Mammalian Actin-binding Protein-1/Hip-55 Interacts with FHL2 and Negatively Regulates Cell Invasion

Abstract

Mammalian actin-binding protein-1 (mAbp1) is an adaptor protein that binds actin and modulates scission during endocytosis. Recent studies suggest that mAbp1 impairs cell invasion; however, the mechanism for the inhibitory effects of mAbp1 remain unclear. We performed a yeast two-hybrid screen and identified the adaptor protein, FHL2, as a novel binding partner that interacts with the N-terminal actin depolymerizing factor homology domain (ADFH) domain of mAbp1. Here we report that depletion of mAbp1 or ectopic expression of the ADFH domain of mAbp1 increased Rho GTPase signaling and breast cancer cell invasion. Moreover, cell invasion induced by the ADFH domain of mAbp1 required the expression of FHL2. Taken together, our findings show that mAbp1 and FHL2 are novel binding partners that differentially regulate Rho GTPase signaling and MTLn3 breast cancer cell invasion.Invasive migration of cancer cells is dependent on a dynamic actin cytoskeleton (1). Key regulators of the actin cytoskeleton include actin-binding proteins like cortactin, which have been implicated in invadopodia and podosome formation and invasive cell migration. Mammalian actin-binding protein-1 (mAbp1, 2 Hip-55, DBNL) has significant structural homology to cortactin and binds actin with an N-terminal ADFH domain (2). Both mAbp1 and cortactin have identified roles during endocytosis and vesicle trafficking (3-5), and both proteins localize to lamellipodia, podosomes, and dorsal ruffles (4, 6, 7). However, in contrast to cortactin, mAbp1 impairs podosome formation and invasive migration of Src-transformed fibroblasts (8).To determine how mAbp1 regulates cell invasion, we performed a yeast two-hybrid screen and identified the fourand-a-half LIM domain protein 2 (FHL2) as a novel mAbp1-binding partner that does not interact with cortactin. FHL2 (also known as DRAL or SLIM3) is a LIM domain protein consisting of four-and-a-half LIM domains. Each LIM domain contains two zinc finger loops that mediate proteinprotein interactions. FHL2 interacts with Ͼ50 proteins (9) including integrins and focal adhesion kinase (FAK) (10, 11), actin (12), and the transcription factors estrogen receptor (ER), activator protein-1 (AP-1), and ␤-catenin (13-18). Interestingly, FHL2 is expressed at low levels in many tissues like breast, placenta, uterus, and the lungs (17, 19) and is up-regulated in cancer. For example, FHL2 expression is increased in many cancers including breast (20, 21), ovarian (22), prostate (23), lung (24), colon (25), brain (26), and skin cancers (27). In primary breast tumors, patients with high levels of FHL2 have a poorer survival rate (21), suggesting that FHL2 may be involved in cancer progression. Furthermore, overexpression of FHL2 in MDA-MB-231 human breast cancer cells enhances colony formation in soft agar, whereas knockdown reduces the ability to form colonies (20).Here we report a novel interaction between the N-terminal ADFH domain of mAbp1 and FHL2 that modulates cell invasion. In the yeast, but not the mammalian form, the N terminu...

Four and a half domain 2 (FHL2) scaffolding protein is a marker of connective tissues of developing digits and regulates fibrogenic differentiation of limb mesodermal progenitors

“…In addition to the transcriptional impact of FHL2 in the expression of fibrous tissue markers, the association of FHL2 with the actin cytoskeleton and findings of gain‐of‐function experiments revealed a role of FHL2 in the organization of the actin cytoskeleton in skeletal progenitors, which is involved in the transport of secreted matrix and cell dynamics. This finding is consistent with the demonstrated functional interaction of FHL2 with Rho signalling molecules (Müller et al, ) and with its ability to bind actin (Coghill et al, ) and actin‐binding proteins (Boateng, Bennin, De Oliveira, & Huttenlocher, ).…”

The role of FHL2 in wound healing and inflammation

“…The granulation tissue of skin wounds expressed less a-SMA in the absence of FHL2 because FHL2 up-regulates, via coactivation of serum response factor (SRF), the expression of a-SMA (9,44). Furthermore, via interaction with actin-and myosinbinding proteins (71,72), FHL2 localizes along actin stress fibers, which are additionally armed by incorporated a-SMA, and supports myofibroblast contractility.…”

Drebrin-like protein regulates body bending ofC. elegansvia suppression of NCA cation leak channels

“…Recently, Boateng and colleagues discovered that the mammalian drebrin-like protein mAbp1 counteracts Rho GTP-ase in breast cancer cells 24 . Our data clearly indicate that an analogous mechanism is at work in C. elegans, because both the RhoGEF suppression and the pan-neuronal expression of the dominant negative RHO-1 decreased the body curvature and the movement velocity of the dbn-1(vit7) mutant.…”

“…Mutations of players downstream of Gq, namely RhoGEF, NCA channels and NCA channel accessory subunits (UNC-79, UNC-80 and NLF-1) have been shown to suppress the effect of activated RHO-1 34,17 . Second, depletion of mammalian drebrin-like protein mAbp1 or ectopic expression of its ADF-H domain increase Rho GTPase signaling in human breast cancer cells 24 . Therefore, it is reasonable to assume that the dbn-1(vit7) mutation in C. elegans might over-activate RHO-1, leading to the observed hyperbending locomotion.…”

“…Drebrin-like protein (known as mAbp1, HIP-55, SH3P7, DBNL, and DBN-1 in C. elegans) is an adaptor protein that mediates between the actin cytoskeleton and proteins of different intracellular pathways 20,21,22,23,24 . Drebrin-like protein is, for example, an important component of the endocytic machinery.…”