Hypoxic preconditioning enhances bone marrow mesenchymal stem cell migration via Kv2.1 channel and FAK activation

Transplantation using stem cells including bone marrow mesenchymal stem cells (BMSCs) is emerging as a potential regenerative therapy after ischemic attacks in the heart and brain. The migration capability of transplanted cells is a critical cellular function for tissue repair. Based on our recent observations that hypoxic preconditioning (HP) has multiple benefits in improving stem cell therapy and that the potassium Kv2.1 channel acts as a promoter for focal adhesion kinase (FAK) activation and cell motility, the present investigation tested the hypothesis that HP treatment can increase BMSC migration via the mechanism of increased Kv2.1 expression and FAK activities. BMSCs derived from green fluorescent protein-transgenic mice were treated under either normoxic (N-BMSC) or hypoxic (0.5% O(2)) (HP-BMSC) conditions for 24 h. Western blot analysis showed HP selectively upregulated Kv2.1 expression while leaving other K(+) channels, such as Kv1.5 and Kv1.4, unaffected. Compared with normoxic controls, significantly larger outward delayed rectifier K(+) currents were recorded in HP-BMSCs. HP enhanced BMSC migration/homing activities in vitro and after intravenous transplantation into rats subjected to permanent myocardial infarction (MI). The HP-promoted BMSC migration was inhibited by either blocking K(+) channels or knocking down Kv2.1. Supporting a relationship among HP, Kv2.1, and FAK activation, HP increased phosphorylation of FAK(397) and FAK(576/577), and this effect was antagonized by blocking K(+) channels. These findings provide novel evidence that HP enhances the ability of BMSCs to migrate and home to the injured region; this effect is mediated through a regulatory role of Kv2.1 on FAK phosphorylation/activation.     

Modulation of Kv2.1 channel gating and TEA sensitivity by distinct domains of SNAP-25

Distinct domains within the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins, STX1A (syntaxin 1A) and SNAP-25 (synaptosome-associated protein-25 kDa), regulate hormone secretion by their actions on the cell's exocytotic machinery, as well as voltage-gated Ca2+ and K+ channels. We examined the action of distinct domains within SNAP-25 on Kv2.1 (voltage gated K+ 2.1) channel gating. Dialysis of N-terminal SNAP-25 domains, S197 (SNAP-25(1-197)) and S180 (SNAP-25(1-180)), but not S206 (full-length SNAP-25(1-206)) increased the rate of Kv2.1 channel activation and slowed channel inactivation. Remarkably, these N-terminal SNAP-25 domains, acting on the Kv2.1 cytoplasmic N-terminus, potentiated the external TEA (tetraethylammonium)-mediated block of Kv2.1. To further examine whether these are effects of the channel pore domain, internal K+ was replaced with Na+ and external K+ was decreased from 4 to 1 mM, which decreased the IC50 of the TEA block from 6.8+/-0.9 mM to >100 mM. Under these conditions S180 completely restored TEA sensitivity (7.9+/-1.5 mM). SNAP-25 C-terminal domains, SNAP-25(198-206) and SNAP-25(181-197), had no effect on Kv2.1 gating kinetics. We conclude that different domains within SNAP-25 can form distinct complexes with Kv2.1 to execute a fine allosteric regulation of channel gating and the architecture of the outer pore structure in order to modulate cell excitability.     

