v-Src-dependent down-regulation of the Ste20-like kinase SLK by casein kinase II

We have previously shown that the Ste20-like kinase SLK is a microtubule-associated protein inducing actin stress fiber disassembly. Here, we show that v-Src expression can down-regulate SLK activity. This down-regulation is independent of focal adhesion kinase but requires v-Src kinase activity and membrane translocation. SLK down-regulation by v-Src is indirect and is accompanied by SLK hyperphosphorylation on serine residues. Deletion analysis revealed that casein kinase II (CK2) sites at position 347/348 are critical for v-Src-dependent modulation of SLK activity. Further studies show that CK2 can directly phosphorylate SLK at these positions and that inhibition of CK2 in v-Src-transformed cells results in normal kinase activity. Finally, CK2 and SLK can be co-localized in fibroblasts spreading on fibronectin-coated substrates, suggesting a mechanism whereby SLK may be regulated at sites of actin remodeling, such as membrane lamellipodia and ruffles, through CK2.     

Diverse effects of RacV12 on cell transformation by Raf: partial inhibition of morphological transformation versus deregulation of cell cycle control

Activated Raf kinases and Rac GTPases were shown to cooperate in the oncogenic transformation of fibroblasts, which is characterised by the disassembly of the cellular actin cytoskeleton, a nearly complete loss of focal adhesion complexes and deregulated cell proliferation. This is surprising since the Rac GTPase induces actin structures and the adhesion of suspended cells to extracellular matrix proteins. NIH 3T3 cells expressing a hydroxytamoxifen-inducible oncogenic c-Raf-1-oestrogen receptor fusion protein (c-Raf-1-BxB-ER, N-BxB-ER cells) undergo morphological transformation upon stimulation of the Raf kinase. We show that treatment with the Rac, Rho and Cdc42 activating Escherichia coli toxin CNF1 or coexpression of an activated RacV12 mutant partially inhibits and reverses the disassembly of cellular actin structures and focal adhesion complexes by oncogenic Raf. Activation of the Rac GTPase restores actin structures and focal adhesion complexes at the cellular boundary, leading to spreading of the otherwise spindle-shaped Raf-transformed cells. Actin stress fibres, however, which are regulated by the function of the Rho GTPase, are disassembled by oncogenic Raf even in the presence of activated Rac and Rho. With respect to the RacV12-mediated spreading of Raf-transformed cells, we postulate an anti-oncogenic function of the activated Rac. Another feature of cell transformation is the deregulation of cell cycle control. NIH 3T3 cells expressing high levels of the c-Raf-1-BxB-ER protein undergo a cell cycle arrest upon stimulation of the oncogenic Raf kinase. Our results show that in N-BxB-ER-RacV12 cells the expression of the activated RacV12 mediates cell proliferation in the presence of high-intensity Raf signals and high levels of the Cdk inhibitor p21(Cip1). These results indicate a pro-oncogenic function of the Rac GTPase with respect to the deregulation of cell cycle control.     

Essential role of SRC suppressed C kinase substrates in Schwann cells adhesion, spreading and migration

Integrin-mediated substrate adhesion of endothelial cells leads to dynamic rearrangement of the actin cytoskeleton. Protein kinase C (PKC) stimulates reorganization of microfilaments and adhesion, while the responses of Schwann cells during adhesion and migration are unknown, so we examined the expression changes of SSeCKS and F-actin in Schwann cells after exposure to fibronectin. Src (sarcoma) suppressed C kinase substrate (SSeCKS) is a PKC substrate that may play an important role in regulating actin cytoskeleton. We found that SSeCKS was localized to focal adhesion sites soon after Schwann cells adhesion and that SSeCKS increased during the process of cell spreading. Using small interfering RNAs specific to SSeCKS, we showed that Schwann cells in which SSeCKS expression was inhibited reduced cellular adhesion, spreading and promoted cellular migration on fibronectin through reorganization of actin stress fibers and blocking formation of focal adhesions. These results demonstrated SSeCKS modulate Schwann cells adhesion, spreading and migration by reorganization of the actin cytoskeleton.     

