Caldesmon phosphorylation in actin cytoskeletal remodeling

Caldesmon is an actin-binding protein that is capable of stabilizing actin filaments against actin-severing proteins, inhibiting actomyosin ATPase activity, and inhibiting Arp2/3-mediated actin polymerization in vitro. Caldesmon is a substrate of cdc2 kinase and Erk1/2 MAPK, and phosphorylation by either of these kinases reverses the inhibitory effects of caldesmon. Cdc2-mediated caldesmon phosphorylation and the resulting dissociation of caldesmon from actin filaments are essential for M-phase progression during mitosis. Cells overexpressing the actin-binding carboxyterminal fragment of caldesmon fail to release the fragment completely from actin filaments during mitosis, resulting in a higher frequency of multinucleated cells. PKC-mediated MEK/Erk/caldesmon phosphorylation is an important signaling cascade in the regulation of smooth muscle contraction. Furthermore, PKC activation has been shown to remodel actin stress fibers into F-actin-enriched podosome columns in cultured vascular smooth muscle cells. Podosomes are cytoskeletal adhesion structures associated with the release of metalloproteases and degradation of extracellular matrix during cell invasion. Interestingly, caldesmon is one of the few actin-binding proteins that is associated with podosomes but excluded from focal adhesions. Caldesmon also inhibits the function of gelsolin and Arp2/3 complex that are essential for the formation of podosomes. Thus, caldesmon appears to be well positioned for playing a modulatory role in the formation of podosomes. Defining the roles of actin filament-stabilizing proteins such as caldesmon and tropomyosin in the formation of podosomes should provide a more complete understanding of molecular systems that regulate the remodeling of the actin cytoskeleton in cell transformation and invasion.     

Changes in the balance between caldesmon regulated by p21-activated kinases and the Arp2/3 complex govern podosome formation

Podosomes are dynamic cell adhesion structures that degrade the extracellular matrix, permitting extracellular matrix remodeling. Accumulating evidence suggests that actin and its associated proteins play a crucial role in podosome dynamics. Caldesmon is localized to the podosomes, and its expression is down-regulated in transformed and cancer cells. Here we studied the regulatory mode of caldesmon in podosome formation in Rous sarcoma virus-transformed fibroblasts. Exogenous expression analyses revealed that caldesmon represses podosome formation triggered by the N-WASP-Arp2/3 pathway. Conversely, depletion of caldesmon by RNA interference induces numerous small-sized podosomes with high dynamics. Caldesmon competes with the Arp2/3 complex for actin binding and thereby inhibits podosome formation. p21-activated kinases (PAK)1 and 2 are also repressors of podosome formation via phosphorylation of caldesmon. Consequently, phosphorylation of caldesmon by PAK1/2 enhances this regulatory mode of caldesmon. Taken together, we conclude that in Rous sarcoma virus-transformed cells, changes in the balance between PAK1/2-regulated caldesmon and the Arp2/3 complex govern the formation of podosomes.     

Conventional protein kinase C mediates phorbol-dibutyrate-induced cytoskeletal remodeling in a7r5 smooth muscle cells

Phorbol dibutyrate (PDBu) induced the formation of podosome-like structures together with partial disassembly of actin stress fibers in A7r5 smooth muscle cells. These podosomes contained alpha-actinin, F-actin, and vinculin and exhibit a tubular, column-like structure arising perpendicularly from the bottom of PDBu-treated cells. The conventional protein kinase C (PKC) antagonist, GO6976, inhibited PDBu-induced cytoskeletal remodeling at 0.1 microM, whereas the novel PKC antagonist, rottlerin, was ineffective at 10 microM. PDBu induced the translocation of the conventional PKC-alpha but not the novel PKC-delta to the sites of podosome formation in A7r5 cells. Although partial disassembly of actin stress fibers was observed in both Y-27632- and PDBu-treated cells, focal adhesions were much reduced in number and size only in Y-27632-treated cells. Furthermore, PDBu restored focal adhesions in Y-27632-treated cells. Live video fluorescence microscopy of alpha-actinin GFP revealed a lag phase of about 20 min prior to the rapid formation and dynamic reorganization of podosomes during PDBu treatment. These findings suggest that conventional PKCs mediate PDBu-induced formation of dynamic podosome-like structures in A7r5 cells, and Rho-kinase is unlikely to be the underlying mechanism. The podosome columns could represent molecular scaffolds where PKC-alpha phosphorylates regulatory proteins necessary for Ca(2+) sensitization in smooth muscle cells.     