Fibronectin promotes cervical cancer tumorigenesis through activating FAK signaling pathway

Cervical cancer is a cancer arising from the cervix, and it is the fourth most common cause of death in women. Overexpression of fibronectin 1 (FN1) was observed in many tumors and associated with the survival and metastasis of cancer cells. However, the mechanism by which FN1 promotes cervical cancer cell viability, migration, adhesion, and invasion, and inhibits cell apoptosis through focal adhesion kinase (FAK) signaling pathway remains to be investigated. Our results demonstrated that FN1 was upregulated in patients with cervical cancer and higher FN1 expression correlated with a poor prognosis for patients with cervical cancer. FN1 knockdown by small interfering RNA (siRNA) inhibited SiHa cell viability, migration, invasion, and adhesion, and promoted cell apoptosis. FN1 overexpression in CaSki cell promoted cell viability, migration, invasion, and adhesion, and inhibited cell apoptosis. Further, phosphorylation of FAK, a main downstream signaling molecule of FN1, and the protein expression of Bcl-2/Bax, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), and N-cadherin was upregulated in CaSki cells with FN1 overexpression, but caspase-3 protein expression was downregulated. The FAK phosphorylation inhibitor PF573228 inhibited FN1 overexpression-induced expression of those proteins in CaSki cells with FN1 overexpression. In vivo experiment demonstrated that FN1 knockdown significantly inhibited FN1 expression, phosphorylation of FAK, and tumor growth in xenograft from the nude mice. These results suggest that FN1 regulates the viability, apoptosis, migration, invasion, and adhesion of cervical cancer cells through the FAK signaling pathway and is a potential therapeutic target in the treatment of cervical cancer.     

The effect of epidermal growth factor receptor variant III on glioma cell migration by stimulating ERK phosphorylation through the focal adhesion kinase signaling pathway

Epidermal growth factor receptor variant III (EGFRvIII), the most common EGFR mutation, is associated with cell migration of glioblastoma multiforme (GBM) cases; however, the mechanism has not been elucidated. In this study, we found that the EGFRvIII-promoted glioma cell migration was closely linked to high levels of tyrosine phosphorylation in focal adhesion kinase (FAK) Y397. We also demonstrated that EGFRvIII formed a complex with FAK, resulting in enhanced tyrosine phosphorylation levels of FAK Y397 and EGFR Y1068. After knockdown of FAK expression via anti-FAK shRNA, the U87ΔEGFR cell migration was significantly inhibited, accompanying with the reduced phosphorylation levels of extracellular signal-regulated kinase (ERK1/2). Furthermore, the role of ERK1/2 in FAK-regulated cell migration was confirmed. Taken together, our results suggest that FAK and its downstream molecule ERK were involved in EGFRvIII-promoted glioma cell migration in U87ΔEGFR cells.     

Focal adhesion kinase protein levels in gut epithelial motility

Mucosal healing requires migration and proliferation. Most studies of focal adhesion kinase (FAK), a protein that regulates motility, proliferation, and apoptosis, have focused on rapid phosphorylation. We reported lower FAK protein levels in motile Caco-2 colon cancer cells and postulated that this reduction in FAK available for activation might impact cell migration and mucosal healing. Therefore, total and active FAK (FAK(397)) immunoreactivity was assessed at the migrating fronts of human Caco-2 and rat IEC-6 intestinal epithelial cells. Caco-2 and IEC-6 motility, quantitated as migration into linear or circular wounds, was examined following FAK protein inhibition by small interfering RNA (siRNA). FAK protein stability and mRNA expression were ascertained by cycloheximide decay, RT-PCR, and in situ hybridization in static and migrating Caco-2 cells. Cells at the migrating front of Caco-2 and IEC-6 monolayers exhibited lower immunostaining for both total and activated FAK than cells immediately behind the front. Western blot analysis also demonstrated diminished FAK protein levels in motile cells by >/=30% in both the differential density seeding and multiple scrape models. siRNA FAK protein inhibition enhanced motility in both the linear scrape (20% in Caco-2) and circular wound (16% in Caco-2 and 19% in IEC-6 cells) models. FAK protein degradation did not differ in motile and static Caco-2 cells and was unaffected by FAK(397) phosphorylation, but FAK mRNA was lower in migrating Caco-2 cells. Thus FAK protein abundance appears regulated at the mRNA level during gut epithelial cell motility and may influence epithelial cell migration coordinately with signals that modify FAK phosphorylation.     

SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover

Integrin signaling is central to cell growth and differentiation, and critical for the processes of apoptosis, cell migration and wound repair. Previous research has demonstrated a requirement for SNARE-dependent membrane traffic in integrin trafficking, as well as cell adhesion and migration. The goal of the present research was to ascertain whether SNARE-dependent membrane trafficking is required specifically for integrin-mediated signaling. Membrane traffic was inhibited in Chinese hamster ovary cells by expression of dominant-negative (E329Q) N-ethylmaleimide-sensitive fusion protein (NSF) or a truncated form of the SNARE SNAP23. Integrin signaling was monitored as cells were plated on fibronectin under serum-free conditions. E329Q-NSF expression inhibited phosphorylation of focal adhesion kinase (FAK) on Tyr397 at early time points of adhesion. Phosphorylation of FAK on Tyr576, Tyr861 and Tyr925 was also impaired by expression of E329Q-NSF or truncated SNAP23, as was trafficking, localization and activation of Src and its interaction with FAK. Decreased FAK-Src interaction coincided with reduced Rac activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-trisphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3K-dependent pathway.     

XIAP is essential for shear stress-enhanced Tyr-576 phosphorylation of FAK

In endothelial cells, X-chromosome linked inhibitor of apoptosis protein (XIAP) regulates cell survival, migration and adhesion. We have recently found that XIAP recruits focal adhesion kinase (FAK) into integrin-associated focal adhesions, controlling cell migration. However, little is understood about the molecular mechanisms by which FAK modulation is controlled by XIAP. In this study, we show that XIAP modulates FAK activity through the control of FAK phosphorylation. In bovine aortic endothelial cells (BAEC), phosphorylation of Tyr-576 in FAK is elevated by laminar shear stress. This elevated phosphorylation appears to be responsible for shear stress-stimulated ERK activation. We found that XIAP knockdown reduces shear stress-enhanced phosphorylation of Tyr-576 and induces shear stress-triggered translocation of FAK into nucleus. Nuclear translocation of FAK reduces contact between FAK and Src, a kinase which phosphorylates Tyr-576. This spatial segregation of FAK from Src decreases Tyr-576 phosphorylation and thus shear-stimulated ERK activation. Taken together, our results demonstrate that XIAP plays a key role in shear stress-stimulated ERK activation by maintaining the Src-accessible location of FAK.     

Suppression of uPAR retards radiation-induced invasion and migration mediated by integrin β1/FAK signaling in medulloblastoma

Background

Despite effective radiotherapy for the initial stages of cancer, several studies have reported the recurrence of various cancers, including medulloblastoma. Here, we attempt to capitalize on the radiation-induced aggressive behavior of medulloblastoma cells by comparing the extracellular protease activity and the expression pattern of molecules, known to be involved in cell adhesion, migration and invasion, between non-irradiated and irradiated cells.

Methodology/Principal Findings

We identified an increase in invasion and migration of irradiated compared to non-irradiated medulloblastoma cells. RT-PCR analysis confirmed increased expression of uPA, uPAR, focal adhesion kinase (FAK), N-Cadherin and integrin subunits (e.g., α3, α5 and β1) in irradiated cells. Furthermore, we noticed a ∼2-fold increase in tyrosine phosphorylation of FAK in irradiated cells. Immunoprecipitation studies confirmed increased interaction of integrin β1 and FAK in irradiated cells. In addition, our results show that overexpression of uPAR in cancer cells can mimic radiation-induced activation of FAK signaling. Moreover, by inhibiting FAK phosphorylation, we were able to reduce the radiation-induced invasiveness of the cancer cells. In this vein, we studied the effect of siRNA-mediated knockdown of uPAR on cell migration and adhesion in irradiated and non-irradiated medulloblastoma cells. Downregulation of uPAR reduced the radiation-induced adhesion, migration and invasion of the irradiated cells, primarily by inhibiting phosphorylation of FAK, Paxillin and Rac-1/Cdc42. As observed from the immunoprecipitation studies, uPAR knockdown reduced interaction among the focal adhesion molecules, such as FAK, Paxillin and p130Cas, which are known to play key roles in cancer metastasis. Pretreatment with uPAR shRNA expressing construct reduced uPAR and phospho FAK expression levels in pre-established medulloblastoma in nude mice.

