Phosphorylation of actin-binding proteins plays a pivotal role in the remodeling of the actin cytoskeleton to regulate cell migration. Palladin is an actin-binding protein that is phosphorylated by growth factor stimulation; however, the identity of the involved protein kinases remains elusive. In this study, we report that palladin is a novel substrate of extracellular signal-regulated kinase (ERK). Suppression of ERK activation by a chemical inhibitor reduced palladin phosphorylation, and expression of active MEK alone was sufficient for phosphorylation. In addition, an in vitro kinase assay demonstrated direct palladin phosphorylation by ERK. We found that Ser77 and Ser197 are essential residues for phosphorylation. Although the phosphorylation of these residues was not required for actin cytoskeletal organization, we found that expression of non-phosphorylated palladin enhanced cell migration. Finally, we show that phosphorylation inhibits the palladin association with Abl tyrosine kinase. Taken together, our results indicate that palladin phosphorylation by ERK has an anti-migratory function, possibly by modulating interactions with molecules that regulate cell migration.
The phosphoinositide 3-kinase (PI 3-K) signaling pathway is frequently deregulated in cancer. Downstream of PI 3-K, Akt1 and Akt2 have opposing roles in breast cancer invasive migration leading to metastatic dissemination. Here we identify palladin, an actin-associated protein, as an Akt1-specific substrate that modulates breast cancer cell invasive migration. Akt1, but not Akt2, phosphorylates palladin at S507 in a domain that is critical for F-actin bundling. Downregulation of palladin enhances migration and invasion of breast cancer cells and induces abnormal branching morphogenesis in 3D cultures. Palladin phosphorylation at S507 is required for Akt1-mediated inhibition of breast cancer cell migration and also for F-actin bundling leading to the maintenance of an organized actin cytoskeleton. These findings identify palladin as an Akt1-specific substrate that regulates cell motility and provide a molecular mechanism that accounts for the functional distinction between Akt isoforms in breast cancer cell signaling to cell migration.
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.
Cancer metastasis involves multiple steps including detachment of the metastatic cells from neighboring cells, the acquisition of motility and invasion to other tissue. All of these steps require the reorganization of the actin cytoskeleton. In this study, we found that the protein palladin, a molecular scaffold with an important function in actin organization, is expressed at higher overall levels in tumors compared to benign breast tissue, and also significantly higher in four invasive breast cancer cell lines when compared to four non-invasive cell lines. In addition, we found that palladin plays a key role in the formation of podosomes. Podosomes are actin-rich structures that function in adhesion and matrix degradation and have been found in many invasive cell types. Our results show that phorbol ester treatment stimulated the formation of palladin-containing podosomes in invasive, but not in non-invasive cell lines. More importantly, palladin knockdown resulted in decreased podosome formation and a significant reduction in transwell migration and invasive motility. Palladin overexpression induced podosome formation in the non-invasive MCF7 cells, which are otherwise unable to form podosomes, suggesting that palladin plays a critical role in the assembly of podosomes. Overall, these results indicate that palladin overexpression contributes to the invasive behavior of metastatic cells.
podosomes; migration; actin; metastasis
Cell adhesion to the extracellular matrix is mediated by adhesion receptors, mainly integrins, which upon interaction with the extracellular matrix, bind to the actin cytoskeleton via their cytoplasmic domains. This association is mediated by a variety of scaffold and signaling proteins, which control the mechanical and signaling activities of the adhesion site. Upon transformation of fibroblasts with active forms of Src (e.g., v-Src), focal adhesions are disrupted, and transformed into dot-like contacts known as podosomes, and consisting of a central actin core surrounded by an adhesion ring. To clarify the mechanism underlying Src-dependent modulation of the adhesive phenotype, and its influence on podosome organization, we screened for the effect of siRNA-mediated knockdown of tyrosine kinases, MAP kinases and phosphatases on the reorganization of the adhesion-cytoskeleton complex, induced by a constitutively active Src mutant (SrcY527F). In this screen, we discovered several genes that are involved in Src-induced remodeling of the actin cytoskeleton. We further showed that knockdown of Src in osteoclasts abolishes the formation of the podosome-based rings and impairs cell spreading, without inducing stress fiber development. Our work points to several genes that are involved in this process, and sheds new light on the molecular plasticity of integrin adhesions.
