Axonal Neuregulin Signals Cells of the Oligodendrocyte Lineage through Activation of HER4 and Schwann Cells through HER2 and HER3

We are interested in the signaling between axons and glia that leads to myelination and maintenance of the myelin internode, and we have focused on the role of neuregulins and their receptors. Neuregulins are a family of ligands that includes heregulin, neu differentiation factor, glial growth factor, and the acetylcholine receptor–inducing activity. Three signal transducing transmembrane receptors for neuregulins, which bear significant homology to the EGF receptor, are currently known: HER2 (erbB2), HER3 (erbB3), and HER4 (erbB4). We have found that oligodendrocite–type II astrocyte (O2A) progenitor cells and mature oligodendrocytes express HER2 and HER4 but no HER3. Schwann cells express HER2 and HER3 but little HER4. In O2A progenitor cells and oligodendrocytes, recombinant neuregulin induces the rapid tyrosine phosphorylation of only HER4. HER2 is not phosphorylated in cells of the oligodendrocyte lineage, but a physical interaction between HER2 and HER4 was detected in coimmunoprecipitation experiments. In Schwann cells, neuregulin induces the phosphorylation of both HER2 and HER3. Coimmunoprecipitation experiments indicate that receptor activation in Schwann cells results in the formation of HER2:HER3 heterodimers. Neuregulin localized immunocytochemically was present on neurites of cultured dorsal root ganglion neurons, and it was released into the medium in a form that promoted receptor tyrosine phosphorylation. Neuregulins therefore meet important criteria expected of molecules involved in axonal-glial signaling. The use of unique neuregulin receptor combinations in oligodendrocytes and Schwann cells likely results in recruitment of different signaling pathways and thus provides a basis for different biological responses.     

Neuregulin Facilitates Nerve Regeneration by Speeding Schwann Cell Migration via ErbB2/3-Dependent FAK Pathway

Background

Adequate migration of Schwann cells (Sc) is crucial for axon-guidance in the regenerative process after peripheral nerve injury (PNI). Considering neuregulin-erbB-FAK signaling is an essential pathway participating in the regulation of Sc migration during development, the present study is aimed to examine whether neuregulin would exert its beneficial effects on adult following PNI and further determine the potential changes of downstream pathway engaged in neuro-regeneration by both in vitro and in vivo approaches.

Methodology and Principal Findings

Cultured RSC96 cells treated with neuregulin were processed for erbB2/3 immunofluorescence and FAK immunoblotings. The potential effects of neuregulin on Sc were assessed by cell adherence, spreading, and migration assays. In order to evaluate the functional significance of neuregulin on neuro-regeneration, the in vivo model of PNI was performed by chronic end-to-side neurorrhaphy (ESN). In vitro studies indicated that after neuregulin incubation, erbB2/3 were not only expressed in cell membranes, but also distributed throughout the cytoplasm and nucleus of RSC96 cells. Activation of erbB2/3 was positively correlated with FAK phosphorylation. Neuregulin also increases Sc adherence, spreading, and migration by 127.2±5.0%, 336.8±3.0%, and 80.0±5.7%, respectively. As for in vivo study, neuregulin significantly accelerates the speed of Sc migration and increases Sc expression in the distal stump of injured nerves. Retrograde labeling and compound muscle action potential recordings (CMAP) also showed that neuregulin successfully facilitates nerve regeneration by eliciting noticeably larger CMAP and promoting quick re-innervation of target muscles.

Conclusions

As neuregulin successfully improves axo-glial interaction by speeding Sc migration via the erbB2/3-FAK pathway, therapeutic use of neuregulin may thus serve as a promising strategy to facilitate the progress of nerve regeneration after PNI.     

Differential regulation of components of the focal adhesion complex by heregulin: role of phosphatase SHP-2.