FAK/src-family dependent activation of the Ste20-like kinase SLK is required for microtubule-dependent focal adhesion turnover and cell migration

Cell migration involves a multitude of signals that converge on cytoskeletal reorganization, essential for development, immune responses and tissue repair. Using knockdown and dominant negative approaches, we show that the microtubule-associated Ste20-like kinase SLK is required for focal adhesion turnover and cell migration downstream of the FAK/c-src complex. Our results show that SLK co-localizes with paxillin, Rac1 and the microtubules at the leading edge of migrating cells and is activated by scratch wounding. SLK activation is dependent on FAK/c-src/MAPK signaling, whereas SLK recruitment to the leading edge is src-dependent but FAK independent. Our results show that SLK represents a novel focal adhesion disassembly signal.     

Integrin-mediated Signals Regulated by Members of the Rho Family of GTPases

The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, we examined the role of Cdc42-, Rac-, and Rho-dependent signaling pathways in regulating the cytoskeleton during integrin-mediated adhesion and cell spreading using dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation, we observed some phosphorylation of the focal adhesion kinase (FAK) and Src, which occurred independently of Rho family GTPases. However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Moreover, we found Rho dependence of the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; we show that this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Furthermore, we observed that Cdc42 controls the integrin-dependent activation of extracellular signal–regulated kinase 2 and of Akt, a kinase whose activity has been demonstrated to be dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, we observed that all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior.     

A novel role for the Ste20 kinase SLK in adhesion signaling and cell migration

With over 60 members, the Sterile 20 family of kinases has been implicated in numerous biological processes, including growth, survival, apoptosis and cell migration. Recently, we have shown that, in addition to cell death, the Ste20-like kinase SLK is required for efficient cell migration in fibroblasts. We have observed that SLK is involved in cell motility through its effect on actin reorganization and microtubule-induced focal adhesion turnover. Scratch wounding of confluent monolayers results in SLK activation. The induction of SLK kinase activity requires the scaffold function of FAK and a MAPK-dependent pathway. However, its recruitment to the leading edge of migrating fibroblasts requires the activity of the Src family kinases. Since SLK is microtubule-associated, it may represent one of the signals delivered to focal contacts that induces adhesions turnover. A speculative model is proposed to illustrate the mechanism of SLK activation and recruitment at the leading edge of migrating cells.     

EGF-induced ERK-activation downstream of FAK requires rac1-NADPH oxidase

Reactive oxygen species (ROS) function as signaling molecules mainly by reversible oxidation of redox-sensitive target proteins. ROS can be produced in response to integrin ligation and growth factor stimulation through Rac1 and its effector protein NADPH oxidase. One of the central roles of Rac1-NADPH oxidase is actin cytoskeletal rearrangement, which is essential for cell spreading and migration. Another important regulator of cell spread is focal adhesion kinase (FAK), a coordinator of integrin and growth factor signaling. Here, we propose a novel role for NADPH oxidase as a modulator of the FAK autophosphorylation site. We found that Rac1-NADPH oxidase enhanced the phosphorylation of FAK at Y397. This site regulates FAK's ability to act as a scaffold for EGF-mediated signaling, including activation of ERK. Accordingly, we found that EGF-induced activation of FAK at Y925, the following activation of ERK, and phosphorylation of FAK at the ERK-regulated S910-site depended upon NADPH oxidase. Furthermore, the inhibition of NADPH oxidase caused excessive focal adhesions, which is in accordance with ERK and FAK being modulators of focal adhesion dissociation. Our data suggest that Rac1 through NADPH oxidase is part of the signaling pathway constituted by FAK, Rac1, and ERK that regulates focal adhesion disassembly during cell spreading.     

Stimulation of fascin spikes by thrombospondin-1 is mediated by the GTPases Rac and Cdc42