Src; podosomes; actin; cytoskeleton; siRNA; screening; cell adhesion; microscopy
We describe here a novel sarcomeric 145-kD protein, myopalladin, which tethers together the COOH-terminal Src homology 3 domains of nebulin and nebulette with the EF hand motifs of α-actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and α-actinin–binding sites are contained in two distinct regions within its COOH-terminal 90-kD domain. Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A. Otey. 2000. J. Cell Biol. 150:643–656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH2-terminal region of myopalladin specifically binds to the cardiac ankyrin repeat protein (CARP), a nuclear protein involved in control of muscle gene expression. Immunofluorescence and immunoelectron microscopy studies revealed that myopalladin also colocalized with CARP in the central I-band of striated muscle sarcomeres. Overexpression of myopalladin's NH2-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin–CARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity. Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via α-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).
α-actinin; nebulin; palladin; myopalladin; CARP
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.
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.
focal adhesion; adherens junction; microfilament; α-actinin; trophoblast
The PI 3-K/Akt pathway is responsible for key aspects of tumor progression, and is frequently hyperactivated in cancer. We have recently identified palladin, an actin-bundling protein that functions to control the actin cytoskeleton, as an Akt1-specific substrate that inhibits breast cancer cell migration. Here we have identified a role for Akt isoforms in the regulation of palladin expression. Akt2, but not Akt1, enhances palladin expression by maintaining protein stability and upregulating transcription. These data reveal that Akt signaling regulates the stability of palladin, and further supports the notion that Akt isoforms have distinct and specific roles in tumorigenesis.
Akt isoform; palladin; protein stability; breast cancer
Alix/AIP1 is an adaptor protein involved in regulating the function of receptor and cytoskeleton-associated tyrosine kinases. Here, we investigated its interaction with and regulation by Src. Tyr319 of Alix bound the isolated Src homology-2 (SH2) domain and was necessary for interaction with intact Src. A proline-rich region in the C terminus of Alix bound the Src SH3 domain, but this interaction was dependent on the release of the Src SH2 domain from its Src internal ligand either by interaction with Alix Tyr319 or by mutation of Src Tyr527. Src phosphorylated Alix at a C-terminal region rich in tyrosines, an activity that was stimulated by the presence of the Alix binding partner SETA/CIN85. Phosphorylation of Alix by Src caused it to translocate from the membrane and cytoskeleton to the cytoplasm and reduced its interaction with binding partners SETA/ CIN85, epidermal growth factor receptor, and Pyk2. As a consequence of this, Src antagonized the negative regulation of receptor tyrosine kinase internalization and cell adhesion by Alix. We propose a model whereby Src antagonizes the effects of Alix by phosphorylation of its C terminus, leading to the disruption of interactions with target proteins.
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.
Lasp-1; alpha-Actinin; VASP; Eps8; Podosomes; Dorsal ruffles; Focal adhesion
We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA) currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl), control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood.
Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation.
This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.
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.
Palladin is an actin cytoskeleton–associated protein which is crucial for cell morphogenesis and motility. Previous studies have shown that palladin is localized to the axonal growth cone in neurons and may play an important role in axonal extension. Previously, we have generated palladin knockout mice which display cranial neural tube closure defect and embryonic lethality before embryonic day 15.5 (E15.5). To further study the role of palladin in the developing nervous system, we examined the innervation of palladin-deficient mouse embryos since the 200 kd, 140 kd, 90–92 kd and 50 kd palladin isoforms were undetectable in the mutant mouse embryo brain. Contrary to the results of previous studies, we found no inhibition of the axonal extension in palladin-deficient mouse embryos. The cortical neurons derived from palladin-deficient mice also showed no significant difference in neurite outgrowth as compared with those from wild-type mice. Moreover, no difference was found in neurite outgrowth of neural stem cell derived-neurons between palladin-deficient mice and wild-type mice. In conclusion, these results suggest that palladin is dispensable for normal neurite outgrowth in mice.
