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1.  STRUCTURE AND FUNCTION OF PALLADIN’S ACTIN BINDING DOMAIN 
Journal of molecular biology  2013;425(18):3325-3337.
Here we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct F-actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and crosslinking. Sedimentation equilibrium assays indicate that the Ig3 domain of palladin does not self-associate. These combined data are consistent with an actin crosslinking mechanism that involves concurrent attachment of two actin filaments by a single palladin molecule by an electrostatic mechanism. Palladin mutations that disrupt actin binding show altered cellular distributions and morphology of actin in cells, revealing a functional requirement for the interaction between palladin and actin in vivo.
doi:10.1016/j.jmb.2013.06.016
PMCID: PMC3759364  PMID: 23806659
2.  STRUCTURAL CHARACTERIZATION OF THE INTERACTIONS BETWEEN PALLADIN AND α-ACTININ 
Journal of molecular biology  2011;413(3):712-725.
The interaction between α-actinin and palladin, two actin-crosslinking proteins, is essential for proper bidirectional targeting of these proteins. As a first step toward understanding the role of this complex in organizing cytoskeletal actin, we have characterized binding interactions between the EF hand domain of α-actinin (Act-EF34) and peptides derived from palladin, and generated a NMR-derived structural model for the Act-EF34/palladin peptide complex. The critical binding site residues are similar to an actinin binding motif previously suggested for the complex between Act-EF34 and titin Z-repeats. The structure-based model of the Act-EF34/palladin peptide complex expands our understanding of binding specificity between the scaffold protein α-actinin and various ligands, which appears to require an α-helical motif containing four hydrophobic residues, common to many α–actinin ligands. We also provide evidence that the Family-X mutation in palladin, associated with a highly penetrant form of pancreatic cancer, does not interfere with α-actinin binding.
doi:10.1016/j.jmb.2011.08.059
PMCID: PMC3226707  PMID: 21925511
3.  Morphology and Viscoelasticity of Actin Networks Formed with the Mutually Interacting Crosslinkers: Palladin and Alpha-actinin 
PLoS ONE  2012;7(8):e42773.
Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not clearly understood. The ABP, palladin, is essential for the maintenance of cell morphology and the regulation of cell movement. Palladin coexists with -actinin in stress fibers and focal adhesions and binds to both actin and -actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we characterized the micro-structure and mechanics of actin networks crosslinked with palladin and -actinin. We first showed that palladin crosslinks actin filaments into bundled networks which are viscoelastic in nature. Our studies also showed that composite networks of -actinin/palladin/actin behave very similar to pure palladin or pure -actinin networks. However, we found evidence that palladin and -actinin synergistically modify network viscoelasticity. To our knowledge, this is the first quantitative characterization of the physical properties of actin networks crosslinked with two mutually interacting crosslinkers.
doi:10.1371/journal.pone.0042773
PMCID: PMC3420904  PMID: 22916157
4.  The Actin Associated Protein Palladin Is Important for the Early Smooth Muscle Cell Differentiation 
PLoS ONE  2010;5(9):e12823.
Palladin, an actin associated protein, plays a significant role in regulating cell adhesion and cell motility. Palladin is important for development, as knockdown in mice is embryonic lethal, yet its role in the development of the vasculature is unknown. We have shown that palladin is essential for the expression of smooth muscle cells (SMC) marker genes and force development in response to agonist stimulation in palladin deficient SMCs. The goal of the study was to determine the molecular mechanisms underlying palladin's ability to regulate the expression of SMC marker genes. Results showed that palladin expression was rapidly induced in an A404 cell line upon retinoic acid (RA) induced differentiation. Suppression of palladin expression with siRNAs inhibited the expression of RA induced SMC differentiation genes, SM α-actin (SMA) and SM22, whereas over-expression of palladin induced SMC gene expression. Chromatin immunoprecipitation assays provided evidence that palladin bound to SMC genes, whereas co-immunoprecipitation assays also showed binding of palladin to myocardin related transcription factors (MRTFs). Endogenous palladin was imaged in the nucleus, increased with leptomycin treatment and the carboxyl-termini of palladin co-localized with MRTFs in the nucleus. Results support a model wherein palladin contributes to SMC differentiation through regulation of CArG-SRF-MRTF dependent transcription of SMC marker genes and as previously published, also through actin dynamics. Finally, in E11.5 palladin null mouse embryos, the expression of SMA and SM22 mRNA and protein is decreased in the vessel wall. Taken together, our findings suggest that palladin plays a key role in the differentiation of SMCs in the developing vasculature.
