This report describes characterization of human palladin, a novel widely expressed component of the actin-containing cytoskeleton. The protein is localized along microfilaments of smooth muscle, epithelial, and glial cells in a periodic manner that is typical for the components of dense bodies of smooth muscle and dense regions in stress fibers. Human palladin and the mouse ortholog (Parast and Otey, 2000
) demonstrate many similar features. Both proteins are expressed in a variety of cell types, and they are localized in a similar periodical punctate pattern along actin filaments. Both mouse and human palladin may be expressed as several isoforms, possibly dependent on the cell type. In both species, at least with the tested antibodies, the major immunoreactive bands migrate at ~95 and 140 kDa.
Palladin contains three Ig-domains and is thus a new member of a family of cytoplasmic proteins with these structural modules. All of the family members are components of the cytoskeleton and most of them associate with sarcomeric myosin. A notable exception is the actin-associated protein myotilin, which shares highest homology with palladin. The Ig-domains may provide rigidity for the proteins and function as a ruler separating structural components at a proper distance (Puius et al., 1998
), but they also provide elasticity for the titin molecule (Linke et al., 1998
; Trombitas et al., 1998
; Witt et al., 1998
). Ig-domains also serve as sites for intermolecular interactions. For instance, in myotilin the Ig-domains serve as a dimerization interface (Storbjörk, Salmikangas, and Carpén, unpublished results), the Ig-domains Z1-Z2 of titin bind to T-cap protein (Gregorio et al., 1998
), and the Ig-domains of MyB-C interact with the A-band super-repeats of titin (Okagaki et al., 1993
Palladin contains two polyproline stretches, the first of which has a consensus binding site for the EVH1 domain present in the Ena/VASP/WASP family of proteins. This FPPPP peptide sequence is found in proteins such as ActA, zyxin, and vinculin that all bind tightly to the EVH1 domain (Prehoda et al., 1999
). The recognition of FPPPP by the EVH1 domain targets Ena/VASP/WASP family members to sites of cytoskeletal remodeling (Gertler et al., 1996
; Symons et al., 1996
). After correct localization, other regions of Ena/VASP/WASP proteins can bind to profilin and Arp2/3 proteins that directly promote actin polymerization. Thus, the structural characteristics suggest that palladin may be involved in the organization of the actin cytoskeleton.
Sequence comparison indicates that palladin is most homologous to myotilin. The homology is not restricted to the Ig-domains, but is extended to the N-terminal sequence. The N-terminal region, which in myotilin is responsible for interaction with α-actinin, contains sequence unique for these two molecules, and thus myotilin and palladin appear to form a subfamily within the Ig-domain–containing cytoskeletal proteins. The subcellular localization in Z-lines and an association with limb girdle muscular dystrophy 1A, a disease characterized ultrastructurally by extensive Z-line streaming, suggest a role for myotilin in the organization of actin-containing thin filaments of the sarcomere (Salmikangas et al., 1999
; Hauser et al., 2000
). It will be interesting to determine whether palladin serves an analogous function in smooth muscle and nonmuscle cells. Further functional similarity between myotilin and palladin is suggested by the fact that both myotilin and mouse palladin associate with α-actinin (Salmikangas et al., 1999
; Parast and Otey, 2000
In addition to the similarities, there are notable differences between myotilin and palladin. mRNA and protein studies indicate that expression of the two proteins is differentially regulated. The expression of myotilin in adult tissues is very restricted; it is mainly seen in striated and cardiac muscle (Salmikangas et al., 1999
). On the other hand, relatively strong palladin expression is seen in a variety of epithelial and mesenchymal tissues, including smooth muscle but, in comparison with myotilin, the expression level in skeletal muscle is low. This notion is also supported by the EST database information. Currently, >400 human palladin cDNAs are available. Only three of them are from skeletal muscle libraries, whereas >90% of the >40 EST myotilin cDNAs are from skeletal muscle, heart, or fetal libraries. A second difference between palladin and myotilin is that myotilin lacks the two polyproline sequences implicated in modulation of actin polymerization. This structural difference may be related to the fact that the organization of sarcomeric actin is strictly regulated, whereas in other cell types that express palladin, dynamic modulation of actin filaments by polymerization/depolymerization is a continuous process.