The protein tyrosine kinase c-Src is regulated by two intramolecular interactions. The repressed state is achieved through the interaction of the Src homology 2 (SH2) domain with the phosphorylated C-terminal tail and the association of the SH3 domain with a polyproline type II helix formed by the linker region between SH2 and the kinase domain. hnRNP K, the founding member of the KH domain protein family, is involved in chromatin remodeling, regulation of transcription, and translation of specific mRNAs and is a target in different signal transduction pathways. In particular, it functions as a specific activator and a substrate of the tyrosine kinase c-Src. Here we address the question how hnRNP K interacts with and activates c-Src. We define the proline residues in hnRNP K in the proline-rich motifs P2 (amino acids [aa] 285 to 297) and P3 (aa 303 to 318), which are necessary and sufficient for the specific activation of c-Src, and we dissect the amino acid sequence (aa 216 to 226) of hnRNP K that mediates a second interaction with c-Src. Our findings indicate that the interaction with c-Src and the activation of the kinase are separable functions of hnRNP K. hnRNP K acts as a scaffold protein that integrates signaling cascades by facilitating the cross talk between kinases and factors that mediate nucleic acid-directed processes.
ABL-family proteins comprise one of the best conserved branches of the tyrosine kinases. Each ABL protein contains an SH3-SH2-TK (Src homology 3–Src homology 2–tyrosine kinase) domain cassette, which confers autoregulated kinase activity and is common among nonreceptor tyrosine kinases. This cassette is coupled to an actin-binding and -bundling domain, which makes ABL proteins capable of connecting phosphoregulation with actin-filament reorganization. Two vertebrate paralogs, ABL1 and ABL2, have evolved to perform specialized functions. ABL1 includes nuclear localization signals and a DNA binding domain through which it mediates DNA damage-repair functions, whereas ABL2 has additional binding capacity for actin and for microtubules to enhance its cytoskeletal remodeling functions. Several types of posttranslational modifications control ABL catalytic activity, subcellular localization, and stability, with consequences for both cytoplasmic and nuclear ABL functions. Binding partners provide additional regulation of ABL catalytic activity, substrate specificity, and downstream signaling. Information on ABL regulatory mechanisms is being mined to provide new therapeutic strategies against hematopoietic malignancies caused by BCR-ABL1 and related leukemogenic proteins.
The vast complexity of platelet-derived growth factor (PDGF)-induced downstream signaling pathways is well known, but the precise roles of critical players still elude us due to our lack of specific and temporal control over their activities. Accordingly, although Src family members are some of the better characterized effectors of PDGFβ signaling, considerable controversy still surrounds their precise functions. To address these questions and limitations, we applied a chemical–genetic approach to study the role of c-Src at the cellular level, in defined signaling cascades; we also uncovered novel phosphorylation targets and defined its influence on transcriptional events. The spectacular control of c-Src on actin reorganization and chemotaxis was delineated by global substrate labeling and transcriptional analysis, revealing multiple cytoskeletal proteins and chemotaxis promoting genes to be under c-Src control. Additionally, this tool revealed the contrasting roles of c-Src in controlling DNA synthesis, where it transmits conflicting inputs via the phosphatidylinositol 3 kinase and Ras pathways. Finally, this study reveals a mechanism by which Src family kinases may control PDGF-mediated responses both at transcriptional and translational levels.