doi:10.1371/journal.pone.0012823
PMCID: PMC2943901  PMID: 20877641
5.  Cytoplasmic Ig-Domain Proteins: Cytoskeletal Regulators with a Role in Human Disease 
Immunoglobulin domains are found in a wide variety of functionally diverse transmembrane proteins, and also in a smaller number of cytoplasmic proteins. Members of this latter group are usually associated with the actin cytoskeleton, and most of them bind directly to either actin or myosin, or both. Recently, studies of inherited human disorders have identified disease-causing mutations in five cytoplasmic Ig-domain proteins: myosin-binding protein C, titin, myotilin, palladin, and myopalladin. Together with results obtained from cultured cells and mouse models, these clinical studies have yielded novel insights into the unexpected roles of Ig domain proteins in mechanotransduction and signaling to the nucleus. An emerging theme in this field is that cytoskeleton-associated Ig domain proteins are more than structural elements of the cell, and may have evolved to fill different needs in different cellular compartments.
doi:10.1002/cm.20385
PMCID: PMC2735333  PMID: 19466753
titin; palladin; MyBP-C; myotilin; myopalladin
6.  Isoform-Specific Upregulation of Palladin in Human and Murine Pancreas Tumors 
PLoS ONE  2010;5(4):e10347.
Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with a characteristic pattern of early metastasis, which is driving a search for biomarkers that can be used to detect the cancer at an early stage. Recently, the actin-associated protein palladin was identified as a candidate biomarker when it was shown that palladin is mutated in a rare inherited form of PDA, and overexpressed in many sporadic pancreas tumors and premalignant precursors. In this study, we analyzed the expression of palladin isoforms in murine and human PDA and explored palladin's potential use in diagnosing PDA. We performed immunohistochemistry and immunoblot analyses on patient samples and tumor-derived cells using an isoform-selective monoclonal antibody and a pan-palladin polyclonal antibody. Immunoblot and real-time quantitative reverse transcription-PCR were used to quantify palladin mRNA levels in human samples. We show that there are two major palladin isoforms expressed in pancreas: 65 and 85–90 kDa. The 65 kDa isoform is expressed in both normal and neoplastic ductal epithelial cells. The 85–90 kDa palladin isoform is highly overexpressed in tumor-associated fibroblasts (TAFs) in both primary and metastatic tumors compared to normal pancreas, in samples obtained from either human patients or genetically engineered mice. In tumor-derived cultured cells, expression of palladin isoforms follows cell-type specific patterns, with the 85–90 kDa isoform in TAFs, and the 65 kDa isoform predominating in normal and neoplastic epithelial cells. These results suggest that upregulation of 85–90 kDa palladin isoform may play a role in the establishment of the TAF phenotype, and thus in the formation of a desmoplastic tumor microenvironment. Thus, palladin may have a potential use in the early diagnosis of PDA and may have much broader significance in understanding metastatic behavior.
doi:10.1371/journal.pone.0010347
PMCID: PMC2859948  PMID: 20436683
7.  The role of palladin in actin organization and cell motility 
European journal of cell biology  2008;87(8-9):517-525.
Palladin is a widely expressed protein found in stress fibers, focal adhesions, growth cones, Z-discs, and other actin-based subcellular structures. It belongs to a small gene family that includes the Z-disc proteins myopalladin and myotilin, all of which share similar Ig-like domains. Recent advances have shown that palladin shares with myotilin the ability to bind directly to F-actin, and to crosslink actin filaments into bundles, in vitro. Studies in a variety of cultured cells suggest that the actin-organizing activity of palladin plays a central role in promoting cell motility. Correlative evidence also supports this hypothesis, as palladin levels are typically upregulated in cells that are actively migrating: in developing vertebrate embryos, in cells along a wound edge, and in metastatic cancer cells. Recently, a mutation in the human palladin gene was implicated in an unusually penetrant form of inherited pancreatic cancer, which has stimulated new ideas about the role of palladin in invasive cancer.
doi:10.1016/j.ejcb.2008.01.010
PMCID: PMC2597190  PMID: 18342394
Lasp-1; alpha-Actinin; VASP; Eps8; Podosomes; Dorsal ruffles; Focal adhesion
8.  Roles of the small GTPases RhoA and Rac1 in cell behavior 
The Rho-family GTPases are proving to have a variety of biological functions apart from their well known effects on the cytoskeleton. Recent work indicates their involvement in signaling between the adhesion receptors integrin and syndecan, effects on the recruitment of beta-catenin to the nucleus, and potential roles in the nucleus as well as the cytoplasm.
doi:10.3410/B1-4
PMCID: PMC2920686  PMID: 20948645
9.  Palladin interacts with SH3 domains of SPIN90 and Src and is required for Src-induced cytoskeletal remodeling 
Experimental cell research  2007;313(12):2575-2585.