Heregulin (HRG) has been implicated in the progression of breast cancer cells to a malignant phenotype, a process that involves changes in cell motility and adhesion. Here we demonstrate that HRG differentially regulates the site-specific phosphorylation of the focal adhesion components focal adhesion kinase (FAK) and paxilin in a dose-dependent manner. HRG at suboptimal doses (0.01 and 0.1 nM) increased adhesion of cells to the substratum, induced phosphorylation of FAK at Tyr-577, -925, and induced formation of well-defined focal points in breast cancer cell line MCF-7. HRG at a dose of 1 nM, increased migratory potential of breast cancer cells, selectively dephosphorylated FAK at Tyr-577, -925, and paxillin at Tyr-31. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by HRG stimulation. FAK associated with HER2 only in response to 0.01 nM HRG. In contrast, 1 nM HRG induced activation and increased association of tyrosine phosphatase SHP-2 with HER2 but decreased association of HER2 with FAK. Expression of dominant-negative SHP-2 blocked HRG-mediated dephosphorylation of FAK and paxillin, leading to persistent accumulation of mature focal points. Our results suggest that HRG differentially regulates signaling from focal adhesion complexes through selective phosphorylation and dephosphorylation and that tyrosine phosphatase SHP-2 has a role in the HRG signaling.     

The deaf and the dumb: the biology of ErbB-2 and ErbB-3

ErbB-2 (also called HER2/neu) and ErbB-3 are closely related to the epidermal growth factor receptor (EGFR/ErbB-1), but unlike EGFR, ErbB-2 is a ligandless receptor, whereas ErbB-3 lacks tyrosine kinase activity. Hence, both ErbB-2 and ErbB-3 are active only in the context of ErbB heterodimers, and ErbB-2. ErbB-3 heterodimers, which are driven by neuregulin ligands, are the most prevalent and potent complexes. These stringently controlled heterodimers are repeatedly employed throughout embryonic development and dictate the establishment of several cell lineages through mesenchyme-epithelial inductive processes and the interactions of neurons with muscle, glia, and Schwann cells. Likewise, the potent combination of signaling pathways engaged by the heterodimers drives an aggressive phenotype of tumors of secretory epithelia, including breast and lung cancers. This review highlights recent structural insights into the mechanism of ligand-induced heterodimer formation, and concentrates on signaling pathways employed by ErbB-2 and ErbB-3 in normal and in malignant cells.     

FAK is required for axonal sorting by Schwann cells

Signaling by laminins and axonal neuregulin has been implicated in regulating axon sorting by myelin-forming Schwann cells. However, the signal transduction mechanisms are unknown. Focal adhesion kinase (FAK) has been linked to alpha6beta1 integrin and ErbB receptor signaling, and we show that myelination by Schwann cells lacking FAK is severely impaired. Mutant Schwann cells could interdigitate between axon bundles, indicating that FAK signaling was not required for process extension. However, Schwann cell FAK was required to stimulate cell proliferation, suggesting that amyelination was caused by insufficient Schwann cells. ErbB2 receptor and AKT were robustly phosphorylated in mutant Schwann cells, indicating that neuregulin signaling from axons was unimpaired. These findings demonstrate the vital relationship between axon defasciculation and Schwann cell number and show the importance of FAK in regulating cell proliferation in the developing nervous system.     

Neuregulin-heparan-sulfate proteoglycan interactions produce sustained erbB receptor activation required for the induction of acetylcholine receptors in muscle

Neuregulins bind to and activate members of the EGF receptor family of tyrosine kinases that initiate a signaling cascade that induces acetylcholine receptor synthesis in the postsynaptic membrane of neuromuscular synapses. In addition to an EGF-like domain, sufficient for receptor binding and tyrosine auto-phosphorylation, many spliced forms also have an IG-like domain that binds HSPGs and maintains a high concentration of neuregulin at synapses. Here, we show that the IG-like domain functions to keep the EGF-like domain at sufficiently high concentrations for a sufficiently long period of time necessary to induce acetylcholine receptor gene expression in primary chick myotubes. Using recombinant neuregulins with and without the IG-like domain, we found that IG-like domain binding to endogenous HSPGs produces a 4-fold increase in receptor phosphorylation. This enhancement of activity was blocked by soluble heparin or by pretreatment of muscle cells with heparitinase. We show that at least 12-24 h of neuregulin exposure was required to turn on substantial acetylcholine receptor gene expression and that the erbB receptors need to be kept phosphorylated during this time. The need for sustained erbB receptor activation may be the reason why neuregulins are so highly concentrated in the extracellular matrix of synapses.     

