The tumor suppressor LATS2 reduces v-Src-induced membrane blebs in a kinase activity-independent manner.

When cells with excess DNA, such as tetraploid cells, undergo cell division, it can contribute to cellular transformation via asymmetrical chromosome segregation-generated genetic diversity. Cell cycle progression of tetraploid cells is suppressed by large tumor suppressor 2 (LATS2) kinase-induced inhibitory phosphorylation of the transcriptional coactivator Yes-associated protein (YAP). We recently reported that the oncogene v-Src induces tetraploidy and promotes cell cycle progression of tetraploid cells by suppressing LATS2 activity. We explore here the mechanism by which v-Src suppresses LATS2 activity and the role of LATS2 in v-Src-expressing cells. LATS2 was directly phosphorylated by v-Src and the proto-oncogene c-Src, resulting in decreased LATS2 kinase activity. This kinase-deficient LATS2 accumulated in a YAP transcriptional activity-dependent manner, and knockdown of either LATS2 or the LATS2-binding partner moesin-ezrin-radixin-like protein (Merlin) accelerated v-Src-induced membrane bleb formation. Upon v-Src expression, the interaction of Merlin with LATS2 was increased possibly due to a decrease in Merlin phosphorylation at Ser518, the dephosphorylation of which is required for the open conformation of Merlin and interaction with LATS2. LATS2 was colocalized with Merlin at the plasma membrane in a manner that depends on the Merlin-binding region of LATS2. The bleb formation in v-Src-expressing and LATS2-knockdown cells was rescued by the reexpression of wild-type or kinase-dead LATS2 but not the LATS2 mutant lacking the Merlin-binding region. These results suggest that the kinase-deficient LATS2 plays a role with Merlin at the plasma membrane in the maintenance of cortical rigidity in v-Src-expressing cells, which may cause tumor suppression.

Phosphorylation of moesin by Jun N-terminal kinase is important for podosome rosette formation in Src-transformed fibroblasts.

Podosomes are actin-based membrane protrusions that facilitate extracellular matrix degradation and motility of invasive cells. Podosomes can self-organize into large rosette-like structures in Src-transformed fibroblasts, osteoclasts and some highly invasive cancer cells. However, the mechanism of this assembly remains obscure. In this study, we show that the suppression of Jun N-terminal kinase (JNK) by the JNK inhibitor SP600125 or short-hairpin RNA inhibited podosome rosette formation in SrcY527F-transformed NIH3T3 fibroblasts. In addition, SrcY527F was less able to induce podosome rosettes in JNK1-null or JNK2-null mouse embryo fibroblasts than in wild-type counterparts. The kinase activity of JNK was essential for promoting podosome rosette formation but not for its localization to podosome rosettes. Moesin, a member of the ERM (ezrin, radixin and moesin) protein family, was identified as a substrate of JNK. We show that the phosphorylation of moesin at Thr558 by JNK was important for podosome rosette formation in SrcY527F-transformed NIH3T3 fibroblasts. Taken together, our results unveil a novel role of JNK in podosome rosette formation through the phosphorylation of moesin.

Induction of membrane circular dorsal ruffles requires co-signalling of integrin-ILK-complex and EGF receptor.

Integrin and receptor tyrosine kinase signalling networks cooperate to regulate various biological functions. The molecular details underlying the integration of both signalling networks remain largely uncharacterized. Here we identify a signalling module composed of a fibronectin-α5ß1-integrin-integrin-linked-kinase (ILK) complex that, in concert with epidermal growth factor (EGF) cues, cooperatively controls the formation of transient actin-based circular dorsal ruffles (DRs) in fibroblasts. DR formation depends on the precise spatial activation of Src at focal adhesions by integrin and EGF receptor signals, in an ILK-dependent manner. In a SILAC-based phosphoproteomics screen we identified the tumour-suppressor Cyld as being required for DR formation induced by α5ß1 integrin and EGF receptor co-signalling. Furthermore, EGF-induced Cyld tyrosine phosphorylation is controlled by integrin-ILK and Src as a prerequisite for DR formation. This study provides evidence for a novel function of integrin-ILK and EGF signalling crosstalk in mediating Cyld tyrosine phosphorylation and fast actin-based cytoskeletal rearrangements.