Palladin and SPIN90 are widely expressed proteins, which participate in modulation of actin cytoskeleton by binding to a variety of scaffold and signaling molecules. Cytoskeletal reorganization can induced by activation of signaling pathways, including the PDGF receptor and Src tyrosine kinase pathways. In this study we have analyzed the interplay between palladin, SPIN90 and Src, and characterized the role of palladin and SPIN90 in PDGF and Src-induced cytoskeletal remodeling. We show that the SH3 domains of SPIN90 and Src directly bind palladin’s poly-proline sequence and the interaction controls intracellular targeting of SPIN90. In PDGF-treated cells, palladin and SPIN90 co-localize in actin rich membrane ruffles and lamellipodia. The effect of PDGF on the cytoskeleton is at least partly mediated by the Src kinase, since PP2, a selective Src kinase family inhibitor, blocked PDGF-induced changes. Furthermore, expression of active Src kinase resulted in coordinated translocation of both palladin and SPIN90 to membrane protrusions. Knock-down of endogenous SPIN90 did not inhibit Src-induced cytoskeletal rearrangement, whereas knock-down of palladin resulted in cytoskeletal disorganization and inhibition of remodeling. Further studies showed that palladin is tyrosine phosphorylated in cells expressing active Src indicating bidirectional interplay between palladin and Src. These results may have implications in understanding the invasive and metastatic phenotype of neoplastic cells induced by Src.
doi:10.1016/j.yexcr.2007.04.030
PMCID: PMC2000818  PMID: 17537434
Palladin; SPIN90; Src; cytoskeleton
10.  Palladin Mutation Causes Familial Pancreatic Cancer and Suggests a New Cancer Mechanism 
PLoS Medicine  2006;3(12):e516.
Background
Pancreatic cancer is a deadly disease. Discovery of the mutated genes that cause the inherited form(s) of the disease may shed light on the mechanism(s) of oncogenesis. Previously we isolated a susceptibility locus for familial pancreatic cancer to chromosome location 4q32–34. In this study, our goal was to discover the identity of the familial pancreatic cancer gene on 4q32 and determine the function of that gene.
Methods and Findings
A customized microarray of the candidate chromosomal region affecting pancreatic cancer susceptibility revealed the greatest expression change in palladin (PALLD), a gene that encodes a component of the cytoskeleton that controls cell shape and motility. A mutation causing a proline (hydrophobic) to serine (hydrophilic) amino acid change (P239S) in a highly conserved region tracked with all affected family members and was absent in the non-affected members. The mutational change is not a known single nucleotide polymorphism. Palladin RNA, measured by quantitative RT-PCR, was overexpressed in the tissues from precancerous dysplasia and pancreatic adenocarcinoma in both familial and sporadic disease. Transfection of wild-type and P239S mutant palladin gene constructs into HeLa cells revealed a clear phenotypic effect: cells expressing P239S palladin exhibited cytoskeletal changes, abnormal actin bundle assembly, and an increased ability to migrate.
Conclusions
These observations suggest that the presence of an abnormal palladin gene in familial pancreatic cancer and the overexpression of palladin protein in sporadic pancreatic cancer cause cytoskeletal changes in pancreatic cancer and may be responsible for or contribute to the tumor's strong invasive and migratory abilities.
The presence of abnormalpalladin in familial pancreatic cancer and its overexpression in sporadic pancreatic cancer leads to cytoskeletal changes and may be responsible for the tumor's invasive and migratory abilities.
Editors' Summary
Background.
Pancreatic cancer is a leading cause of cancer-related death in the US. Because it causes few symptoms in its early stages, pancreatic cancer is rarely detected until it has spread (metastasized) around the body. Pancreatic tumors can occasionally be removed surgically but the usual treatment is radio- or chemotherapy, and neither of these is curative; most patients die within a year of diagnosis. As in other cancers, the cells in pancreatic tumors have acquired genetic changes (mutations) that allow them to divide uncontrollably (normal cells divide only to repair damaged tissue). Other mutations alter the shape of the cells and allow them to migrate into (invade) other areas of the body. These mutations usually arise randomly—the cells in the human body are bombarded by chemicals and other agents that can damage their DNA—and cause “sporadic” pancreatic cancer. But some people inherit mutated genes that increase their susceptibility to pancreatic cancer. These people are recognizable because pancreatic cancer is more common in their families than in the general population.