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.     

Rescue of the cardiac defect in ErbB2 mutant mice reveals essential roles of ErbB2 in peripheral nervous system development.

ErbB2 receptor tyrosine kinase plays a role in neuregulin signaling and is expressed in the developing nervous system. We genetically rescued the cardiac defect of erbB2 null mutant embryos, which otherwise died at E11. These rescued erbB2 mutant mice die at birth and display a severe loss of both motor and sensory neurons. Motor and sensory axons are severely defasciculated and aberrantly projected within their final target tissues. Schwann cells are completely absent in the peripheral nerves. Schwann cell precursors are present within the DRG and proliferate normally, but their ability to migrate is decreased. Acetylcholine receptors cluster within the central band of the mutant diaphragm muscle. However, these clusters are dispersed and morphologically different from those in control muscle. Our results reveal an important role for erbB2 during normal peripheral nervous system development.     

Cloning and neuronal expression of a type III newt neuregulin and rescue of denervated, nerve-dependent newt limb blastemas by rhGGF2

Urodele amphibians are the only vertebrates that can regenerate their limbs throughout their life. The critical feature of limb regeneration is the formation of a blastema, a process that requires an intact nerve supply. Nerves appear to provide an unidentified factor, known as the neurotrophic factor (NTF), which stimulates cycling of blastema cells. One candidate NTF is glial growth factor (GGF), a member of the neuregulin (NRG) growth factor family. NRGs are both survival factors and mitogens to glial cells, including Schwann cells. All forms of NRGs contain an EGF-like domain that is sufficient to activate NRG receptors erbB2, erbB3, and erbB4. To investigate the involvement of neuregulin in newt limb regeneration, we cloned and characterized one neuregulin isoform, a neuregulin with a cysteine-rich domain (CRD-NRG), from newt (Notophthalmus viridescens) spinal cord. Results of in situ hybridization showed that the newt CRD-NRG is highly expressed in dorsal root ganglia and spinal cord neurons that innervate the limbs. We also demonstrated the biological activity of recombinant human GGF2 (rhGGF2) in urodele limb regeneration. When rhGGF2 was injected into denervated, nerve-dependent axolotl blastemas, the labeling index (LI) of blastema cells was maintained at a level near to that of control, innervated blastemas, whereas without rhGGF2 the LI decreased significantly. In another experiment, rhGGF2 was delivered into denervated, nerve-dependent blastemas either by direct infusion into blastemas or by injection into the intraperitoneal cavity. The denervated blastemas were rescued into a regeneration response.     

Expression,localization, and regulation of the neuregulin receptor ErbB3 in mouse heart

Neuregulins (NRG) belong to the EGF family of growth factors, which are ligands of the ErbB receptors. Their expression in the adult heart is essential, especially when the heart is submitted to cardiotoxic stress such as that produced by anthracyclines. It is considered that ErbB4 is the only NRG receptor expressed by the adult heart. Upon binding, ErbB4 may dimerize with ErbB2 to generate signals inside cells. However, here we show the presence of ErbB3 in the mouse heart from birth to adulthood by Western blotting and real‐time RT‐PCR. The expression level of ErbB3 mRNA was lower than that of ErbB2 or ErbB4, but was more stable throughout postnatal development. In isolated heart myocytes, ErbB3 localized to the Z‐lines similarly to ErbB1. Perfusion of isolated hearts with NRG‐1β induced phosphorylation of ErbB3, as well as ErbB2 and ErbB4. In adult mice, both ErbB2 and ErbB3, but not ErbB1 or ErbB4, were rapidly down‐regulated upon the induction of heart hypertrophy. In conclusion, our results demonstrate that ErbB3, in addition to ErbB4, is a receptor for neuregulin‐1β in the adult mouse heart. J. Cell. Physiol. 226: 450–455, 2011. © 2010 Wiley‐Liss, Inc.     