Conclusion/Significance

Taken together, our results show that radiation enhances uPAR-mediated FAK signaling and by targeting uPAR we can inhibit radiation-activated cell adhesion and migration both in vitro and in vivo.     

Collagen IV regulates Caco-2 cell spreading and p130Cas phosphorylation by FAK-dependent and FAK-independent pathways

We previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src-dependent p130(Cas) phosphorylation and stimulates focal adhesion kinase (FAK). However, the role of FAK and the related kinase, Pyk2, in Caco-2 spreading and migration is unclear. FAK- or Pyk2-specific siRNAs reduced protein levels by 90%. However, when detached cells were replated on collagen IV neither individual nor combined FAK and Pyk2 siRNAs affected the cell spreading rate. As combined FAK and Pyk2 siRNAs increased p130(Cas) protein levels, we cotransfected cells with 1 nm p130(Cas) siRNA to partially reduce p130(Cas) protein to control levels. Although p130(Cas) Tyr(P)(249) phosphorylation was reduced by 60%, cell spreading was unaffected. Combined siRNA reduction of FAK, Pyk2 and p130(Cas) increased cell spreading by 20% compared to p130(Cas) siRNA alone, suggesting that FAK and Pyk2 negatively regulate spreading in addition to stimulating spreading via p130(Cas). FAK-binding mutant SH3 domain-deleted rat p130(Cas) was not phosphorylated after adhesion and, unlike full-length p130(Cas), did not restore spreading after human-specific p130(Cas) siRNA knockdown of endogenous p130(Cas). Together, these data suggest that FAK positively regulates Caco-2 spreading on collagen IV via p130(Cas) phosphorylation, but also suggests that FAK may negatively regulate spreading through other mechanisms and the presence of additional FAK-independent pathways regulating p130(Cas).     

FAK phosphorylation at Ser-843 inhibits Tyr-397 phosphorylation, cell spreading and migration

Multiple stimuli promote the tyrosine phosphorylation and activation of focal adhesion kinase (FAK), which ultimately facilitates migration. Little is known about the effect of adhesion-dependent signals and cytoskeleton organization on the regulation of FAK phosphorylation at serine sites, or about the role of FAK serine phosphorylation in cell migration. Here, we show that FAK phosphorylation at Ser-843 is strikingly increased when adherent cells are removed from the substratum and held in suspension or by treatment of adherent cells with cytochalasin D, conditions that disrupt the F-actin cytoskeleton and promote focal adhesion disassembly. Notably, the increase in Ser-843 phosphorylation was accompanied by a concomitant sharp decrease in Tyr-397 phosphorylation. To further examine the cause-effect relationship between these two phosphorylation sites we generated Ser-843 phosphorylation-deficient and phosphorylation-mimicking FAK mutants. We found that mutation of Ser-843 to aspartic acid (FAK[S843D]) markedly decreased FAK Tyr-397 phosphorylation in integrin-stimulated cells. While the migratory defect of FAK-deficient fibroblasts was rescued by stable re-expression of WT FAK or FAK[S843A], stable re-expression of FAK[S843D] failed to restore the ability of the cells to migrate into the denuded area of a wound. Our results indicate that increased FAK phosphorylation at Ser-843 represses FAK phosphorylation at Tyr-397, thus suggesting a mechanism of cross-talk between these phosphorylation sites that could regulate FAK-mediated cell shape and migration.     

Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells.