Why Was This Study Done?
The identification of the genes that are mutated in familial pancreatic cancer might provide insights into how both inherited and sporadic cancer develops in the pancreas. Such information could suggest ways to detect pancreatic cancers earlier than is currently possible and could identify new therapeutic targets for this deadly disease. Previous work by the researchers who did this study localized a gene responsible for inherited pancreatic cancer to a small region of Chromosome 4 in a family in which pancreatic cancer is very common (Family X). In this study, the researchers identified which of the genes in this region is likely to be responsible for the susceptibility to pancreatic cancer of Family X.
What Did the Researchers Do and Find?
The researchers made a DNA microarray (a small chip spotted with DNA sequences) of the 243 genes in the chromosomal region linked to pancreatic cancer in Family X. They used this to examine gene expression in dysplastic pancreatic tissue from a Family X member (pancreatic dysplasia is a precancerous lesion that precedes cancer), in normal pancreatic tissue, and in samples from sporadic pancreatic cancers. The most highly overexpressed (compared to normal tissue) gene in both the Family X tissue and the sporadic cancers encoded a protein called palladin. Palladin is a component of the cytoskeleton (a structure that helps to control cell shape and motility) and it organizes other cytoskeletal components. Next, the researchers quantified the expression of palladin RNA in an independent set of normal and cancerous pancreatic samples, and in precancerous pancreatic tissue taken from Family X members and from people who inherit pancreatic cancer but who were not in Family X. This analysis indicated that palladin was overexpressed early in sporadic and inherited pancreatic cancer development. Sequencing of the palladin gene then uncovered a mutation in palladin that was present in Family X members with pancreatic cancer or precancerous lesions but not in unaffected members. This specific mutation, which probably affects palladin's interaction with another cytoskeletal protein called alpha-actinin, was not found in sporadic cancers although many sporadic cancer cell lines had abnormal expression of alpha-actinin protein in addition to palladin protein. Finally, the researchers showed that the introduction of mutated palladin into a human cell line growing in the laboratory increased its migration rate and disrupted its cytoskeleton.
What Do These Findings Mean?
These results strongly suggest that mutated palladin is involved in the development of familial pancreatic cancer. Because genes tend to be inherited in groups, there is still chance that a mutation in a nearby gene could be responsible for the increased susceptibility to pancreatic cancer in Family X. However, the data showing palladin overexpression in sporadic tumors and alterations of cell behavior in the laboratory after introduction of the mutated gene make this unlikely. To prove the involvement of palladin in pancreatic cancer, palladin mutations must now be identified in other familial cases and the overexpression of palladin in sporadic cancers must be explained. The results here nevertheless provide an intriguing glimpse into a potential new mechanism for cancer development in the pancreas and possibly other tissues, one in which abnormalities in palladin function or expression (or in the proteins with which it associates) drive some of the changes in cell migration, shape, and size that characterize cancer cells.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030516
US National Cancer Institute, information on pancreatic cancer for patients and health professionals
MedlinePlus encyclopedia entry on pancreatic carcinoma
Cancer Research UK, information for patients about pancreatic cancer
Johns Hopkins University, information on pancreatic cancer that includes details on familial cancer
CancerQuest, information provided by Emory University about how cancer develops
doi:10.1371/journal.pmed.0030516
PMCID: PMC1751121  PMID: 17194196
11.  Simultaneous Stretching and Contraction of Stress Fibers In Vivo 
Molecular Biology of the Cell  2004;15(7):3497-3508.
To study the dynamics of stress fiber components in cultured fibroblasts, we expressed α-actinin and the myosin II regulatory myosin light chain (MLC) as fusion proteins with green fluorescent protein. Myosin activation was stimulated by treatment with calyculin A, a serine/threonine phosphatase inhibitor that elevates MLC phosphorylation, or with LPA, another agent that ultimately stimulates phosphorylation of MLC via a RhoA-mediated pathway. The resulting contraction caused stress fiber shortening and allowed observation of changes in the spacing of stress fiber components. We have observed that stress fibers, unlike muscle myofibrils, do not contract uniformly along their lengths. Although peripheral regions shortened, more central regions stretched. We detected higher levels of MLC and phosphorylated MLC in the peripheral region of stress fibers. Fluorescence recovery after photobleaching revealed more rapid exchange of myosin and α-actinin in the middle of stress fibers, compared with the periphery. Surprisingly, the widths of the myosin and α-actinin bands in stress fibers also varied in different regions. In the periphery, the banding patterns for both proteins were shorter, whereas in central regions, where stretching occurred, the bands were wider.
doi:10.1091/mbc.E03-09-0696
PMCID: PMC452600  PMID: 15133124
12.  Characterization of Palladin, a Novel Protein Localized to Stress Fibers and Cell Adhesions 
The Journal of Cell Biology  2000;150(3):643-656.