Phorbol ester-induced podosomes in normal human bronchial epithelial cells.

Spreading and migration of the basal cells neighboring a wound is essential for airway epithelial repair. To gain insight into the molecular mechanisms that govern these cellular processes, we asked whether normal human airway epithelial cells can form podosomes, a cellular structure discovered from cancer and mesenchymal cells that controls migration and invasion. Herein, we report that phorbol-12, 13-dibutyrate (PDBu), a protein kinase C activator, induced reorganization of cytoskeletal structure in primary normal human bronchial epithelial cells, and in normal human airway epithelial BEAS2B cells. Z-stack scanning confocal microscopy showed that PDBu-induced podosome-like structures contain actin-rich columns that arise from the ventral surface of the cell, and also revealed the presence of circular ruffles/waves at the dorsal cell surface. The molecular components of these cytoskeletal structures were determined with immunofluorescent staining. Using in situ zymography, we demonstrated that PDBu-induced podosomes were capable of degrading fibronectin-gelatin-sucrose matrix. PDBu also increased epithelial cell invasion across Transwell chamber. Podosomes and circular dorsal ruffles may be important for epithelial cell migration and invasion, thus contributing to respiratory epithelial repair and regeneration.

Proximal, selective, and dynamic interactions between integrin alphaIIbbeta3 and protein tyrosine kinases in living cells.

Stable platelet aggregation, adhesion, and spreading during hemostasis are promoted by outside-in alphaIIbbeta3 signals that feature rapid activation of c-Src and Syk, delayed activation of FAK, and cytoskeletal reorganization. To evaluate these alphaIIbbeta3-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 alphaIIbbeta3 interacts with c-Src at the periphery of nonadherent CHO cells. After plating cells on fibrinogen, complexes of alphaIIbbeta3-c-Src, alphaIIbbeta3-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 alphaIIbbeta3 and c-Src, but not with Syk, in focal complexes and adhesions. All of these interactions require the integrin beta3 cytoplasmic tail. Thus, alphaIIbbeta3 interacts proximally, if not directly, with tyrosine kinases in a coordinated, selective, and dynamic manner during sequential phases of alphaIIbbeta3 signaling to the actin cytoskeleton.

Integrin linked kinase (ILK) regulates podosome maturation and stability in dendritic cells.

Podosomes are integrin-based adhesions fundamental for stabilisation of the leading lamellae in migrating dendritic cells (DCs) and for extracellular matrix (ECM) degradation. We have previously shown that soluble factors and chemokines such as SDF 1-a trigger podosome initiation whereas integrin ligands promote podosome maturation and stability in DCs. The exact intracellular signalling pathways that regulate the sequential organisation of podosomal components in response to extracellular cues remain largely undetermined. The Wiskott Aldrich Syndrome Protein (WASP) mediates actin polymerisation and the initial recruitment of integrins and associated proteins in a circular configuration surrounding the core of filamentous actin (F-actin) during podosome initiation. We have now identified integrin linked kinase (ILK) surrounding the podosomal actin core. We report that DC polarisation in response to chemokines and the assembly of actin cores during podosome initiation require PI3K-dependent clustering of the Wiskott Aldrich Syndrome Protein (WASP) in puncta independently of ILK. ILK is essential for the clustering of integrins and associated proteins leading to podosome maturation and stability that are required for degradation of the subjacent extracellular matrix and the invasive motility of DCs across connective tissue barriers. We conclude that WASP regulates DCs polarisation for migration and initiation of actin polymerisation downstream of PI3K in nascent podosomes. Subsequently, ILK mediates the accumulation of integrin-associated proteins during podosome maturation and stability for efficient degradation of the subjacent ECM during the invasive migration of DCs.

Secretory and endo/exocytic trafficking in invadopodia formation: the MT1-MMP paradigm.