Tyrosine phosphorylation of cytoskeletal proteins occurs during integrin-mediated cell adhesion to extracellular matrix proteins. We have investigated the role of tyrosine phosphorylation in the migration and initial spreading of human umbilical vein endothelial cells (HUVEC). Elevated phosphotyrosine concentrations were noted in the focal adhesions of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots of extracts of wounded HUVEC monolayers demonstrated increased phosphorylation at 120-130 kDa when compared with extracts of intact monolayers. The pp125FAK immunoprecipitated from wounded monolayers exhibited increased kinase activity as compared to pp125FAK from intact monolayers. The time to wound closure in HUVEC monolayers was doubled by tyrphostin AG 213 treatment. The same concentration of AG 213 interfered with HUVEC focal adhesion and stress fiber formation. AG 213 inhibited adhesion-associated tyrosine phosphorylation of pp125FAK in HUVEC. Tyrphostins AG 213 and AG 808 inhibited pp125FAK activity in in vitro kinase assays. pp125FAK immunoprecipitates from HUVEC treated with both of these inhibitors also had kinase activity in vitro that was below levels seen in untreated HUVEC. These findings suggest that tyrosine phosphorylation of cytoskeletal proteins may be important in HUVEC spreading and migration and that pp125FAK may mediate phosphotyrosine formation during these processes.     

Endogenous KCNE subunits govern Kv2.1 K+ channel activation kinetics in Xenopus oocyte studies

Kv2.1 is a voltage-gated potassium (Kv) channel that generates delayed rectifier currents in mammalian heart and brain. The biophysical properties of Kv2.1 and other ion channels have been characterized by functional expression in heterologous systems, and most commonly in Xenopus laevis oocytes. A number of previous oocyte-based studies of mammalian potassium channels have revealed expression-level-dependent changes in channel properties, leading to the suggestion that endogenous oocyte factors regulate channel gating. Here, we show that endogenous oocyte potassium channel KCNE ancillary subunits xMinK and xMiRP2 slow the activation of oocyte-expressed mammalian Kv2.1 channels two-to-fourfold. This produces a sigmoidal relationship between Kv2.1 current density and activation rate in oocyte-based two-electrode voltage clamp studies. The effect of endogenous xMiRP2 and xMinK on Kv2.1 activation is diluted at high Kv2.1 expression levels, or by RNAi knockdown of either endogenous subunit. RNAi knockdown of both xMiRP2 and xMinK eliminates the correlation between Kv2.1 expression level and activation kinetics. The data demonstrate a molecular basis for expression-level-dependent changes in Kv channel gating observed in heterologous expression studies.     

RNA interference elucidates the role of focal adhesion kinase in HLA class I-mediated focal adhesion complex formation and proliferation in human endothelial cells

Ligation of class I molecules by anti-HLA Ab stimulates an intracellular signaling cascade resulting in endothelial cell (EC) survival and proliferation, and has been implicated in the process of chronic allograft rejection and transplant-associated vasculopathy. In this study, we used small interfering RNA blockade of focal adhesion kinase (FAK) protein to determine its role in class I-mediated organization of the actin cytoskeleton, cell survival, and cell proliferation in primary cultures of human aortic EC. Knockdown of FAK appreciably inhibited class I-mediated phosphorylation of Src at Tyr(418), p85 PI3K, and Akt at both Thr(308) and Ser(473) sites. FAK knockdown also reduced class I-mediated phosphorylation of paxillin at Try(118) and blocked class I-induced paxillin assembly into focal contacts. FAK small interfering RNA completely abrogated class I-mediated formation of actin stress fibers. Interestingly, FAK knockdown did not modify fibroblast growth factor receptor expression induced by class I ligation. However, FAK knockdown blocked HLA class I-stimulated cell cycle proliferation in the presence and absence of basic fibroblast growth factor. This study shows that FAK plays a critical role in class I-induced cell proliferation, cell survival, and focal adhesion assembly in EC and may promote the development of transplant-associated vasculopathy.     