The Tanapoxvirus 15L Protein Is a Virus-Encoded Neuregulin That Promotes Viral Replication in Human Endothelial Cells

Studies on large double-stranded DNA (dsDNA) viruses such as poxviruses have been helpful in identifying a number of viral and cellular growth factors that contribute to our broad understanding of virus-host interaction. Orthopoxviruses and leporipoxviruses are among the most studied viruses in this aspect. However, tanapoxvirus (TPV), a member of the genus Yatapoxvirus, still remains largely unexplored, as the only known hosts for this virus are humans and monkeys. Here, we describe the initial characterization of an epidermal growth factor (EGF)-like growth factor mimicking human neuregulin from TPV, expressed by the TPV-15L gene. Assays using a baculovirus-expressed and tagged TPV-15L protein demonstrated the ability to phosphorylate neuregulin receptors. Neuregulins represent a large family of EGF-like growth factors that play important roles in embryonic endocardium development, Schwann and oligodendrocyte survival and differentiation, localized acetylcholine receptor expression at the neuromuscular junction, and epithelial morphogenesis. Interestingly, certain neuregulin molecules are able to target specific tissues through interactions with heparin sulfate proteoglycans via an immunoglobulin (Ig)-like domain. Analyses of TPV-15L revealed no Ig-like domain, but it retains the ability to bind heparin and phosphorylate neuregulin receptors, providing compelling evidence that TPV-15L is a functional mimetic of neuregulin. TPV-15L knockout virus experiments demonstrate that the virus replicates in human umbilical vein endothelial cells less efficiently than wild-type TPV-Kenya, indicating that this is a nonessential protein for virus viability but can serve a stimulatory role for replication in some cultured cells. However, the precise role of this protein in host-virus interaction still remains to be deduced.     

The FAK–Arp2/3 interaction promotes leading edge advance and haptosensing by coupling nascent adhesions to lamellipodia actin

Cell migration is initiated in response to biochemical or physical cues in the environment that promote actin-mediated lamellipodial protrusion followed by the formation of nascent integrin adhesions (NAs) within the protrusion to drive leading edge advance. Although FAK is known to be required for cell migration through effects on focal adhesions, its role in NA formation and lamellipodial dynamics is unclear. Live-cell microscopy of FAK^−/− cells with expression of phosphorylation deficient or a FERM-domain mutant deficient in Arp2/3 binding revealed a requirement for FAK in promoting the dense formation, transient stabilization, and timely turnover of NA within lamellipodia to couple actin-driven protrusion to adhesion and advance of the leading edge. Phosphorylation on Y397 of FAK promotes dense NA formation but is dispensable for transient NA stabilization and leading edge advance. In contrast, transient NA stabilization and advance of the cell edge requires FAK–Arp2/3 interaction, which promotes Arp2/3 localization to NA and reduces FAK activity. Haptosensing of extracellular matrix (ECM) concentration during migration requires the interaction between FAK and Arp2/3, whereas FAK phosphorylation modulates mechanosensing of ECM stiffness during spreading. Taken together, our results show that mechanistically separable functions of FAK in NA are required for cells to distinguish distinct properties of their environment during migration.     

ErbB transmembrane tyrosine kinase receptors are expressed by sensory and motor neurons projecting into sciatic nerve.