Cell adhesion to extracellular matrix is an important physiological stimulus for organization of the actin-based cytoskeleton. Adhesion to the matrix glycoprotein thrombospondin-1 (TSP-1) triggers the sustained formation of F-actin microspikes that contain the actin-bundling protein fascin. These structures are also implicated in cell migration, which may be an important function of TSP-1 in tissue remodelling and wound repair. To further understand the function of fascin microspikes, we examined whether their assembly is regulated by Rho family GTPases. We report that expression of constitutively active mutants of Rac or Cdc42 triggered localization of fascin to lamellipodia, filopodia, and cell edges in fibroblasts or myoblasts. Biochemical assays demonstrated prolonged activation of Rac and Cdc42 in C2C12 cells adherent to TSP-1 and activation of the downstream kinase p21-activated kinase (PAK). Expression of dominant-negative Rac or Cdc42 in C2C12 myoblasts blocked spreading and formation of fascin spikes on TSP-1. Spreading and spike assembly were also blocked by pharmacological inhibition of F-actin turnover. Shear-loading of monospecific anti-fascin immunoglobulins, which block the binding of fascin to actin into cytoplasm, strongly inhibited spreading, actin cytoskeletal organization and migration on TSP-1 and also affected the motility of cells on fibronectin. We conclude that fascin is a critical component downstream of Rac and Cdc42 that is needed for actin cytoskeletal organization and cell migration responses to thrombospondin-1.     

Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin.

Experiments were carried out to test the hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which has been secretd spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblasts attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence analysis using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared, and most fibronectin was subsequently observed in spots at the cell substratum interface (60 min). A fibrillar pattern of fibronectin appeared later (2-8 hr). The sites beneath the cells could be visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A second fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kideny cells) was used to assay biologically for the presence or absence of the factor secreted by human fibroblasts on the substratum. Human fibroblasts were found to secrete an adhesion factor for baby hamster kidney cells into the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that the factor secreted by human fibroblasts was cross-reactive with cold-in-soluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was also found to be an attachment and spreading factor for human fibroblasts in experiments measuring human fibroblast adhesion to fibronectin footprints of human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also found to be an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, it seems that the diffusely organized fibronectin on the substratum constitutes the sites to which cell attachment occurs. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.     

EphrinA1-induced cytoskeletal re-organization requires FAK and p130(cas)

Ephrins and Eph receptors are involved in axon guidance and cellular morphogenesis. An interaction between ephrin and Eph receptors elicits neuronal growth-cone collapse through cytoskeletal disassembly. When NIH3T3 cells were plated onto an ephrinA1-coated surface, the cells both adhered and spread. Adhesion and spreading proceeded concomitantly with changes in both the actin and microtubule cytoskeleton. EphA2, focal adhesion kinase (FAK) and p130(cas) were identified as the major ephrin-dependent phosphotyrosyl proteins during the ephrin-induced morphological changes. Mouse embryonic fibroblasts (MEFs) derived from FAK(-/-) and p130(cas-/-) mice had severe defects in ephrinA1-induced cell spreading, which were reversed after re-expression of FAK or p130(cas), respectively. Expression of a constitutively active EphA2 induced NIH3T3 cells to undergo identical, but ligand-independent, morphological changes. These data show that ephrinA1 can induce cell adhesion and actin cytoskeletal changes in fibroblasts in a FAK- and p130(cas)-dependent manner, through activation of the EphA2 receptor. The finding that ephrin Eph signalling can result in actin cytoskeletal assembly, rather than disassembly, has many implications for ephrin Eph responses in other cell types.     

Genetic deletion of Rac1 GTPase reveals its critical role in actin stress fiber formation and focal adhesion complex assembly

Rac1 is an intracellular signal transducer regulating a variety of cell functions. Previous studies by overexpression of dominant-negative or constitutively active mutants of Rac1 in clonal cell lines have established that Rac1 plays a key role in actin lamellipodia induction, cell-matrix adhesion, and cell anoikis. In the present studies, we have examined the cellular behaviors of Rac1 gene-targeted primary mouse embryonic fibroblasts (MEFs) after Cre recombinase-mediated deletion of Rac1 gene. Rac1-null MEFs became contracted and elongated in morphology and were defective in lamellipodia formation, cell spreading, cell-fibronectin adhesion, and focal contact formation in response to platelet-derived growth factor or serum. Unexpectedly, deletion of Rac1 also abolished actin stress fibers in the cells without detectable alteration of endogenous RhoA activity. Although the expression and/or activation status of focal adhesion complex components such as Src, FAK, and vinculin were not affected by Rac1 deletion, the number and size of adhesion plaques were significantly reduced, and the molecular complex between Src, FAK, and vinculin was dissembled in Rac1-null cells. Overexpression of an active RhoA mutant or ROK failed to rescue the stress fiber and adhesion plaque defects of the Rac1-null cells. Although Rac1 deletion caused a significant reduction in phospho-PAK1, -AKT, and -ERK under serum stimulation, reconstitution of active PAK1, but not AKT or MEK1, was able to rescue the actin cytoskeleton and adhesion phenotypes of the Rac1-deficient cells. Furthermore, Rac1 deletion led to a marked increase in spontaneous apoptosis that could be rescued by active PAK1, AKT, or MEK1 expression. Our results obtained from gene-targeted primary MEFs indicate that Rac1 is essential not only for lamellipodia induction but also for the RhoA-regulated actin stress fiber and focal adhesion complex formation and that Rac1 is involved in cell survival regulation through anoikis-dependent as well as -independent mechanisms.     