Invadopodia are actin-rich, adhesive protrusions that extend into and remodel the extracellular matrix. They are associated with high levels of pericellular proteolysis and correlate with the invasive capacity of a variety of tumour cells. Invadopodia have, thus, been proposed to recapitulate key events of the metastatic process. Although our understanding of the patho-physiology of invadopodia is still in its infancy, the molecular components and signalling pathways leading to their formation have received increasing attention. Recent studies have revealed that diverse membrane polarized secretory and endo/exocytic trafficking pathways converge at these structures for the delivery, in a temporally controlled and spatially confined manner, of key proteolytic enzymes. Here, we will focus our attention on MT1-MMP, a paradigmatic metalloprotease that is primarily responsible for the proteolytic activity of invadopodia. We propose that the biosynthetic/secretory pathway might be critical for the polarized delivery of MT1-MMP to invadopodia that form as "default response" whenever cells have to deal with extracellular matrix (ECM) of variable composition and stiffness. Conversely, "inducible" endo/exocytic trafficking routes might primarily control the delivery of MT1-MMP to invadopodia when cells need to respond in a fast and transient manner to soluble motogenic factors, rather than the insoluble ECM.

Dissecting activation of the PAK1 kinase at protrusions in living cells.

The p21-activated kinase (PAK) 1 kinase, an effector of the Cdc42 and Rac1 GTPases, regulates cell protrusions and motility by controlling actin and adhesion dynamics. Its deregulation has been linked to human cancer. We show here that activation of PAK1 is necessary for protrusive activity during cell spreading. To investigate PAK1 activation dynamics at live protrusions, we developed a conformational biosensor, based on fluorescence resonance energy transfer. This novel PAK1 biosensor allowed the spatiotemporal visualization of PAK1 activation during spreading of COS-7 cells and during motility of normal rat kidney cells. By using this imaging approach in COS-7 cells, the following new insights on PAK1 regulation were unveiled. First, PAK1 acquires an intermediate semi-open conformational state upon recruitment to the plasma membrane. This semi-open PAK1 species is selectively autophosphorylated on serines in the N-terminal regulatory region but not on the critical threonine 423 in the catalytic site. Second, this intermediate PAK1 state is hypersensitive to stimulation by Cdc42 and Rac1. Third, interaction with PIX proteins contributes to PAK1 stimulation at membrane protrusions, in a GTPase-independent way. Finally, trans-phosphorylation events occur between PAK1 molecules at the membrane possibly playing a relevant role for its activation. This study leads to a model for the complex and accurate regulation of PAK1 kinase in vivo at cell protrusions.

ERK5 promotes Src-induced podosome formation by limiting Rho activation.

Increased Src activity, often associated with tumorigenesis, leads to the formation of invasive adhesions termed podosomes. Podosome formation requires the function of Rho family guanosine triphosphatases and reorganization of the actin cytoskeleton. In addition, Src induces changes in gene expression required for transformation, in part by activating mitogen-activated protein kinase (MAPK) signaling pathways. We sought to determine whether MAPK signaling regulates podosome formation. Unlike extracellular signal-regulated kinase 1/2 (ERK1/2), ERK5 is constitutively activated in Src-transformed fibroblasts. ERK5-deficient cells expressing v-Src exhibited increased RhoA activation and signaling, which lead to cellular retraction and an inability to form podosomes or induce invasion. Addition of the Rho-kinase inhibitor Y27632 to ERK5-deficient cells expressing v-Src led to cellular extension and restored podosome formation. In Src-transformed cells, ERK5 induced the expression of a Rho GTPase-activating protein (RhoGAP), RhoGAP7/DLC-1, via activation of the transcription factor myocyte enhancing factor 2C, and RhoGAP7 expression restored podosome formation in ERK5-deficient cells. We conclude that ERK5 promotes Src-induced podosome formation by inducing RhoGAP7 and thereby limiting Rho activation.

Src-dependent phosphorylation of ASAP1 regulates podosomes.

Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.

P-glycoprotein and 'lipid rafts': some ambiguous mutual relationships (floating on them, building them or meeting them by chance?).