Induced Focal Adhesion Kinase (FAK) Expression in FAK-Null Cells Enhances Cell Spreading and Migration Requiring Both Auto- and Activation Loop Phosphorylation Sites and Inhibits Adhesion-Dependent Tyrosine Phosphorylation of Pyk2

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in integrin-mediated control of cell behavior. Following cell adhesion to components of the extracellular matrix, FAK becomes phosphorylated at multiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosphorylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and FAK catalytic activity may be elevated through phosphorylation of these residues by associated Src family kinase. Recent studies have implicated FAK as a positive regulator of cell spreading and migration. To further study the mechanism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetracycline repression system to achieve inducible expression of either wild-type FAK or phosphorylation site mutants in fibroblasts derived from FAK-null mouse embryos. Using these Tet-FAK cells, we demonstrated that both the FAK autophosphorylation and activation loop sites are critical for maximum adhesion-induced FAK activation and FAK-enhanced cell spreading and migration responses. Negative effects on cell spreading and migration, as well as decreased phosphorylation of the substrate p130(Cas), were observed upon induced expression of the FAK autophosphorylation site mutant. These negative effects appear to result from an inhibition of integrin-mediated signaling by the FAK-related kinase Pyk2/CAKbeta/RAFTK/CadTK.     

Growth factors stimulate kidney proximal tubule cell migration independent of augmented tyrosine phosphorylation of focal adhesion kinase

Migration of human proximal tubule cells (HKC-5) was stimulated by epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). Integrin signaling via phosphorylation of focal adhesion kinase (FAK) appears to play a central role in cell migration. Once stimulated, FAK undergoes autophosphorylation at tyrosine (Y) 397, followed by phosphorylation of several sites including Y576/Y577 which increases FAK's kinase activity, as well as at Y407, Y861, and Y925. EGF, HGF, and IGF-1 stimulate FAK phosphorylation in various cells. We showed that endothelin stimulated phosphorylation of Y397 in fibroblasts but not HKC-5 cells. After EGF stimulation, HKC-5 cells showed no change in tyrosine phosphorylation at FAK Y397, 407, 576, 861, or 925. Similarly, HGF and IGF-1 did not stimulate the phosphorylation of FAK Y397 in HKC-5 cells. Further, after inhibition of FAK expression by siRNA, cell migration was similar to cells treated with non-target siRNA and responded to EGF with increased migration. Thus, in proximal tubule cells, stimulation of cell migration by growth factors was independent of augmented FAK tyrosine phosphorylation.     

Phosphorylation of focal adhesion kinase (pp125(FAK)) is increased in human keratinocytes induced to migrate by extracellular matrices

During the healing process of skin wounds, human keratinocytes migrate across a provisional matrix of the wound bed. The mechanisms by which keratinocytes migrate on connective tissue are not known. In this study, we examined the role of focal adhesion kinase (FAK), an 125 kDa protein that co-localizes with focal adhesions in cells plated on extracellular matrix. We induced human keratinocytes into various states of migration by plating them on extracellular matrices that minimally, moderately, or strongly induce cellular migration, and then examined the expression of FAK at the protein level and its degree of tyrosine phosphorylation using Western immunoblotting and immunoprecipitation. In highly migratory human keratinocytes, we found that three proteins were predominantly tyrosine phosphorylated, one of them being FAK. Tyrosine phosphorylation of FAK tightly correlated with the level of cellular motility but not cell attachment to the matrix. Time course experiments demonstrated that in highly motile keratinocytes, tyrosine phosphorylation of FAK peaked at 12 h, the time when maximal migration on the matrix ensues. In contrast to FAK, the beta1 integrin subunit of human keratinocytes that configures with the alpha2, alpha3, and alpha5 integrin subunits to form integrin receptors for matrix, did not display tyrosine phosphorylation linked to motility. Using anti-sense oligonucleotides to FAK, we demonstrate that FAK is required for human keratinocyte migration, but not for focal adhesion formation.     