14-3-3beta-Rac1-p21 activated kinase signaling regulates Akt1-mediated cytoskeletal organization, lamellipodia formation and fibronectin matrix assembly

Akt1 belongs to the three-gene Akt family and functions as a serine-threonine kinase regulating phosphorylation of an array of substrates and mediating cellular processes such as cell migration, proliferation, survival, and cell cycle. Our previous studies have established the importance of Akt1 in angiogenesis and absence of Akt1 resulted in impaired integrin activation, adhesion, migration, and extracellular matrix assembly by endothelial cells and fibroblasts. In this study, we identify the downstream signaling pathways activated by Akt1 in the regulation of these cellular events. We demonstrate here that Akt1 is necessary for the growth factor stimulated activation of 14-3-3beta-Rac1-p21 activated kinase (Pak) pathway in endothelial cells and fibroblasts. While activation of Akt1 resulted in translocation of Rac1 to membrane ruffles, enhanced Rac1 activity, Pak1 phosphorylation, and lamellipodia formation, resulting in enhanced adhesion and assembly of fibronectin, inhibition of Akt1 resulted in inhibition of these processes due to impaired Rac1-Pak signaling. Formation of lamellipodia, adhesion, and fibronectin assembly by myristoylated Akt1 expression in NIH 3T3 fibroblasts was inhibited by co-expression with either dominant negative Rac1 or dominant negative Pak1. In contrast, impaired lamellipodia formation, adhesion, and fibronectin assembly by dominant negative-Akt1 expression was rescued by co-expression with either constitutively active-Rac1 or -Pak1. Moreover, previously reported defects in adhesion and extracellular matrix assembly by Akt1(-/-) fibroblasts could be rescued by expression with either active-Rac1 or -Pak1, implying the importance of Rac1-Pak signaling in growth factor stimulated cytoskeletal assembly, lamellipodia formation and cell migration in endothelial cells and fibroblasts downstream of Akt1 activation.     

The Ste20-like kinase SLK is required for cell cycle progression through G2

We have previously shown that the Ste20-like kinase SLK is a microtubule-associated protein that can regulate actin reorganization during cell adhesion and spreading (Wagner, S., Flood, T. A., O'Reilly, P., Hume, K., and Sabourin, L. A. (2002) J. Biol. Chem. 277, 37685-37692). Because of its association with the microtubule network, we investigated whether SLK plays a role in cell cycle progression, a process that requires microtubule dynamics during mitosis. Consistent with microtubule association in exponentially growing cells, our results showed that SLK co-localizes with the mitotic spindle in cells undergoing mitosis. Expression of a kinase-inactive mutant or SLK small interfering RNAs inhibited cell proliferation and resulted in an accumulation of quiescent cells stimulated to re-enter the cell cycle in the G2 phase. Cultures expressing the mutant SLK displayed a normal pattern of cyclin D, E, and B expression but failed to down-regulate cyclin A levels, suggesting that they cannot proceed through M phase. In addition, these cultures displayed low levels of both phospho-H3 and active p34/cdc2 kinase. Overexpression of active SLK resulted in ectopic spindle assembly and the induction of cell cycle re-entry of Xenopus oocytes, suggesting that SLK is required for progression through G2 upstream of H1 kinase activation.     