Here, we describe the identification of a novel phosphoprotein named palladin, which colocalizes with α-actinin in the stress fibers, focal adhesions, cell–cell junctions, and embryonic Z-lines. Palladin is expressed as a 90–92-kD doublet in fibroblasts and coimmunoprecipitates in a complex with α-actinin in fibroblast lysates. A cDNA encoding palladin was isolated by screening a mouse embryo library with mAbs. Palladin has a proline-rich region in the NH2-terminal half of the molecule and three tandem Ig C2 domains in the COOH-terminal half. In Northern and Western blots of chick and mouse tissues, multiple isoforms of palladin were detected. Palladin expression is ubiquitous in embryonic tissues, and is downregulated in certain adult tissues in the mouse. To probe the function of palladin in cultured cells, the Rcho-1 trophoblast model was used. Palladin expression was observed to increase in Rcho-1 cells when they began to assemble stress fibers. Antisense constructs were used to attenuate expression of palladin in Rcho-1 cells and fibroblasts, and disruption of the cytoskeleton was observed in both cell types. At longer times after antisense treatment, fibroblasts became fully rounded. These results suggest that palladin is required for the normal organization of the actin cytoskeleton and focal adhesions.
PMCID: PMC2175193  PMID: 10931874
focal adhesion; adherens junction; microfilament; α-actinin; trophoblast
13.  A Role for P21-Activated Kinase in Endothelial Cell Migration 
The Journal of Cell Biology  1999;147(4):831-844.
The serine/threonine p21-activated kinase (PAK) is an effector for Rac and Cdc42, but its role in regulating cytoskeletal organization has been controversial. To address this issue, we investigated the role of PAK in migration of microvascular endothelial cells. We found that a dominant negative (DN) mutant of PAK significantly inhibited cell migration and in-creased stress fibers and focal adhesions. The DN effect mapped to the most NH2-terminal proline-rich SH3-binding sequence. Observation of a green fluorescent protein-tagged α-actinin construct in living cells revealed that the DN construct had no effect on membrane ruffling, but dramatically inhibited stress fiber and focal contact motility and turnover. Constitutively active PAK inhibited migration equally well and also increased stress fibers and focal adhesions, but had a somewhat weaker effect on their dynamics. In contrast to their similar effects on motility, DN PAK decreased cell contractility, whereas active PAK increased contractility. Active PAK also increased myosin light chain (MLC) phosphorylation, as indicated by staining with an antibody to phosphorylated MLC, whereas DN PAK had little effect, despite the increase in actin stress fibers. These results demonstrate that although PAK is not required for extension of lamellipodia, it has substantial effects on cell adhesion and contraction. These data suggest a model in which PAK plays a role coordinating the formation of new adhesions at the leading edge with contraction and detachment at the trailing edge.
PMCID: PMC2156168  PMID: 10562284
Rac; Cdc42; cell motility; cytoskeleton; contractility
14.  A Role for the Cytoskeleton-associated Protein Palladin in Neurite Outgrowth 
Molecular Biology of the Cell  2001;12(9):2721-2729.
The outgrowth of neurites is a critical step in neuronal maturation, and it is well established that the actin cytoskeleton is involved in this process. Investigators from our laboratory recently described a novel protein named palladin, which has been shown to play an essential role in organizing the actin cytoskeleton in cultured fibroblasts. We investigated the expression of palladin in the developing rat brain by Western blot and found that the E18 brain contained a unique variant of palladin that is significantly smaller (∼85 kDa) than the common form found in other developing tissues (90–92 kDa). Because the expression of a tissue-specific isoform suggests the possibility of a cell type-specific function, we investigated the localization and function of palladin in cultured cortical neurons. Palladin was found preferentially targeted to the developing axon but not the dendrites and was strongly localized to the axonal growth cone. When palladin expression was attenuated by transfection with antisense constructs in both the B35 neuroblastoma cell line and in primary cortical neurons, a reduction in the expression of palladin resulted in a failure of neurite outgrowth. These results implicate palladin as a critical component of the developing nervous system, with an important role in axonal extension.
PMCID: PMC59707  PMID: 11553711

Results 1-14 (14)