Involvement of palladin and α-actinin in targeting of the Abl/Arg kinase adaptor ArgBP2 to the actin cytoskeleton

Abstract

Palladin and α-actinin are major components of stress fiber dense bodies, cardiomyocyte Z-discs and neuronal synapses. They function as structural molecules and cytoskeletal regulators but also as docking sites to other proteins. Both antisense and transient overexpression experiments have shown that palladin plays an important role in the regulation of actin cytoskeleton. ArgBP2 is a multi-domain scaffolding protein which shares both the tissue distribution and subcellular localization with palladin. ArgBP2 is directly linked to intracellular signaling cascades by its interaction with Abl family kinases, Pyk2 and the ubiquitin ligase Cbl. It has several actin associated binding partners and has been shown to regulate cytoskeletal dynamics. Here, we show by in vivo and in vitro methods that palladin's amino-terminal poly-proline sequences directly interact with the first carboxy-terminal SH3 domain of ArgBP2. We further demonstrate a direct interaction between α-actinin and the amino-terminal segment of ArgBP2. Immunoprecipitation and targeting assays suggest that a three-way complex of the proteins occurs in vivo. The interactions provide an explanation to the previously observed Z-disc-specific localization of ArgBP2 and indicate interplay between signaling adaptors and structural proteins of the Z-disc.

Introduction

The striated muscle Z-disc is a complex protein network that stably crosslinks actin filaments to mediate force transmission along serially aligned sarcomeres [1]. A major component of the Z-disc is α-actinin, a ubiquitously expressed actin crosslinking protein [2]. The general structure of α-actinin is composed of an N-terminal actin-binding domain (ABD), a central rod containing four spectrin-like repeats and a calmodulin-like domain at the C-terminus [3], [4]. Many of the characterized Z-disc components bind α-actinin. These include α-actinin-associated LIM protein (ALP), FATZ (calsarcin-2/myozenin), ZASP, cypher, myotilin, palladin, myopalladin, actin and the giant protein titin [2]. The Z-disc is laterally coupled to extracellular matrix via a specialized protein network at the costameres [5]. Adjacent cardiomyocytes are also connected longitudinally by intercellular contacts to ensure mechanical and electrochemical coupling. This function is mediated by yet another specialized protein network, the intercalated disc, which contains three types of cell–cell contacts, namely adherens junctions, desmosomes and gap junctions [6]. The molecular composition and function of costameres and intercalated discs differ considerably, but some proteins like vinculin are found in both types of structures.

In addition to α-actinin, members of the palladin–myotilin–myopalladin family are important structural elements of the sarcomere Z-discs. The three proteins are characterized by conserved IgI type domains in the C-terminal part of the protein [7], [8], [9], [10]. Although the family members show structural similarity, there are also important differences. mRNA and protein studies have suggested the presence of several palladin isoforms due to more extensive alternative splicing as compared to myopalladin and myotilin. Furthermore, the N-terminal sequence of the major palladin isoform (PP isoform) has two proline-rich regions, which serve as binding sites for Ena/VASP proteins and profilin [11]. The proline-rich sequences are also known to act as ligands for SH3 domains that recognize the PXXP motif. SH3 domain-containing proteins are often involved in intracellular signaling [12]. Myotilin and myopalladin are mainly expressed in striated muscle, whereas palladin is more widely expressed both in mesenchymal and epithelial cells [2], [8]. Both myotilin and myopalladin play an important role in maintaining the integrity of the sarcomere. Myotilin mutations lead to two forms of muscle disorders, limb-girdle muscular dystrophy 1A (LGMD1A) and myofibrillar myopathy (also termed desmin-related myopathy/desmin storage disease) [13], [14], [15]. All members of the protein family directly interact with α-actinin [10], [16], [17]. Myotilin binds also actin and filamin-C [18], myopalladin interacts with nebulin and CARP [10], and palladin with the ERM-family protein ezrin [8], VASP and profilin [11], [46]. In cardiomyocytes, palladin localizes to Z-discs and in non-muscle cells to stress fiber dense bodies, the non-muscle equivalents of the Z-discs [19] and in focal adhesions. Thus, α-actinin and palladin have a highly overlapping distribution both in striated muscle and in non-muscle cells.

The Z-disc is not only an important structural element of the sarcomere, it also plays a role in (cardio)myocyte signaling [20]. To perform this function, components of different signaling pathways need to be located in the Z-disc, either transitionally or stably. Among such proteins is ArgBP2, a member of a cytoskeleton associated adaptor protein family which also includes vinexin and CAP/ponsin [21], [22]. All members of the family have an N-terminal SoHo domain and three C-terminal SH3 domains (SH3-1, SH3-2 and SH3-3). These proteins are ubiquitously expressed, and ArgBP2 is especially abundant in cardiac muscle. All three proteins localize to focal adhesions, but ArgBP2 is also prominent in stress fiber dense bodies and sarcomeric Z-discs [21]. There is accumulating evidence that the protein family members could regulate cytoskeletal organization and intracellular signaling. This is highlighted by the variety of protein–protein interactions characterized for the individual members of the protein family. Vinexin interacts with vinculin, Sos and flotillin, and CAP/Ponsin binds to vinculin, Grb4, ataxin 7, c-Cbl and the insulin receptor. ArgBP2 was first identified as an Arg (Abelson related gene, Abl2) interacting protein, but it also interacts with other proteins mainly involved in signaling pathways [22], [23]. None of the previously described partners can, however, fully explain the targeting of ArgBP2 to stress fiber dense bodies and cardiomyocyte Z-discs. In this study, we show that both the N-terminal part and the SH3 domains of ArgBP2 contain information sufficient for Z-disc targeting. Furthermore, we identify Z-disc associated partners for each region, α-actinin mediating binding to N-terminus and palladin to the first SH3 domain.