P-glycoprotein (P-gp) is an active membrane transporter responsible for cell detoxification against numerous amphiphilic compounds, leading to multidrug resistance in tumor cells. It displays entangled connections with its membrane environment since it recognizes its substrates within the cytosolic leaflet and it also translocates some endogenous lipids to the exoplasmic leaflet. Regarding its relationships with membrane microdomains, 'lipid rafts', a literature analysis concludes that (i) P-gp also exists in rafts and non-raft membrane domains, depending on the cell considered, the experimental conditions and the method used to test it; (ii) cholesterol has a positive influence on P-gp function, and this may be a direct effect of the free cholesterol present in membrane or an indirect effect mediated by the cholesterol-enriched microdomains; (iii) when present in rafts, P-gp interacts with protein partners regulating its activity; (iv) P-gp is a lipid translocase that handles the raft-constituting lipids with particular efficiency, and it also influences membrane trafficking in the cell.

Modeling the cascade of enzymatic reactions in liposomes including successive free radical peroxidation, reduction, and hydrolysis of phospholipid polyenoic acyls for studying the effect of these processes on the structural-dynamic parameters of the membranes.

Studies of the effect of primary products of free radical lipid peroxidation (LPO) on the structural-dynamic parameters of natural lipid-protein supramolecular complexes (biomembranes and blood serum lipoproteins) using standard inducers of radical processes in vitro (azo-initiators, transition metal ions, flavin oxidases, etc.) are impossible because of simultaneous production of numerous secondary LPO products that can induce structural changes. The data obtained suggest that phospholipid liposome microviscosity, as assessed by the extent of eximerization of the fluorescent probe pyrene, may significantly differ when oxidation is induced by animal C-15 lipoxygenase (yielding acylhydroperoxides only) and Fe(2+)-ascorbate system (resulting in simultaneous accumulation of primary and secondary LPO products). It is also shown that liver glutathione S-transferase can effectively reduce hydroperoxy-acyls in phospholipid liposomes and liver microsomes without their preliminary hydrolysis with phospholipase A(2). An enzymatic system is proposed for a cascade of enzymatic reactions simulating lipohydroperoxide metabolism in living cells, including successive free radical oxidation of phosphatidylcholine polyenoic acyls, reduction of their hydroperoxy-derivatives, and hydrolysis of fatty acid residues in the course of catalysis mediated by animal C-15 lipoxygenase, glutathione S-transferase, and phospholipase A(2), respectively.

Rho GTPases link cytoskeletal rearrangements and activation processes induced via the tetraspanin CD82 in T lymphocytes.

Activation of T lymphocytes requires the engagement of the T-cell receptor and costimulation molecules through cell-to-cell contacts. The tetraspanin CD82 has previously been shown to act as a cytoskeleton-dependent costimulation molecule. We show here that CD82 engagement leads to the tyrosine phosphorylation and association of both the Rho GTPases guanosine exchange factor Vav1 and adapter protein SLP76, suggesting that Rho GTPases participate in CD82 signaling. Indeed, broad inactivation of all Rho GTPases, or a specific blockade of RhoA, Rac1 or Cdc42, inhibited the morphological changes linked to CD82 engagement but failed to modulate the inducible association of CD82 with the actin network. Rho GTPase inactivation, as well as actin depolymerization, reduced the ability of CD82 to phosphorylate Vav and SLP76 and to potentiate the phosphorylation of two early TcR signaling intermediates: the tyrosine kinases ZAP70 and membrane adapter LAT. Taken together, this suggests that an amplification loop, via early Vav and SLP76 phosphorylations and Rho-GTPases activation, is initiated by CD82 association with the cytoskeleton, which permits cytoskeletal rearrangements and costimulatory activity. Moreover, the involvement of CD82 in the formation of the immunological synapse is strongly suggested by its accumulation at the site of TcR engagement. This novel link between a tetraspanin and the Rho GTPase cascade could explain why tetraspanins, which are known to form heterocomplexes, are involved in cell activation, adhesion, growth and metastasis.