FHL3 Is an Actin-binding Protein That Regulates α-Actinin-mediated Actin Bundling

Abstract

Four and a half LIM domain (FHL) proteins are members of the LIM protein superfamily. Several FHL proteins function as co-activators of CREM/CREB transcription factors and the androgen receptor. FHL3 is highly expressed in skeletal muscle, but its function is unknown. FHL3 localized to the nucleus in C2C12 myoblasts and, following integrin engagement, exited the nucleus and localized to actin stress fibers and focal adhesions. In mature skeletal muscle FHL3 was found at the Z-line. Actin was identified as a potential FHL3 binding partner in yeast two-hybrid screening of a skeletal muscle library. FHL3 complexed with actin both in vitro and in vivo as shown by glutathione S-transferase pulldown assays and co-immunoprecipitation of recombinant and endogenous proteins. FHL3 promoted cell spreading and when overexpressed in spread C2C12 cells disrupted actin stress fibers. Increased FHL3 expression was detected in highly motile cells migrating into an artificial wound, compared with non-motile cells. The molecular mechanism by which FHL3 induced actin stress fiber disassembly was demonstrated by low speed actin co-sedimentation assays and electron microscopy. FHL3 inhibited ␣-actinin-mediated actin bundling. These studies reveal FHL3 as a significant regulator of actin cytoskeletal dynamics in skeletal myoblasts.The LIM superfamily of proteins is defined by the presence of one or more LIM domains, which represent a cysteinerich double zinc finger motif denoted by the sequence (CX 2 -CX 17-19 HX 2 C)X 2 (CX 2 CX 16 -20 CX 2 (H/D/C)) (1). The LIM zinc finger does not interact with DNA but functions as a proteinprotein binding module (2, 3). LIM proteins localize to the nucleus and scaffold the assembly of transcription factors and thereby regulate transcription. In the cytoplasm LIM proteins facilitate the complex association of signaling proteins with the actin cytoskeleton (3).A subset of LIM proteins are the LIM-only proteins, which comprise solely multiple copies of LIM domains. The family of four and a half LIM domain (FHL) 1 proteins all contain a single N-terminal LIM-type zinc finger followed by four sequential LIM domains and no other modular domains (4). This group of proteins includes FHL1 (also called SLIM1) and its alternatively spliced isoforms SLIMMER and KyoT2, FHL2 (also called DRAL for "down-regulated in rhabdomyosarcoma" or SLIM3), FHL3 (also called SLIM2), FHL4, and ACT. FHL1, FHL2, and FHL3 are highly expressed in striated muscle, whereas ACT is expressed only in the testis (4 -12).FHL1 is strongly implicated in the pathogenesis of human cardiomyopathy. Microarray analysis has demonstrated FHL1 mRNA levels are decreased in failing human hearts with dilated cardiomyopathy and significantly increased in hypertrophic cardiomyopathy (13,14). Mouse models of hypertrophic cardiomyopathy induced by transverse aortic constriction also show elevated expression of FHL1, suggesting FHL1 may play a significant role in regulating the heart muscle cytoarchitecture (15). In skeletal myoblasts FHL1 localizes in...

The LIM-only Proteins FHL2 and FHL3 Interact with α- and β-Subunits of the Muscle α7β1 Integrin Receptor

“…Confocal microscopy analysis of FHL2 in cardiac and FHL3 in skeletal muscle sections (tibialis anterior) showed a costameric distribution pattern for both FHL proteins and for ␣ 7 ␤ 1 integrin (Fig. 2, C and D), which reflects the already published distribution of these proteins in Z-lines (20,40,42). Whereas FHL2 and FHL3 were mostly located at Z-discs, the ␣ 7 ␤ 1 receptor showed the highest distribution at the lateral plasmalemma of muscle fibers, exactly were FHL and integrin molecules colocalize (Fig.…”

“…A dual binding of FHL2 and FHL3 to both ␣-and ␤-integrin chains might provide a stronger connection of the receptor to the cell interior than only a single interaction. A binding of FHL2 and FHL3 to actin molecules has recently been published (42), which would suggest that the LIM-only proteins directly link the cytoskeleton to the ECM via binding to integrins and actin. In contrast, our yeast two-hybrid experiments with monomeric ␣ or ␤ G-actin molecules failed to confirm this observation.…”

Dephosphorylation-dependent Inhibitory Activity of Juxtanodin on Filamentous Actin Disassembly

“…3C and 4E). Some F-actin-binding proteins stabilize actin cytoskeleton by bundling pre-existing actin filaments, such as Fascin and ␣-actinin (36,37). However, as mentioned above, JN has no F-actin bundling activity.…”

SLIMMER (FHL1B/KyoT3) Interacts with the Proapoptotic Protein Siva-1 (CD27BP) and Delays Skeletal Myoblast Apoptosis

“…Protein concentration was determined using a Bio-Rad DC protein assay kit, and lysates were analyzed by SDS-PAGE and Western blotting. Frozen longitudinal and transverse sections of mouse gastrocnemius muscle were prepared, fixed, stained, and viewed as described (17,26,29).…”