A novel role for the Ste20 kinase SLK in adhesion signaling and cell migration

With over 60 members, the Sterile 20 family of kinases has been implicated in numerous biological processes, including growth, survival, apoptosis and cell migration. Recently, we have shown that, in addition to cell death, the Ste20-like kinase SLK is required for efficient cell migration in fibroblasts. We have observed that SLK is involved in cell motility through its effect on actin reorganization and microtubule-induced focal adhesion turnover. Scratch wounding of confluent monolayers results in SLK activation. The induction of SLK kinase activity requires the scaffold FAK and a MAPK-dependent pathway. However, its recruitment to the leading edge of migrating fibroblasts requires the activity of the Src family kinases. Since SLK is microtubule-associated, it may represent one of the signals delivered to focal contacts that induces adhesions turnover. A speculative model is proposed to illustrate the mechanism of SLK activation and recruitment at the leading edge of migrating cells.Key words: cell migration, cell adhesion, SLK, microtubules, adhesion turnoverCell migration is involved in multiple biological processes such as development, tissue regeneration, immune surveillance and tumor metastasis. Numerous studies reported a multitude of cellular and molecular players that take part in the signaling networks that regulate cell migration.^1,2 Recently, we reported the participation of a new member, the Ste20 serine/threonine kinase SLK, in the regulation of cell migration. We have shown that SLK is a novel adhesion disassembly signal that is activated and recruited downstream of the FAK/Src complex following scratch wound-induced migration.³ Furthermore, SLK-dependent signals are required to mediate microtubule-dependent focal adhesion tunrnover.³ These findings provide new insights into the mechanisms of cell migration and adhesion dynamics.Since sterile 20 protein (Ste20p) acts as a MAP4K in yeast, it was suggested that mammalian homologues of Ste20p also function as MAP4K.⁴ Several members of the Ste20 family of kinases have been identified in mammals and implicated in various biological processes such as stress responses, cell death and cytoskeletal reorganization.⁵ We and others previously identified a novel Ste20-related kinase termed SLK, which is a part of a signaling pathway mediating c-Jun terminal kinase 1 (JNK1) activation and apoptosis in cultured fibroblasts.^6–8 In addition, recent reports showed that SLK is involved in C2C12 myoblast differentiation and plays a role in cell cycle progression.^9,10 SLK is ubiquitously expressed, but during embryogenesis it is highly enriched in muscle and neuronal tissues.¹¹ It has been shown that SLK is associated with the microtubule cytoskeleton and we have demonstrated that SLK-induced disassembly of actin stress fibers can be inhibited by dominant negative Rac1.^12–14Recently, SLK was identified as a member of a new signaling pathway that induce vasodilatation in response to angiotensin II type 2 receptor activation.¹⁵ It was reported that SLK negatively regulates RhoA-dependent functions by phosphorylation of RhoA at Ser188.¹⁵ These findings suggest that SLK represents a novel relaxation signal involved in cytoskeletal remodeling and cell migration.We have observed that SLK is recruited to the leading edge of migrating fibroblasts by a mechanism involving c-Src signaling.³ The molecular mechanism regulating SLK recruitment is still unclear but is likely to implicate the association of SLK with another protein. The translocation of SLK could involve a microtubule-dependent mechanism leading to its redistribution to peripheral adhesions, using actin stress fibers as tracks. The Rho GTPases have been shown to be important in the targeting of signaling components, such as c-Src, to specific adhesion sites.^16,17 Whether SLK recruitment to the leading edge requires the Rho GTPases remains to be investigated. The Rho-mDia pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and is responsible for delivering APC/Cdc42 and c-Src to their respective sites of action.¹⁸ One attractive possibility is that mDia facilitates SLK-microtubule translocation in a c-Src dependent manner.Integrin molecules which link the extracellular matrix to the intracellular machinery are key players in initiating polarized cell migration into the wound. We investigated SLK activity in a scratch-induced migration model and have been able to decipher various signaling components regulating SLK activation.³ Using knockdown and dominant negative approaches, we showed that SLK is required for microtubule-dependent focal adhesion turnover and cell migration downstream of the FAK/Src complex.³The molecular mechanisms by which microtubules contribute to cell migration have been intensively studied. Geiger''s group provided the first demonstration that cytoskeletal modulation, such as microtubule disruption, triggers integrin-dependent signaling in the absence of external growth factor stimulation.¹⁹ The authors suggested that the involvement of microtubules in adhesion dependent signaling is related to microtubule interaction with the contractile actin-myosin system.¹⁹ By using a nocodazole washout system, it was shown that FAK and the GTPase dynamin are required for microtubule-induced focal adhesion disassembly.²⁰Adhesion turnover involves a number of adapters and signaling molecules, most of which are engaged in FAK signaling pathways.²¹ FAK stimulates adhesion disassembly through a signaling pathway that includes extracellular signal-regulated kinase (ERK) and myosin light chain kinase (MLCK).²² Our data have shown that SLK is activated downstream of FAK/Src/MAPK signaling, suggesting that SLK may be a new target of this pathway that leads to adhesion disassembly. Furthermore, if RhoA is a bona fide substrate for SLK in fibroblasts, then by phosphorylating and inhibiting RhoA, SLK could tilt the Rho/Rac antagonistic interplay toward relaxation and adhesion disassembly. Downstream targets of FAK and Src kinase activity often regulate the recruitment of adapter and structural protein complexes to adhesions.²² The integration of molecules such as zyxin, α-actinin or paxillin into focal contacts can lead to their stabilization and maturation into focal adhesions.²² Interestingly, depending on their phosphorylation state, these components can promote adhesion destabilization and turnover. Therefore, it is tempting to speculate that activated SLK at the leading edge may phosphorylate key signaling components to induce adhesion turnover.A recent study has shown that the frequency of microtubule catastrophes is higher at focal adhesion sites and this event leads to a local release of microtubule regulatory proteins, such as GEF-H1 and APC.²³ Signaling molecules that are released from the microtubules at adhesions could directly associate with molecular factors concentrated at the adhesion plaques, such as Src, PAK and Arp2/3. Furthermore, it was speculated that microtubule catastrophe could be associated with phosphorylated paxillin-dependent protein complexes.²³ One possibility is that through the microtubule, SLK is delivered to focal contacts or adhesions where it serves as a scaffold for disassembling signals. Alternatively, SLK may be phsophorylating key signaling molecules, which ultimately leads to adhesion destabilization and turnover.Overall, our recent findings suggest that SLK is novel regulator of focal adhesion turnover and cell migration (Fig. 1). The molecular mechanisms regulating SLK activity and SLK-dependent adhesion turnover remain to be uncovered and await the identification of SLK substrates.Open in a separate windowFigure 1Model for SLK activation and recruitment at the leading edge. A proportion of SLK is microtubule-associated, likely through a microtubule-binding protein (X). Following activation of the FAK/c-Src complex, signaling through the MAPK pathway can activate and recruit the microtubule-SLK complex, inducing adhesion turnover by destabilization of the actin network or focal contacts/adhesions through an unknown mechanism. (C) denotes a cargo protein coupling the microtubule to polymerized actin. Nocodazole treatment fails to recruit SLK resulting in stable adhesions.     

Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase

Imaging studies implicate microtubule targeting of focal adhesions in focal adhesion disassembly, although the molecular mechanism is unknown. Here, we develop a model system of focal adhesion disassembly based on the finding that microtubule regrowth after nocodazole washout induces disassembly of focal adhesions, and that this disassembly occurs independently of Rho and Rac, but depends on focal adhesion kinase (FAK) and dynamin. During disassembly, dynamin interacts with FAK and colocalizes with focal adhesions. Inhibition of dynamin prevents migration of cells with a focal adhesion phenotype. Our results show that focal adhesion disassembly involves microtubules, dynamin and FAK, and is not simply the reversal of focal adhesion formation.     

Caspase 3 cleavage of the Ste20-related kinase SLK releases and activates an apoptosis-inducing kinase domain and an actin-disassembling region

We have demonstrated that a novel Ste20-related kinase, designated SLK, mediates apoptosis and actin stress fiber dissolution through distinct domains generated by caspase 3 cleavage. Overexpression of SLK in C2C12 myoblasts stimulated the disassembly of actin stress fibers and focal adhesions and induced apoptosis, as determined by annexin V binding and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling analysis. SLK was cleaved by caspase 3 in vitro and in vivo during c-Myc-, tumor necrosis factor alpha, and UV-induced apoptosis. Furthermore, cleavage of SLK released two domains with distinct activities: an activated N-terminal kinase domain that promoted apoptosis and cytoskeletal rearrangements and a C-terminus domain that disassembled actin stress fibers. Moreover, our analysis has identified a novel conserved region (termed the AT1-46 homology domain) that efficiently promotes stress fiber disassembly. Finally, transient transfection of SLK also activated the c-Jun N-terminal kinase signaling pathway. Our results suggest that caspase-activated SLK represents a novel effector of cytoskeletal remodeling and apoptosis.     