c-Src non-receptor tyrosine kinase is an important component of the platelet-derived growth factor (PDGF) receptor signaling pathway. c-Src has been shown to mediate the mitogenic response to PDGF in fibroblasts. However, the exact components of PDGF receptor signaling pathway mediated by c-Src remain unclear. Here, we used stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to identify Src family kinase substrates involved in PDGF signaling. Using SILAC, we were able to detect changes in tyrosine phosphorylation patterns of 43 potential c-Src kinase substrates in PDGF receptor signaling. This included 23 known c-Src kinase substrates, of which 16 proteins have known roles in PDGF signaling while the remaining 7 proteins have not previously been implicated in PDGF receptor signaling. Importantly, our analysis also led to identification of 20 novel Src family kinase substrates, of which 5 proteins were previously reported as PDGF receptor signaling pathway intermediates while the remaining 15 proteins represent novel signaling intermediates in PDGF receptor signaling. In validation experiments, we demonstrated that PDGF indeed induced the phosphorylation of a subset of candidate Src family kinase substrates – Calpain 2, Eps15 and Trim28 – in a c-Src-dependent fashion.
Kinase; Mass spectrometry; Phosphoproteomics; Phosphorylation; Signal transduction; SILAC
Palladin is an actin associated protein serving as a cytoskeleton scaffold, and actin cross linker, localizing at stress fibers, focal adhesions, and other actin based structures. Recent studies showed that palladin plays a critical role in smooth muscle differentiation, migration, contraction, and more importantly contributes to embryonic development. This review will focus on the functions and possible mechanisms of palladin in smooth muscle and in pathological conditions such as cardiovascular diseases and cancers.
Palladin; Smooth muscle; Actin cytoskeleton; Migration; Differentiation
The mRNA endonuclease PMR1 initiates mRNA decay by forming a selective complex with its translating substrate mRNA. Previous work showed that the ability of PMR1 to target to polysomes and activate decay depends on the phosphorylation of a tyrosine residue at position 650. The current study shows that c-Src is responsible for activating this mRNA decay pathway. c-Src was recovered with immunoprecipitated PMR1 and it phosphorylates PMR1 in vitro and in vivo. The interaction with c-Src involves 2 domains of PMR1; Y650 and a series of proline-rich SH3 peptides in the N-terminus. In cells with little c-Src PMR1 targeting to polysomes is induced by constitutively active c-Src but not by inactive forms of the kinase. Similarly only active c-Src induces PMR1-mediated mRNA decay. Finally we show that EGF rapidly induces c-Src phosphorylation of PMR1, providing a direct link between tyrosine kinase-mediated signal transduction and mRNA decay.
mRNA decay; PMR1; mRNA endonuclease; signal transduction; c-Src; tyrosine kinase
The mechanisms through which the small GTPases Rac1 and Cdc42 regulate the formation of membrane ruffles, lamellipodia, and filopodia are currently unknown. The p21-activated kinases (PAKs) are direct targets of active Rac and Cdc42 which can induce the assembly of polarized cytoskeletal structures when expressed in fibroblasts, suggesting that they may play a role in mediating the effects of these GTPases on cytoskeletal dynamics.
We have examined the subcellular localization of endogenous PAK1 in fibroblast cell lines using specific PAK1 antibodies. PAK1 is detected in submembranous vesicles in both unstimulated and stimulated fibroblasts that colocalize with a marker for fluid-phase uptake. In cells stimulated with PDGF, in v-Src–transformed fibroblasts, and in wounded cells, PAK1 redistributed into dorsal and membrane ruffles and into the edges of lamellipodia, where it colocalizes with polymerized actin. PAK1 was also colocalized with F-actin in membrane ruffles extended as a response to constitutive activation of Rac1. PAK1 appears to precede F-actin in translocating to cytoskeletal structures formed at the cell periphery. The association of PAK1 with the actin cytoskeleton is prevented by the actin filament-disrupting agent cytochalasin D and by the phosphatidylinositol 3-kinase inhibitor wortmannin. Co-immunoprecipitation experiments demonstrate an in vivo interaction of PAK1 with filamentous (F)-actin in stimulated cells. Microinjection of a constitutively active PAK1 mutant into Rat-1 fibroblasts overexpressing the insulin receptor (HIRcB cells) induced the formation of F-actin- and PAK1-containing structures reminiscent of dorsal ruffles. These data indicate a close correlation between the subcellular distribution of endogenous PAK1 and the formation of Rac/Cdc42-dependent cytoskeletal structures and support an active role for PAK1 in regulating cortical actin rearrangements.
