A dynamic network of actin-associated proteins modulates the structural and dynamic properties of the actin-based cytoskeleton. Several actin binding proteins function as scaffolds that interact with a number of proteins to regulate a wide range of cellular processes, including cell growth, differentiation, adhesion, and motility. α-Actinin is an actin-crosslinking protein that has been shown to function as a platform for the assembly of multi-protein complexes1
and is uniquely positioned as an anchor between the actin cytoskeleton and the cytoplasmic domains of several cell surface adhesion proteins.2
Due to the substantial overlap in subcellular distribution and the emerging functions of palladin in actin filament regulation, the α-actinin-palladin interaction has gained special interest.
Palladin is an actin-associated protein, cloned independently in the Otey3
labs, that localizes to many actin-containing structures, including stress fibers, focal adhesions, cell-cell junctions and Z-discs. Palladin binds directly to actin regulating proteins (VASP,5
) and actin cross-linking proteins (α-actinin,10
). Multiple studies in both cultured cells and knockout mice suggest that palladin’s actin-organizing activity plays a central role in promoting cell motility.3, 12
Correlative evidence also supports this hypothesis, as palladin levels are upregulated in cells that are actively migrating such as developing vertebrate embryos,13
in cells along a wound-edge,14
and in metastatic cancer cells.15, 16, 17, 18
Our recent results suggest that palladin occupies an unusual functional niche, as it is a molecular scaffold for multiple actin-binding proteins,19
an actin-crosslinking protein,20
and a regulator of transcriptional activity.21
Similar to α-actinin, palladin has emerged as a key player in organizing actin arrays within migrating cells, through both direct and indirect molecular mechanisms.
Palladin is ubiquitous in developing vertebrate tissues and is also expressed in many adult tissues.3
It exists as multiple isoforms that are expressed in tissue-specific patterns.22, 23
In addition, palladin has two close relatives that are expressed in a restricted pattern: myopalladin is found only in heart and skeletal muscle24
and myotilin is expressed mostly in skeletal muscle.25
All three family members bind directly to α-actinin, although apparently via different sites of interaction.10, 24, 25, 26
The α-actinin interaction site of both palladin and myotilin lies in a homologous region with no obvious domain structure or sequence homology to other α-actinin binding partners. Despite shared sequence homology at this region, myopalladin was previously shown to bind α-actinin via its C-terminal Ig domains.24
All three palladin family members bind to the EF-hand repeats 3–4 of α-actinin's C-terminal domain, which is also where titin's Z-repeat 7 interacts with α–actinin.10
Multiple recent studies suggest that disregulation of palladin expression may play a key role in the invasive cell motility that characterizes metastatic cancer cells as well as in the development of cardiovascular diseases.15, 27, 28, 29
Additionally, palladin was directly implicated in a rare inherited form of pancreatic cancer.16
In that study, a point mutation (P239S) in palladin that falls within the α-actinin binding site was identified in an inherited form of highly penetrant pancreatic cancer, suggesting that alteration of palladin/α-actinin interactions may have direct effects on cell behaviors such as motility.
Palladin’s binding partner α-actinin has also been implicated in the metastasis of multiple cancers. Similar to palladin, α-actinin exists in humans as multiple isoforms, including two that are expressed in muscle (actinin-2 and actinin-3) and two that are expressed in non-muscle cells (actinin-1 and actinin-4). Overexpression of one or both non-muscle isoforms of α-actinin has been detected in high-grade sarcomas and in cancers of the esophagus, lung, breast and colon.18, 30, 31, 32, 33, 34, 35, 36
Findings to date suggest that α-actinin can regulate the actin cytoskeleton and increase cell motility; however the specific role of α-actinin in pathological cell motility has not yet been determined.
The direct binding interaction between α-actinin and palladin, their high degree of co-localization in podosomes and other actin-based structures, and the fact that they are both upregulated in invasive cancers suggest that these proteins may have a shared function in motility and adhesion that may be disregulated in cancer cells. It is noteworthy that palladin binds to a region of α-actinin that was previously shown to be involved in auto-inhibitory contacts that regulate α-actinin interactions, suggesting a possible role of palladin to direct or target actinin and/or regulate the ability of α-actinin to bind and cross-link F-actin.37, 38
In fact M. Rönty et al.
show that this interaction is critical for bidirectional targeting of both proteins to actin bundles using transfection-based targeting assays.10
Therefore we examined the complex between α-actinin and palladin in light of the previously observed high degree of co-localization between palladin and α-actinin in various subcellular structures.3, 4, 10
Palladin and α-actinin are both functionally and physically linked to both normal and pathological cell motility, however the precise molecular role of this complex in organizing the actin cytoskeleton is unknown. To advance our understanding of the biological significance of the interaction between palladin and α-actinin in cell motility and invasion, we have undertaken the first detailed structure-function analysis of the α-actinin-palladin complex.
Here we present a structural model of α–actinin bound to a palladin peptide. NMR spectroscopy was employed to assign backbone resonances of the Act-EF34 domain, in the presence and absence of a palladin peptide. Orientation constraints and mutagenesis data were then obtained on the complex to generate a structure-based model. We show that the conformation of α-actinin bound to palladin is very similar to that bound to titin Z-repeats, suggesting a similar binding mode, and postulate that ligands that recognize α–actinin may present a common minimal binding motif within a recognition helix, where specificity is dictated by the hydrophobic ‘1-4-5-8’ motif.39