FAK potentiates Rac1 activation and localization to matrix adhesion sites: a role for betaPIX

FAK, a cytoplasmic protein tyrosine kinase, is activated and localized to focal adhesions upon cell attachment to extracellular matrix. FAK null cells spread poorly and exhibit altered focal adhesion turnover. Rac1 is a member of the Rho-family GTPases that promotes membrane ruffling, leading edge extension, and cell spreading. We investigated the activation and subcellular location of Rac1 in FAK null and FAK reexpressing fibroblasts. FAK reexpressers had a more robust pattern of Rac1 activation after cell adhesion to fibronectin than the FAK null cells. Translocation of Rac1 to focal adhesions was observed in FAK reexpressers, but seldom in FAK null cells. Experiments with constitutively active L61Rac1 and dominant negative N17Rac1 indicated that the activation state of Rac1 regulated its localization to focal adhesions. We demonstrated that FAK tyrosine-phosphorylated betaPIX and thereby increased its binding to Rac1. In addition, betaPIX facilitated the targeting of activated Rac1 to focal adhesions and the efficiency of cell spreading. These data indicate that FAK has a role in the activation and focal adhesion translocation of Rac1 through the tyrosine phosphorylation of betaPIX.     

Chemokines fail to up-regulate beta 1 integrin-dependent adhesion in human Th2 T lymphocytes

Th1 and Th2 cells are functionally distinct subsets of CD4+ T lymphocytes whose tissue-specific homing to sites of inflammation is regulated in part by the differential expression of P- and E-selectin ligands and selected chemokine receptors. Here we investigated the expression and function of beta 1 integrins in Th1 and Th2 cells polarized in vitro. Th1 lymphocytes adhere transiently to the extracellular matrix ligands laminin 1 and fibronectin in response to chemokines such as RANTES and stromal cell-derived factor-1, and this process is paralleled by the activation of the Rac1 GTPase and by a rapid burst of actin polymerization. Selective inhibitors of phosphoinositide-3 kinase prevent efficiently all of the above processes, whereas the protein kinase C inhibitor bisindolylmaleimide prevents chemokine-induced adhesion without affecting Rac1 activation and actin polymerization. Notably, chemokine-induced adhesion to beta 1 integrin ligands is markedly reduced in Th2 cells. Such a defect cannot be explained by a reduced sensitivity to chemokine stimulation in this T cell subset, nor by a defective activation of the signaling cascade involving phosphoinositide-3 kinase, Rac1, and actin turnover, as all these processes are activated at comparable levels by chemokines in the two subsets. We propose that reduced beta 1 integrin-mediated adhesion in Th2 cells may restrain their ability to invade and/or reside in sites of chronic inflammation, which are characterized by thickening of basement membranes and extensive fibrosis, requiring efficient interaction with organized extracellular matrices.     

CCN2 (connective tissue growth factor) promotes fibroblast adhesion to fibronectin

In vivo, CCN2 (connective tissue growth factor) promotes angiogenesis, osteogenesis, tissue repair, and fibrosis, through largely unknown mechanisms. In vitro, CCN2 promotes cell adhesion in a variety of systems via integrins and heparin sulfate proteoglycans (HSPGs). However, the physiological relevance of CCN2-mediated cell adhesion is unknown. Here, we find that HSPGs and the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade are required for adult human dermal fibroblasts to adhere to CCN2. Endogenous CCN2 directly binds fibronectin and the fibronectin receptors integrins alpha4 beta1 and alpha5 and syndecan 4. Using Ccn2-/- mouse embryonic fibroblasts, we show that loss of endogenous CCN2 results in impaired spreading on fibronectin, delayed alpha-smooth muscle actin stress fiber formation, and reduced ERK and focal adhesion kinase phosphorylation. These results suggest that a physiological role of CCN2 is to potentiate the ability of fibroblasts to spread on fibronectin, which may be important in modulating fibroblast adhesion to the provisional matrix during tissue development and wound healing. These results are consistent with the notion that a principal function of CCN2 is to modulate receptor/ligand interactions in vivo.