Phosphorylation of cortactin by p21-activated kinase

Cortactin is an F-actin binding protein that is enriched in dynamic cytoskeletal organelles such as podosomes, membrane ruffles, and lamellipodia. We have shown previously that Src-phosphorylation of cortactin is not required for its translocation to phorbol-ester induced podosomes in A7r5 aortic smooth muscle cells, but may be important for stability and turnover of podosomes. However, little is known of the role of Ser/Thr kinases in the regulation of cortactin. Here, we report that p21-associated kinase (PAK), which plays a crucial role in the formation of podosome and membrane ruffles, is able to phosphorylate cortactin in vitro. The predominant phosphorylation site is located at Ser113 in the first actin-binding repeat. Phosphorylation by PAK is not required for the translocation of cortactin to podosomes, lamellipodia, or membrane ruffles in A7r5 smooth muscle cells. However, binding of cortactin to F-actin is significantly reduced by PAK-phosphorylation. Taken together, these results suggest a role for PAK-phosphorylation of cortactin in the regulation of the dynamics of branched actin filaments in dynamic cytoskeletal organelles.     

Effects of tyrosine phosphorylation of cortactin on podosome formation in A7r5 vascular smooth muscle cells

Cortactin, a predominant substrate of Src family kinases, plays an important role in Arp2/3-dependent actin polymerization in lamellipodia and membrane ruffles and was recently shown to be enriched in podosomes induced by either c-Src or phorbol ester. However, the mechanisms by which cortactin regulates podosome formation have not been determined. In this study, we showed that cortactin is required for podosome formation, using siRNA knockdown of cortactin expression in smooth muscle A7r5 cells. Treatment with phorbol ester or expression of constitutively active c-Src induced genesis of cortactin-containing podosomes as well as increase in phosphorylation of cortactin at Y421 and Y466, the Src phosphorylation sites on cortactin. The Src kinase inhibitor SU-6656 significantly inhibited formation of podosomes induced by phorbol ester and phosphorylation of cortactin, whereas PKC inhibitor did not affect podosome formation in c-Src-transfected cells. Unexpectedly, expression of cortactin mutants containing Y421F, Y421D, Y466F, or Y466D mutated sites did not affect podosome formation or cortactin translocation to podosomes, although endogenous tyrosine-phosphorylated cortactin at Y421 and Y466 was present in podosomes. Our data indicate that 1) PKC acts upstream of Src in phosphorylation of cortactin and podosome formation in smooth muscle cells; 2) expression of cortactin is essential for genesis of podosomes; 3) phosphorylation at Y421 and Y466 is not required for translocation of cortactin to podosomes, although phosphorylation at these sites appears to be enriched in podosomes; and 4) tyrosine phosphorylation of cortactin may be involved in regulation of stability and turnover of podosomes, rather than targeting this protein to the site of podosome formation. actin cytoskeleton; Src; protein kinase C     

Mechanism of fluoride-induced MAP kinase activation in pulmonary artery endothelial cells