In vitro induced differentiation of peripheral blood monocytes into dendritic cells provides a model to study the correlation between changes in cell morphology and cytoskeletal elements. A detailed understanding of the events that control the ultrastructural alterations is still lacking, but apparently, alterations in the expression of cytoskeletal components play an important role. Previous studies have demonstrated that neoexpression of fascin, an actin bundling protein, occurs during dendritic cell differentiation (Mosialos et al., 1996
; Ross et al., 1998
). Our results show that the expression of palladin is also up-regulated during the maturation process. In immature cells, palladin is localized in podosomes, dynamic actin-containing adhesion structures that are regulated by WASP (Linder et al., 1999
), and in mature dendritic cells, along the delicate actin filaments. The regulated expression and subcellular localization raise the possibility that palladin is involved in the control of morphological and cytoskeletal changes associated with dendritic cell maturation. Experiments with the use of fibroblasts and Rcho-1 trophoblast cells indicate a role in cytoskeletal organization for palladin also in other cell types. In those cells, antisense treatment specifically suppresses palladin expression and concomitantly leads to disruption of stress fibers and rounding of treated cells (Parast and Otey, 2000
Our results suggest that in palladin, Ig-domains 2–3 are responsible for the interaction with ezrin, and that they contain binding sites for at least two additional, yet unknown proteins. The fact that palladin does not interact with native full-length ezrin indicates that activation of ezrin is required for the interaction. Native ezrin molecules are in a dormant state due to intramolecular binding of the N-terminal and C-terminal association domain (Gary and Bretscher, 1993
). Activation via phosphorylation and/or phosphatidylinositol bisphosphate binding disrupts the intramolecular association and unmasks binding sites for actin and several other molecules. Deletion constructs, including those used in these experiments, mimic the activated form of ezrin (Grönholm et al., 1999
; Mangeat et al., 1999
). Other characterized binding partners for ezrin include cell surface adhesion molecules, which bind to the N-terminal domain, molecules involved in cell signaling, and cytoskeletal components (Vaheri et al., 1997
; Bretscher, 1999
; Mangeat et al., 1999
). The cytoskeletal components include actin, other ERM proteins, tubulin and, based on these studies, palladin. Although a variety of binding partners for ezrin has been revealed, only one of them, the RIIα subunit of protein kinase A (Dransfield et al., 1997
) is known to interact with the α-helical region.
The in vivo significance of the interaction between palladin and ezrin requires further studies. It is probable that the interaction occurs only in specialized cell types, such as smooth muscle cells, in which the two proteins colocalize. Palladin is associated with actin fibers in all cell types studied, whereas ezrin in epithelial, glial, and most other cell types is a component of the cortical actin cytoskeleton. Although the C-terminal constructs of ezrin and radixin have been shown to localize to stress fibers (Algrain et al., 1993
; Henry et al., 1995
), such localization has not been previously demonstrated for full-length ezrin. There are several possibilities for the unexpected localization of ezrin in smooth muscle cells. We regard major modification of ezrin as an unlikely explanation, because no differences were detected in the mobility of immunoreactive ezrin from HISM cells and other cell types. Other possibilities include the lack of relevant binding partners at the cell membrane of smooth muscle cells, differences in the signaling activity in smooth muscle and epithelial cells, which would affect the subcellular localization of ezrin, or differences in the cytoskeletal composition between smooth muscle cells and epithelial cells. Among various tissues, palladin expression is especially high in smooth muscle. It remains to be seen whether the high expression of palladin in these cells is involved in the microfilament association of ezrin.
From a functional perspective, the coexistence of palladin and ezrin in smooth muscle cytoskeleton is intriguing. Smooth muscle contractions are responsible for many vital functions of the body, including bowel movement and control of blood pressure. The contractile system is regulated by the Rho family of small GTP-binding proteins and by VASP, which coordinates the assembly of smooth muscle acto-myosin filaments, and is a major target for inhibitory vasoactive agents that regulate vessel wall tension and blood pressure. Ezrin and other ERM proteins are known to function as upstream and downstream effectors of Rho activity (Matsui et al., 1998
; Maekawa et al., 1999
). Interestingly, palladin contains the FPPPP peptide consensus sequence, which serves as the binding site for EVH1 domain in Ena/VASP/WASP protein family (Prehoda et al., 1999
). In vascular smooth muscle cells, VASP localizes in proximity of microfilaments and dense bodies (Markert et al., 1996
), and it may thus interact with palladin. If such an interaction indeed takes place, the ezrin–palladin complex could bring together and coordinate two important signaling pathways, i.e., the Rho-pathway and the VASP-mediated control of the acto-myosin system.