The actin filament-associated protein AFAP-110 is an adaptor protein that modulates changes in actin filament integrity.

The actin filament-associated protein of 110 kDa (AFAP-110) was first identified as an SH3/SH2 binding partner for the nonreceptor tyrosine kinase, Src. Subsequent data have demonstrated that AFAP-110 can interact with other Src family members. AFAP-110 contains additional protein binding modules including two pleckstrin homology domains, a leucine zipper motif and a target sequence for serine/threonine phosphorylation. AFAP-110 interacts with actin filaments directly via a carboxy terminal actin-binding domain. Thus AFAP-110 may function as an adaptor protein by linking Src family members and/or other signaling proteins to actin filaments. AFAP-110 also has an intrinsic capability to alter actin filament integrity that can be revealed upon conformational changes associated with phosphorylation or mutagenesis. Recent data has indicated that AFAP-110 may also serve to activate cSrc in response to this conformational change as well. Thus, AFAP-110 may function in several ways by (1) acting as an adaptor protein that links signaling molecules to actin filaments, (2) serving as a platform for the construction of larger signaling complexes, (3) serving as an activator of Src family kinases in response to cellular signals that alter its conformation and (4) directly effecting actin filament organization as an actin filament cross-linking protein. Here, we will review the structure and function of AFAP-110 as well as potential binding partners and effectors of AFAP-110's ability to alter actin filament integrity.

The intrinsic ability of AFAP-110 to alter actin filament integrity is linked with its ability to also activate cellular tyrosine kinases.

The actin filament-associated protein of 110 kDa (AFAP-110) is a Src binding partner that represents a potential modulator of actin filament integrity in response to cellular signals. Previous reports have demonstrated that AFAP-110 is capable of directly binding and altering actin filaments. Deletion of the leucine zipper motif of AFAP-110 (AFAP-110(Deltalzip)) has been shown to induce a phenotype which resembles Src-transformed cells, by repositioning actin filaments into rosettes. This deletion also mimics a conformational change in AFAP-110 that is detected in Src-transformed cells. The results presented here indicate that unlike AFAP-110, AFAP-110(Deltalzip) is capable of activating cellular tyrosine kinases, including Src family members, and that AFAP-110(Deltalzip) itself is hyperphosphorylated. The newly tyrosine phosphorylated proteins and activated Src-family members appear to be associated with actin-rich lamellipodia. A point mutation that alters the SH3-binding motif of AFAP-110(Deltalzip) prevents it from activating tyrosine kinases and altering actin filament integrity. In addition, a deletion within a pleckstrin homology (PH) domain of AFAP-110(Deltalzip) will also revert its effects upon actin filaments. Lastly, dominant-positive RhoA(V14) will block the ability of AFAP-110(Deltalzip) from inducing actin filament rosettes, but does not inhibit Src activation. Thus, conformational changes in AFAP-110 enable it to activate cellular kinases in a mechanism requiring SH3 and/or PH domain interactions. We hypothesize that cellular signals which alter AFAP-110 conformation, enable it to activate cellular kinases such as cSrc, which then direct changes in actin filament integrity in a Rho-dependent fashion.

Src can regulate carboxy terminal interactions with AFAP-110, which influence self-association, cell localization and actin filament integrity.

The SH2 and SH3 binding partner AFAP-110 is a tyrosine phosphorylated substrate of Src. AFAP-110 has been hypothesized to link Src to actin filaments, which may contribute to the effects of Src upon actin filament integrity. However, it has been unclear what effect activated Src (Src527F) has upon AFAP-110 structure or function and whether AFAP-110 plays a role in actin filament integrity. We report here that the carboxy terminal 127 amino acids of AFAP-110 are comprised of an alpha-helical region that contains a leucine zipper motif. This indicated the potential of AFAP-110 to self-associate. Expression of the carboxy terminus as a fusion protein (GST-cterm) will permit affinity absorption of cellular AFAP-110. The integrity of the alpha-helical leucine zipper motif in GST-cterm is required for affinity absorption, but binding is not due to a classical leucine zipper interaction. Co-expression of Src527F, unlike cSrc, will abrogate affinity absorption of AFAP-110 with GST-cterm. These data indicate that Src527F has affected a change in the carboxy terminal structure that renders AFAP-110 unavailable for affinity absorption. Superose chromatography demonstrate that AFAP-110 will fractionate as a monomer or multimer, indicating AFAP-110 can be detected in a self-associated form in cell lysates. Co-expression of Src527F resulted in AFAP-110 fractionating with a molecular weight that predicts only a multimeric population. Deletional mutagenesis also indicate a biological role for the carboxy terminus in cellular localization and actin filament integrity. Deletion of the entire carboxy terminal alpha-helix (84 amino acids) will not permit AFAP-110 to efficiently colocalize with actin filaments or the cell membrane. Deletion of only the leucine zipper region of the carboxy terminal alpha-helix (44 amino acids) from AFAP-110 (AFAPAdeltazip) demonstrate that both AFAPdeltalzip and actin filaments are repositioned into rosette-like structures, similar to the effects of Src527F, while co-expression of AFAP-110 with cSrc will not affect actin filaments. These data indicate that AFAP-110 can play an important role in modulating actin filament integrity through carboxy terminal interactions that can be affected by Src527F.