In this study, we demonstrate that challenge of endothelial cells (EC) with NaF, a recognized G protein activator and protein phosphatase inhibitor, leads to a significant Erk activation, with increased phosphorylation of the well-known Erk substrate caldesmon. Inhibition of the Erk MAPK, MEK, by U0126 produces a marked decrease in NaF-induced caldesmon phosphorylation. NaF transiently increases the activity of the MEK kinase known as Raf-1 (approximately 3- to 4-fold increase over basal level), followed by a sustained Raf-1 inhibition (approximately 3- to 4-fold decrease). Selective Raf-1 inhibitors (ZM-336372 and Raf-1 inhibitor 1) significantly attenuate NaF-induced Erk and caldesmon phosphorylation. Because we have previously shown that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) participates in Erk activation in thrombin-challenged cells, we next explored if CaMKII is involved in NaF-induced EC responses. We found that in NaF-treated EC, CaMKII activity increases in a time-dependent manner with maximal activity at 10 min (approximately 4-fold increase over a basal level). Pretreatment with KN93, a specific CaMKII inhibitor, attenuates NaF-induced barrier dysfunction and Erk phosphorylation. The Rho inhibitor C3 exotoxin completely abolishes NaF-induced CaMKII activation. Collectively, these data suggest that sequential activation of Raf-1, MEK, and Erk is modulated by Rho-dependent CaMKII activation and represents important NaF-induced signaling response. Caldesmon phosphorylation occurring by an Erk-dependent mechanism in NaF-treated pulmonary EC may represent a link between NaF stimulation and contractile responses of endothelium.     

Lasp-1 regulates podosome function

Eukaryotic cells form a variety of adhesive structures to connect with their environment and to regulate cell motility. In contrast to classical focal adhesions, podosomes, highly dynamic structures of different cell types, are actively engaged in matrix remodelling and degradation. Podosomes are composed of an actin-rich core region surrounded by a ring-like structure containing signalling molecules, motor proteins as well as cytoskeleton-associated proteins. Lasp-1 is a ubiquitously expressed, actin-binding protein that is known to regulate cytoskeleton architecture and cell migration. This multidomain protein is predominantely present at focal adhesions, however, a second pool of Lasp-1 molecules is also found at lamellipodia and vesicle-like microdomains in the cytosol.In this report, we show that Lasp-1 is a novel component and regulator of podosomes. Immunofluorescence studies reveal a localization of Lasp-1 in the podosome ring structure, where it colocalizes with zyxin and vinculin. Life cell imaging experiments demonstrate that Lasp-1 is recruited in early steps of podosome assembly. A siRNA-mediated Lasp-1 knockdown in human macrophages affects podosome dynamics as well as their matrix degradation capacity. In summary, our data indicate that Lasp-1 is a novel component of podosomes and is involved in the regulation of podosomal function.     

MEK/ERK pathway mediates PKC activation‐induced recruitment of PKCζ and MMP‐9 to podosomes

Podosomes are adhesive structures on the ventral surface of cells that invade and degrade the extracellular matrix. Recently, we reported that phorbol 12,13‐dibutyrate (PDBu), a protein kinase C (PKC) activator, induced podosome formation in normal human bronchial epithelial (NHBE) cells, and atypical PKCζ regulated MMP‐9 recruitment to podosomes for its release and activation. The objective of this study was to explore signaling pathways that are involved in PKC activation‐induced podosome formation and matrix degradation. Herein, we found that PDBu increased phosphorylation of PI3K p85, Akt, Src, ERK1/2, and JNK. Inhibitors for PI3K, Akt, and Src suppressed PDBu‐induced podosome formation and matrix degradation. In contrast, blockers for MEK/ERK or JNK did not inhibit podosome formation but reduced proteolytic activity of podosomes. Inhibition of PKCζ activity with its pseudosubstrate peptide (PS)‐inhibited PDBu‐induced phosphorylation of MEK/ERK and JNK. On the other hand, inhibition of MEK/ERK or JNK pathway did not affect PKCζ phosphorylation, but reduced the recruitment of PKCζ and MMP‐9 to podosomes. We conclude that PKCζ may regulate MEK/ERK and JNK phosphorylation and in turn activated MEK/ERK and JNK may regulate the proteolytic activity of PDBu‐induced podosomes by influencing the recruitment of PKCζ and MMP‐9 to podosomes. J. Cell. Physiol. 228: 416–427, 2013. © 2012 Wiley Periodicals, Inc.     

