. Used with permission from Kim et al. (2012).

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  FIGURE 3:. Models for CARMIL1 and CARMIL2 function within cells. (A) CARMIL1 is transported to the membrane via myosin-IE. CARMIL1 interacts with the membrane via PH domain and MBD. At the membrane, CARMIL1 recruits CP and relieves it from inhibition by V-1. Released V-1 then activates NF-κB signaling. CARMIL1 also activates Trio, which leads to an increase in Rac1 activation and Arp2/3-mediated actin assembly. (B) CARMIL2 is transported to the membrane via an interaction with vimentin filaments, where it then associates with the membrane via its MBD. In migrating cells, the CARMIL2 model is similar to CARMIL1 in that the CBR interacts with CP bound by V-1. This activates CP, allowing for barbed-end capping. (C) During CD28 costimulation to activate T-cells, membrane-localized CARMIL2 interacts with the adaptor protein GRB2, leading to downstream activation of CARMA1, PKCθ, and NF-κB activation.