Palladin Is an Actin Cross-linking Protein That Uses Immunoglobulin-like Domains to Bind Filamentous Actin

Daniel K. Arneman,Carol A. Otey,M. Joseph Costello,Richard D.S. Dixon,Sharon L. Campbell,Naresh R. Sundaresan,Andrew S. Rachlin

doi:10.1074/jbc.m707694200

Daniel K. Arneman, Carol A. Otey + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m707694200

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Abstract

Palladin is a recently described phosphoprotein that plays an important role in cell adhesion and motility. Previous studies have shown that palladin overexpression results in profound changes in actin organization in cultured cells. Palladin binds to the actin-associated proteins alpha-actinin, vasodilator-stimulated phosphoprotein, profilin, Eps8, and ezrin, suggesting that it may affect actin organization indirectly. To determine its molecular function in generating actin arrays, we purified palladin and asked if it is also capable of binding to F-actin directly. In co-sedimentation and differential sedimentation assays, palladin was found to both bind and cross-link actin filaments. This bundling activity was confirmed by fluorescence and electron microscopy. Palladin fragments were then purified and used to determine the sequences necessary to bind and bundle F-actin. The Ig3 domain of palladin bound to F-actin, and a palladin fragment containing Ig3, Ig4, and the region linking these domains was identified as a fragment that was able to bundle F-actin. Because palladin has multiple Ig domains, and only one of them binds to F-actin, this suggests that different Ig domains may be specialized for distinct biological functions. In addition, our results suggest a potential role for palladin in generating specialized, actin-based cell morphologies via both direct actin cross-linking activity and indirect scaffolding activity.

Highlights

Polymerization of actin at specific sites [7]
Palladin is known to co-localize with ␣-actinin and vasodilator-stimulated phosphoprotein (VASP) to dense regions along actin stress fibers [23]; whether palladin is able to interact with F-actin directly has not been previously determined
Our results show that the widely expressed, actin-associated protein palladin functions as an actin cross-linking protein in vitro, and that the Ig3 domain of palladin is involved in F-actin binding

Summary

EXPERIMENTAL PROCEDURES

Identification and Cloning of Palladin Immunoglobulin Domains—The three tandem Ig domains of the 90-kDa palladin isoform were identified using a BLAST search and were initially characterized as C2-type Ig domains. Actin Co-sedimentation Assay—A solution of 6.4 mg/ml actin purified from rabbit muscle acetone powder was diluted 2-fold with an equal volume of 2ϫ F-actin buffer (20 mM Tris pH 8.0, 200 mM KCl, 5 mM MgCl2, and 4 mM DTT) and allowed to polymerize at room temperature for 30 min. Purified palladin was centrifuged at 150,000 ϫ g for 20 min to pellet any insoluble protein immediately before the co-sedimentation samples were prepared. Fluorescence Microscopy of Actin Filament Bundles—A solution of 6.4 mg/ml actin purified from rabbit muscle acetone powder was polymerized at 74 ␮M concentration by adding an equal volume of 2ϫ F-actin buffer (20 mM Tris, pH 8.0, 100 mM KCl, and 5 mM MgCl2). Both templates produced excellent homology models, the homology model based on the titin I1 domain produced a higher sequence-structure self-consistency score (0.74) and was selected as the better template to generate the homology model

RESULTS

DISCUSSION

ADDITIONS AND CORRECTIONS