Cortactin regulates podosome formation: roles of the protein interaction domains

Cortactin, a multi-domain scaffolding protein involved in actin polymerization, is enriched in podosomes induced by phorbol ester in vascular smooth muscle cells. We generated several functional and truncation mutants of cortactin to probe the roles of various protein interaction domains in the regulation of the dynamics of podosome formation. At the onset of podosome genesis, cortactin clustered near the ends of stress fibers that appeared to act as nucleation platforms onto which the actin polymerization machinery assembled. Translocation of cortactin to these pre-podosome clusters required the intact N-WASp-binding SH3 domain. Overexpression of the C-terminal third of cortactin containing the intact SH3 domain inhibited podosome formation presumably by sequestering of N-WASp and prevented cortactin clustering. Subsequent assembly of the actin-rich core of podosomes required translocation of additional cortactin to the actin core, a process that required the actin-binding repeats, but not the Arp2/3-binding N-terminal acidic region nor the SH3 domain. These results suggest that the SH3 domain and the actin-binding repeat region are involved, respectively, in the early and late stages of podosome formation process.     

Caldesmon suppresses cancer cell invasion by regulating podosome/invadopodium formation

The podosome and invadopodium are dynamic cell-adhesion structures that degrade the extracellular matrix (ECM) and promote cell invasion. We recently reported that the actin-binding protein caldesmon is a pivotal regulator of podosome formation. Here, we analyzed the caldesmon's involvement in podosome/invadopodium-mediated invasion by transformed and cancer cells. The ectopic expression of caldesmon reduced the number of podosomes/invadopodia and decreased the ECM degradation activity, resulting in the suppression of cell invasion. Conversely, the depletion of caldesmon facilitated the formation of podosomes/invadopodia and cell invasion. Taken together, our results indicate that caldesmon acts as a potent repressor of cancer cell invasion.     

The kinesin KIF1C and microtubule plus ends regulate podosome dynamics in macrophages

Microtubules are important for the turnover of podosomes, dynamic, actin-rich adhesions implicated in migration and invasion of monocytic cells. The molecular basis for this functional dependency, however, remained unclear. Here, we show that contact by microtubule plus ends critically influences the cellular fate of podosomes in primary human macrophages. In particular, we identify the kinesin KIF1C, a member of the Kinesin-3 family, as a plus-end-enriched motor that targets regions of podosome turnover. Expression of mutation constructs or small interfering RNA-/short hairpin RNA-based depletion of KIF1C resulted in decreased podosome dynamics and ultimately in podosome deficiency. Importantly, protein interaction studies showed that KIF1C binds to nonmuscle myosin IIA via its PTPD-binding domain, thus providing an interface between the actin and tubulin cytoskeletons, which may facilitate the subcellular targeting of podosomes by microtubules. This is the first report to implicate a kinesin in podosome regulation and also the first to describe a function for KIF1C in human cells.     

EGFR phosphorylation-dependent formation of cell-cell contacts by Ras/Erks cascade inhibition

Cell-cell contacts play important roles in the homeostasis of normal epithelium and in the steps of metastasis of tumor cells, although signaling mechanisms to regulate cell-cell contacts are unclear. In this study, we observed that phenotype of no cell-cell contacts in rat intestinal epithelial cell subline (RIE1-Sca) correlated with increased Erk1/2 signaling activity, compared to that of parental RIE1 cells growing in colonies. Furthermore, cell-cell contacts between RIE1-Sca cells were reformed by treatment with a specific MEK inhibitor (U0126), with translocation of ZO1 and beta-catenin to cell-cell contacts, without changes of their expression levels. U0126 treatment also increased EGFR phosphorylation in a ligand-independent manner. Pretreatment with EGFR kinase inhibitor abolished U0126 treatment-mediated EGFR phosphorylation, and expression of dominant negative H-Ras N17 allowed EGFR phosphorylation and cell-cell contacts even without U0126 treatment. Furthermore, the expression of a nonphosphorylatable EGFR Y5F mutant abolished U0126-mediated cell-cell contacts. U0126 treatment also caused less efficient wound healing by keeping monolayer integrity intact, compared to control untreated cells. This U0126-mediated reduction in wound healing was further altered either by pretreatment of EGFR kinase inhibitor or expression of H-Ras N17 or EGFR Y5F. Taken together, this study supports a unique mechanism of cell-cell contact formation through MEK/Erks inhibition-mediated EGFR phosphorylation.     