Adult spinal cord motor and dorsal root ganglion (DRG) sensory neurons express multiple neuregulin-1 (NRG-1) isoforms that act as axon-associated factors promoting neuromuscular junction formation and Schwann cell proliferation and differentiation. NRG-1 isoforms are also expressed by muscle and Schwann cells, suggesting that motor and sensory neurons are themselves acted on by NRG-1 isoforms produced by their peripheral targets. To test this hypothesis, we examined the expression of the NRG-1 receptor subunits erbB2, erbB3, and erbB4 in rat lumbar DRG and spinal cord. All three erbB receptors are expressed in these tissues. Sciatic nerve transection, an injury that induces Schwann cell expression of NRG-1, alters erbB expression in DRG and cord. Virtually all DRG neurons are erbB2- and erbB3-immunoreactive, with erbB4 also detectable in many neurons. In spinal cord white matter, erbB2 and erbB4 antibodies produce dense punctate staining, whereas the erbB3 antibody primarily labels glial cell bodies. Spinal cord dorsal and ventral horn neurons, including alpha-motor neurons, exhibit erbB2, erbB3, and erbB4 immunoreactivity. Spinal cord ventral horn also contains a population of small erbB3+/S100beta+/GFAP- cells (GFAP-negative astrocytes or oligodendrocytes). We conclude that sensory and motor neurons projecting into sciatic nerve express multiple erbB receptors and are potentially NRG-1 responsive.     

Cloning and neuronal expression of a type III newt neuregulin and rescue of denervated,nerve‐dependent newt limb blastemas by rhGGF2

Urodele amphibians are the only vertebrates that can regenerate their limbs throughout their life. The critical feature of limb regeneration is the formation of a blastema, a process that requires an intact nerve supply. Nerves appear to provide an unidentified factor, known as the neurotrophic factor (NTF), which stimulates cycling of blastema cells. One candidate NTF is glial growth factor (GGF), a member of the neuregulin (NRG) growth factor family. NRGs are both survival factors and mitogens to glial cells, including Schwann cells. All forms of NRGs contain an EGF‐like domain that is sufficient to activate NRG receptors erbB2, erbB3, and erbB4. To investigate the involvement of neuregulin in newt limb regeneration, we cloned and characterized one neuregulin isoform, a neuregulin with a cysteine‐rich domain (CRD‐NRG), from newt (Notophthalmus viridescens) spinal cord. Results of in situ hybridization showed that the newt CRD‐NRG is highly expressed in dorsal root ganglia and spinal cord neurons that innervate the limbs. We also demonstrated the biological activity of recombinant human GGF2 (rhGGF2) in urodele limb regeneration. When rhGGF2 was injected into denervated, nerve‐dependent axolotl blastemas, the labeling index (LI) of blastema cells was maintained at a level near to that of control, innervated blastemas, whereas without rhGGF2 the LI decreased significantly. In another experiment, rhGGF2 was delivered into denervated, nerve‐dependent blastemas either by direct infusion into blastemas or by injection into the intraperitoneal cavity. The denervated blastemas were rescued into a regeneration response. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 150–158, 2000     

Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase.

Focal adhesion kinase (pp125FAK or FAK) and paxillin colocalize with integrins in structures called focal adhesions. pp125FAK plays an important role in the transmission of integrin-induced cytoplasmic signals. Paxillin has also been implicated in cell signaling by virtue of its association with the protein tyrosine kinases pp60src and Csk (C-terminal Src kinase) as well as with the adapter/oncoprotein p47gag-crk. In this report we show that endogenous pp125FAK and paxillin form a stable complex both in vivo and in vitro and that this interaction is direct, requiring only pp125FAK and paxillin. The paxillin binding site on pp125FAK has been localized to the carboxy-terminal 148 residues of pp125FAK, but appears to be distinct from the previously identified focal adhesion-targeting sequence also present in the carboxy-terminal domain of pp125FAK. The interaction of paxillin and pp125FAK is independent of the adhesion of cells to the extracellular matrix, as the association can be detected in suspension cells as well as those attached to fibronectin.     