RANK ligand (RANKL), by mechanisms unknown, directly activates osteoclasts to resorb bone. Because c-Src is key to organizing the cell's cytoskeleton, we asked if the tyrosine kinase also mediates RANKL-stimulated osteoclast activity. RANKL induces c-Src to associate with RANK369–373 in an αvβ3-dependent manner. Furthermore, RANK369–373 is the only one of six putative TRAF binding motifs sufficient to generate actin rings and activate the same cytoskeleton-organizing proteins as the integrin. While c-Src organizes the cell's cytoskeleton in response to the cytokine, it does not participate in RANKL-stimulated osteoclast formation. Attesting to their collaboration, αvβ3 and activated RANK coprecipitate, but only in the presence of c-Src. c-Src binds activated RANK via its Src homology 2 (SH2) domain and αvβ3 via its SH3 domain, suggesting the kinase links the two receptors. Supporting this hypothesis, deletion or inactivating point mutation of either the c-Src SH2 or SH3 domain obviates the RANK/αvβ3 association. Thus, activated RANK prompts two distinct signaling pathways; one promotes osteoclast formation, and the other, in collaboration with c-Src-mediated linkage to αvβ3, organizes the cell's cytoskeleton.
Stable platelet aggregation, adhesion, and spreading during hemostasis are promoted by outside-in αIIbβ3 signals that feature rapid activation of c-Src and Syk, delayed activation of FAK, and cytoskeletal reorganization. To evaluate these αIIbβ3–tyrosine kinase interactions at nanometer proximity in living cells, we monitored bioluminescence resonance energy transfer between GFP and Renilla luciferase chimeras and bimolecular fluorescence complementation between YFP half-molecule chimeras. These techniques revealed that αIIbβ3 interacts with c-Src at the periphery of nonadherent CHO cells. After plating cells on fibrinogen, complexes of αIIbβ3–c-Src, αIIbβ3–Syk, and c-Src–Syk are observed in membrane ruffles and focal complexes, and the interactions involving Syk require Src activity. In contrast, FAK interacts with αIIbβ3 and c-Src, but not with Syk, in focal complexes and adhesions. All of these interactions require the integrin β3 cytoplasmic tail. Thus, αIIbβ3 interacts proximally, if not directly, with tyrosine kinases in a coordinated, selective, and dynamic manner during sequential phases of αIIbβ3 signaling to the actin cytoskeleton.
signaling; Src; Syk; FAK; bioluminescence
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.
titin; palladin; MyBP-C; myotilin; myopalladin
Myotilin, palladin, and myopalladin form a novel small subfamily of cytoskeletal proteins that contain immunoglobulin-like domains. Myotilin is a thin filament-associated protein localized at the Z-disk of skeletal and cardiac muscle cells. The direct binding to F-actin, efficient cross-linking of actin filaments, and prevention of induced disassembly of filaments are key roles of myotilin that are thought to be involved in structural maintenance and function of the sarcomere. Missense mutations in the myotilin-encoding gene cause dominant limb girdle muscular dystrophy type 1A and spheroid body myopathy and are the molecular defect that can cause myofibrillar myopathy. Here we describe the generation and analysis of mice that lack myotilin, myo−/− mice. Surprisingly, myo−/− mice maintain normal muscle sarcomeric and sarcolemmal integrity. Also, loss of myotilin does not cause alterations in the heart or other organs of newborn or adult myo−/− mice. The mice develop normally and have a normal life span, and their muscle capacity does not significantly differ from wild-type mice even after prolonged physical stress. The results suggest that either myotilin does not participate in muscle development and basal function maintenance or other proteins serve as structural and functional compensatory molecules when myotilin is absent.