The Novel Adaptor Protein Tks4 (SH3PXD2B) Is Required for Functional Podosome Formation

Metastatic cancer cells have the ability to both degrade and migrate through the extracellular matrix (ECM). Invasiveness can be correlated with the presence of dynamic actin-rich membrane structures called podosomes or invadopodia. We showed previously that the adaptor protein tyrosine kinase substrate with five Src homology 3 domains (Tks5)/Fish is required for podosome/invadopodia formation, degradation of ECM, and cancer cell invasion in vivo and in vitro. Here, we describe Tks4, a novel protein that is closely related to Tks5. This protein contains an amino-terminal Phox homology domain, four SH3 domains, and several proline-rich motifs. In Src-transformed fibroblasts, Tks4 is tyrosine phosphorylated and predominantly localized to rosettes of podosomes. We used both short hairpin RNA knockdown and mouse embryo fibroblasts lacking Tks4 to investigate its role in podosome formation. We found that lack of Tks4 resulted in incomplete podosome formation and inhibited ECM degradation. Both phenotypes were rescued by reintroduction of Tks4, whereas only podosome formation, but not ECM degradation, was rescued by overexpression of Tks5. The tyrosine phosphorylation sites of Tks4 were required for efficient rescue. Furthermore, in the absence of Tks4, membrane type-1 matrix metalloproteinase (MT1-MMP) was not recruited to the incomplete podosomes. These findings suggest that Tks4 and Tks5 have overlapping, but not identical, functions, and implicate Tks4 in MT1-MMP recruitment and ECM degradation.     

PAK4 and alphaPIX determine podosome size and number in macrophages through localized actin regulation

Podosomes are actin-rich adhesion structures typical for monocytic cells and are implicated in migration and invasion. Major modes of podosome regulation include RhoGTPase signaling and actin regulatory pathways. However, it is not clearly understood how these signals induce highly localized changes in podosome formation and dynamics. Here, we show that the RhoGTPase effector PAK4, a member of the p21 associated kinase family, and its regulator alphaPIX (PAK-interacting exchange factor), are central to podosome formation in primary human macrophages. Immunofluorescence, biochemical and microarray data indicate that PAK4 acts as physiological regulator of podosomes in this system. Accordingly, transfection of a specific shRNA, as well as expression of PAK4 truncation mutants, resulted in reduced numbers of podosomes per cell. Moreover, expression of kinase active or inactive PAK4 mutants enhanced or reduced the size of individual podosomes, respectively, indicating a modulatory influence of PAK4 kinase activity on podosome size. Similar to the results gained with PAK4, cellular/overexpressed PIX was shown to be closely associated with podosomes. Moreover, both overexpression of alphaPIX wt and a mutant lacking the SH3 domain led to coalescence of podosomes. In sum, we propose that PAK4 and alphaPIX can induce highly localized changes in actin dynamics and thereby regulate size and number of podosomes in primary human macrophages.     

Plectin deposition at podosome rings requires myosin contractility

Metalloproteinase-dependent tissue invasion requires the formation of podosomes and invadopodia for localized matrix degradation. Actin cytoskeleton remodeling via Arp2/3-mediated actin polymerization is essential for podosome formation, and dynamic microtubules have an important role in maintaining podosome turnover in macrophages and osteoclasts. Little is known, however, about the involvement of the intermediate filament cytoskeleton in formation, stabilization, and turnover of podosomes. Here we show that vimentin intermediate filaments colocalize with the early sites of podosome formation at the stress fiber - focal adhesion interface in cultured vascular smooth muscle cells, but do not directly contribute to podosome formation, or stabilization. In unstimulated A7r5 cells the cytolinker protein plectin poorly colocalized with vimentin and the microdomains, but following induction by phorbol ester accumulated in the rings that surround the podosomes. In plectin-deficient A7r5 cells actin stress fiber remodelling is reduced in response to PDBu, and small podosomes remain localized at stable actin stress fibres. Pharmacological inhibition of actomyosin contractility by blebbistatin leads to an aberrant localization of podosomes away from the cell periphery and induces failure of plectin to surround the outer perimeter of these invasive adhesions. Taken together, we conclude that plectin is involved in growth and maturation of podosomes by reducing focal adhesion and stress fiber turnover, and that actomyosin-dependent contractility is required for the peripheral localization and specific deposition of plectin at the podosome rings.     

