Analysis of the role of the leucine zipper motif in regulating the ability of AFAP‐110 to alter actin filament integrity

Abstract

AFAP-110 has an intrinsic ability to alter actin filament integrity as an actin filament crosslinking protein. This capability is regulated by a carboxy terminal leucine zipper (Lzip) motif. The Lzip motif facilitates self-association stabilizing the AFAP-110 multimers. Deletion of the Lzip motif (AFAP-110(Deltalzip)) reduces the stability of the AFAP-110 multimer and concomitantly increases its ability to crosslink actin filaments, in vitro, and to activate cSrc and alter actin filament integrity, in vivo. We sought to determine how the Lzip motif regulates AFAP-110 function. Substitution of the c-Fos Lzip motif in place of the AFAP-110 Lzip motif (AFAP-110(fos)) was predicted to preserve the alpha-helical structure while changing the sequence. To alter the structure of the alpha-helix, a leucine to proline mutation was generated in the AFAP-110 alpha-helical Lzip motif (AFAP-110(581P)), which largely preserved the sequence. The helix mutants, AFAP-110(Deltalzip), AFAP-110(fos), and AFAP-110(581P), demonstrated reduced multimer stability with an increased capacity to crosslink actin filaments, in vitro, relative to AFAP-110. An analysis of opposing binding sites indicated that the carboxy terminus/Lzip motif can contact sequences within the amino terminal pleckstrin homology (PH1) domain indicating an auto-inhibitory mechanism for regulating multimer stability and actin filament crosslinking. In vivo, only AFAP-110(Deltalzip) and AFAP-110(581P) were to activate cSrc and to alter cellular actin filament integrity. These data indicate that the intrinsic ability of AFAP-110 to crosslink actin filaments is dependent upon both the sequence and structure of the Lzip motif, while the ability of the Lzip motif to regulate AFAP-110-directed activation of cSrc and changes in actin filament integrity in vivo is dependent upon the structure or presence of the Lzip motif. We hypothesize that the intrinsic ability of AFAP-110 to crosslink actin filaments or activate cSrc are distinct functions.

Identification of AFAP1L1 as a prognostic marker for spindle cell sarcomas

“…AFAP-110 contains several protein-binding motifs at its amino terminus and functions as an adapter protein for actin filaments (Qian et al, 2000;Baisden et al, 2001b;Qian et al, 2004). It is also required to control protein kinase Ca-mediated activation of c-Src and the subsequent formation of podosomes .…”

XB130: A novel adaptor protein in cancer signal transduction

“…The carboxy terminal actin filament-binding domain and the Lzip of AFAP1 enable it to crosslink actin filaments (37). Following activation by the serine/threonine kinase PKCα, AFAP1 is directed to perinuclear region of the cell with cSrc and activates cSrc by binding to the SH3 domain via its N-terminal SH3-binding motif, thus leading to the cSrc downstream activation that affects cell adhesion, invasion and motility (25).…”

Phosphorylation of AFAP-110 affects podosome lifespan in A7r5 cells

“…Actin filament-associated protein 1 (AFAP1; also known and hereafter referred to as actin-filament-associated protein of 110 kDa, AFAP-110) belongs to the functional group of actin-binding proteins that regulate actin-filament crosslinking through its ability to undergo dynamic changes in multimerization (Qian et al, 2004). AFAP-110 is composed of a functional actin-binding domain (Qian et al, 2000) and several protein-binding motifs, including two SH2 and one SH3 (Guappone and Flynn, 1997) binding motifs, two pleckstrin homology (PH) domains and a leucine-zipper (LZ) motif (Qian et al, 1998;Qian et al, 2004).…”

“…AFAP-110 is composed of a functional actin-binding domain (Qian et al, 2000) and several protein-binding motifs, including two SH2 and one SH3 (Guappone and Flynn, 1997) binding motifs, two pleckstrin homology (PH) domains and a leucine-zipper (LZ) motif (Qian et al, 1998;Qian et al, 2004). In quiescent cells AFAP-110 colocalizes to actin stress filaments (Flynn et al, 1993).…”

“…Mutation of the proline residue at position 71 to Ala (P71A) within the SH3-binding motif abolishes Src binding to AFAP-110 and prevents AFAP-110 from directing Src activation. The N-terminal PH domain (PH1) was found to bind to the protein kinase C (PKC) α, β, γ and λ isoforms and, upon PKCα activation, AFAP-110 subsequently becomes phosphorylated on Ser/Thr residues (Qian et al, 2002;Qian et al, 2004). Phosphorylation of AFAP-110 will induce a conformational change that releases autoinhibitory intramolecular interactions and correlates with an acquired ability to activate Src, alter actin filament crosslinking and induce podosome formation .…”