Monoclonal antibodies directed against AFAP-110 recognize species-specific and conserved epitopes.

The actin filament-associated protein, AFAP-110, is a Src SH2/SH3 binding partner that can modulate changes in actin filament structure. AFAP-110 contains a carboxy terminal motif that facilitates actin filament interactions, as well as amino terminal protein binding motifs, including an SH3 binding motif, two SH2 binding motifs, and two Pleckstrin homology domains. Two monoclonal antibodies (MAbs) were developed that recognized epitopes in either the amino terminus (MAb 4C3) or the carboxy terminus (anti-AFAP-110) of AFAP-110. Site-directed mutations that change key proline residues to alanine in the SH3 binding motif and an adjacent proline-rich motif abrogated MAb 4C3 binding. These same mutations have been shown to prevent SH3 interactions between AFAP-110 and Src527F. These data indicate that MAb 4C3 recognizes an epitope that is part of the SH3 binding motif. Interestingly, MAb 4C3 is not efficiently reactive with mammalian homologs of AFAP-110. Sequence analysis of a putative cDNA clone that encodes the amino terminus of the human AFAP-110 isoform predicted a one amino acid difference within this epitope, indicating a mechanism for species-specific binding by MAb 4C3. A second, MAb anti-AFAP-110, recognizes AFAP-110 across species and binds to an epitope within the carboxy terminus. This epitope includes the 5th heptad repeat of the carboxy terminal, leucine zipper motif (amino acids 592-598)--a motif that facilitates self-associations and may regulate the function of AFAP-110. These MAbs will be useful for analyzing the effects of AFAP-110 upon cell morphology and actin filament integrity. In addition, the avian-specific MAb 4C3 may be useful for studying the effects of avian AFAP-110 constructs expressed in mammalian cells, by providing an internal epitope tag.

The carboxy terminus of AFAP-110 modulates direct interactions with actin filaments and regulates its ability to alter actin filament integrity and induce lamellipodia formation.

The actin filament-associated protein AFAP-110 is an SH2/SH3 binding partner for Src. AFAP-110 contains several protein-binding motifs in its amino terminus and has been hypothesized to function as an adaptor molecule that could link signaling proteins to actin filaments. Recent studies using deletional mutagenesis demonstrated that AFAP-110 can alter actin filament integrity in SV40 transformed Cos-1 cells. Thus, AFAP-110 may be positioned to modulate the effects of Src upon actin filaments. In this report, we sought to determine whether (a) AFAP-110 could interact with actin filaments directly and (b) deletion mutants could affect actin filament integrity and cell shape in untransformed fibroblast cells. The data demonstrate that the carboxy terminus of AFAP-110 is both necessary and sufficient for actin filament association, in vivo and in vitro. Analysis of the carboxy terminus revealed a mean 40% similarity with other known actin-binding motifs, indicating a mechanism for binding to actin filaments. AFAP-110 can also induce lamellipodia formation. Contiguous with the alpha-helical, actin-binding motif is an alpha-helical, leucine zipper motif. Deletion of the leucine zipper motif (AFAP(Deltalzip)) followed by cellular expression enabled AFAP(Deltalzip) to alter actin filament integrity and cell shape in untransformed cells as evidenced by the induction of lamellipodia formation. We hypothesize that AFAP-110 may be an important signaling protein that can directly modulate changes in actin filament integrity and induce lamellipodia formation.