The podosome cap: past,present, perspective

Podosomes are prominent actin-based adhesion structures in a variety of cell types. They feature an extensive repertoire of functions, which requires exquisite spatiotemporal fine-tuning. Accordingly, podosomes consist of hundreds of different components, which fulfill specific structural and regulatory functions. Moreover, it has become apparent that podosome architecture is more intricate than previously believed. The classical model of an actin-rich core surrounded by a ring structure containing adhesion plaque proteins thus had to be expanded, and several additional substructures have been described, most notably the podosome cap on top of the actin-rich core. Here, we discuss the known components of the podosome cap, the history of their detection and their potential regulatory roles in podosome turnover and function. We also point out strategies for identifying further cap components and present a new model for the podosome cap as a multilayered module that fine-tunes actomyosin contractility, a central requirement for podosome architecture, dynamics and function.     

The circle of life: Phases of podosome formation,turnover and reemergence

Podosomes are highly dynamic actin-rich structures in a variety of cell types, especially monocytic cells. They fulfill multiple functions such as adhesion, mechanosensing, or extracellular matrix degradation, thus allowing cells to detect and respond to a changing environment. These abilities are based on an intricate architecture that enables podosomes to sense mechanical properties of their substratum and to transduce them intracellularly in order to generate an appropriate cellular response. These processes are enabled through the tightly orchestrated interplay of more than 300 different components that are dynamically recruited during podosome formation and turnover. In this review, we discuss the different phases of the podosome life cycle and the current knowledge on regulatory factors that impact on the genesis, activity, dissolution and reemergence of podosomes. We also highlight mechanoregulatory processes that become important during these different stages, on the level of individual podosomes, and also at podosome sub- and superstructures.     

Podosomes: adhesion hot-spots of invasive cells

Podosomes are highly dynamic, actin-rich adhesion structures of monocyte-derived cells, certain transformed fibroblasts and carcinoma cells and have recently also been discovered in an increasing number of other cell types. Because they are found mainly in motile cells and control the activity of matrix metalloproteases, podosomes are thought to contribute to tissue invasion and matrix remodeling. Importantly, podosomes are physiologically relevant organelles because they can be found in ex vivo models of invasive cells. Regulators of podosome turnover include tyrosine kinases, RhoGTPases, actin regulators and the microtubule system. Podosomes might also serve as an attractive model to study how integration of various signaling pathways controls actin dynamics. Here, we summarize and discuss the known structural, regulatory and functional features of podosomes, our aim being to stimulate further research into these unique structures.     

CSF-1 and PI 3-kinase regulate podosome distribution and assembly in macrophages

Podosomes are actin-rich adhesive foci found in several cell types, including macrophages. They have a core containing actin and actin-binding proteins and a peripheral ring of integrins and associated proteins. We show that podosomes are abundant in polarized mouse bone marrow-derived macrophages (BMM) and are found primarily in lamellae. We investigated the effects of CSF-1, which induces membrane ruffling, cell spreading, and subsequent polarization and migration, on podosome formation. CSF-1 induces a transient increase in podosome number and enhances the formation of circular arrays of podosomes. Conversely, CSF-1 withdrawal leads to a reduction in podosomes and a decrease in polarized cells. The PI 3-kinase inhibitor LY294002 induces loss of podosomes together with rapid retraction of lamellae and loss of polarity. Our results indicate that CSF-1 acts via PI 3-kinase to enhance podosome assembly and that this is linked to macrophage polarization.