Beta2-integrin, LFA-1, and TCR/CD3 synergistically induce tyrosine phosphorylation of focal adhesion kinase (pp125(FAK)) in PHA-activated T cells

Complete T cell activation requires not only a first signal via TCR/CD3 engagement but also a costimulatory signal through accessory receptors such as CD2, CD28, or integrins. Focal adhesion kinase, pp125(FAK) (FAK), was previously shown to be localized in focal adhesions in fibroblasts and to be involved in integrin-mediated cellular activation. Although signaling through beta1- or beta3-integrins induces tyrosine phosphorylation of FAK, there has been no evidence that activation of T cells through the beta2-integrin, LFA-1, involves FAK. We report here that crosslinking of LFA-1 induces tyrosine phosphorylation of FAK in PHA-activated T cells. Moreover, cocrosslinking with anti-LFA-1 mAb and suboptimal concentration of anti-CD3 mAb markedly increases tyrosine phosphorylation of FAK in an antibody-concentration-dependent and time-kinetics-dependent manner compared with stimulation through CD3 alone, which correlates well with enhanced proliferation of PHA-activated T cells. Furthermore, LFA-1beta costimulation with CD3 induces tyrosine phosphorylation of Syk associated with FAK. These results indicate, for the first time, that signals mediated by LFA-1 can regulate FAK, suggesting that LFA-1-mediated T cell costimulation may be involved in T cell activation at least partially through FAK.     

Bivalence of EGF-like ligands drives the ErbB signaling network.

Signaling by epidermal growth factor (EGF)-like ligands is mediated by an interactive network of four ErbB receptor tyrosine kinases, whose mechanism of ligand-induced dimerization is unknown. We contrasted two existing models: a conformation-driven activation of a receptor-intrinsic dimerization site and a ligand bivalence model. Analysis of a Neu differentiation factor (NDF)-induced heterodimer between ErbB-3 and ErbB-2 favors a bivalence model; the ligand simultaneously binds both ErbB-3 and ErbB-2, but, due to low-affinity of the second binding event, ligand bivalence drives dimerization only when the receptors are membrane anchored. Results obtained with a chimera and isoforms of NDF/neuregulin predict that each terminus of the ligand molecule contains a distinct binding site. The C-terminal low-affinity site has broad specificity, but it prefers interaction with ErbB-2, an oncogenic protein acting as a promiscuous low-affinity subunit of the three primary receptors. Thus, ligand bivalence enables signal diversification through selective recruitment of homo- and heterodimers of ErbB receptors, and it may explain oncogenicity of erbB-2/HER2.     

Neuregulin signaling via ErbB receptor assemblies in the nervous system

Neuregulins (NRG) play important roles in the development, maintenance, and repair of the nervous system, with influences on neuronal migration, synaptogenesis, receptor subunit composition, and the proliferation/survival of oligodendrocytes and Schwann cells. However, the precise detail of how the NRGs signal through ErbB receptors, particularly at central synapses, is incomplete. The receptor kinase domain provides sites for association with adaptor proteins. In addition, evidence from recent reports suggests that ErbB2/4 receptors, through their C-terminal amino acids, can form specific associations with scaffolding proteins. The existence of such assemblies expands the range of signaling cascades available to the NRGs.     

Nrg1/ErbB signaling networks in Schwann cell development and myelination

Neuregulin-1 (Nrg1) provides a key axonal signal that regulates Schwann cell proliferation, migration and myelination through binding to ErbB2/3 receptors. The analysis of a number of genetic models has unmasked fundamental mechanisms underlying the specificity of the Nrg1/ErbB signaling axis. Differential expression of Nrg1 isoforms, Nrg1 processing, and ErbB receptor localization and trafficking represent important regulatory themes in the control of Nrg1/ErbB function. Nrg1 binding to ErbB2/3 receptors results in the activation of intracellular signal transduction pathways that initiate changes in Schwann cell behavior. Here, we review data that has defined the role of key Nrg1/ErbB signaling components like Shp2, ERK1/2, FAK, Rac1/Cdc42 and calcineurin in development of the Schwann cell lineage in vivo. Many of these regulators receive converging signals from other cues that are provided by Notch, integrin or G-protein coupled receptors. Signaling by multiple extracellular factors may act as key modifiers and allow Schwann cells at different developmental stages to respond in distinct manners to the Nrg1/ErbB signal.     

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.