Dynamic modulation of the kv2.1 channel by SRC-dependent tyrosine phosphorylation

The voltage-gated K(+) channel Kv2.1 is expressed as a highly phosphorylated protein in most central neurons, where it plays a key role in regulating neuronal membrane excitability. Previous studies have shown that Kv2.1 channel activity is upregulated by Src-mediated phosphorylation through an unknown mechanism. However, a systematic analysis of the molecular mechanism of Kv2.1 channel phosphorylation by Src is lacking. Here, we show that tyrosine phosphorylation by Src plays a fundamental role in regulating Kv2.1-mediated K(+) current enhancement. We found that the level of expression of the Kv2.1 protein is increased by Src kinase. Using mass spectrometric proteomic techniques, we identified two novel phosphotyrosine sites, Y686 and Y810, in the cytoplasmic domains of Kv2.1. We found that Src-dependent phosphorylation at these sites affects Kv2.1 through distinct regulatory mechanisms. Whereas phosphorylation at Y686 regulates Kv2.1 activity similarly to the known site Y124, phosphorylation at Y810 plays a significant role in regulating the intracellular trafficking of Kv2.1 channels. Our results show that these two novel tyrosine phosphorylation sites of Kv2.1 are crucial to regulating diverse aspects of Kv2.1 channel function and provide novel insights into molecular mechanisms for the regulation of Src-dependent modulation of Kv2.1 channels.     

Fibroblast growth factor-2 modulates melanoma adhesion and migration through a syndecan-4-dependent mechanism

Fibroblast growth factor-2 (FGF-2), the most abundant growth factor produced by melanoma cells but not by normal melanocytes, is an important regulator of cell proliferation, migration and differentiation. In this study we show that M5 human metastatic melanoma cells’ ability to migrate is significantly enhanced by exogenously added FGF-2 while, neutralization of endogenous FGF-2 stimulates their adhesion. Previously, we have demonstrated that FGF-2 distinctly modulates the synthesis of individual glycosaminoglycans/proteoglycans (GAGs/PGs) subclasses, changing both their amounts and distribution in M5 cells. Here, treatment with FGF-2 strongly reduces the expression levels of the heparan sulfate-containing proteoglycan, syndecan-4. Syndecan-4 is a focal adhesion component in a range of cell types, adherent to several different matrix molecules, including fibronectin (FN). The reduction in syndecan-4 expression by utilizing specific siRNA discriminately increased melanoma cell motility and decreased their attachment on FN, demonstrating a regulatory role of syndecan-4 on these cell functions. Syndecan-4 has previously been demonstrated to regulate focal adhesion kinase (FAK) phosphorylation. In this study FGF-2 was shown to downregulate FAK Y397-phosphorylation during FN-mediated M5 cell adhesion, promoting their migration. The observed decrease in FAK Y397 activation was correlated to syndecan-4 expression levels. Thus, a balance in syndecan-4 expression perpetrated by FGF-2 may be required for optimal M5 cell migration.These results suggest that essential in melanoma progression FGF-2, specifically regulates melanoma cell ability to migrate through a syndecan-4-dependent mechanism.     

PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration

We investigated the molecular and cellular actions of receptor protein tyrosine phosphatase (PTP) alpha in integrin signaling using immortalized fibroblasts derived from wild-type and PTP alpha-deficient mouse embryos. Defects in PTP alpha-/- migration in a wound healing assay were associated with altered cell shape and focal adhesion kinase (FAK) phosphorylation. The reduced haptotaxis to fibronectin (FN) of PTP alpha-/- cells was increased by expression of active (but not inactive) PTP alpha. Integrin-mediated formation of src-FAK and fyn-FAK complexes was reduced or abolished in PTP alpha-/- cells on FN, concomitant with markedly reduced phosphorylation of FAK at Tyr397. Reintroduction of active (but not inactive) PTP alpha restored FAK Tyr-397 phosphorylation. FN-induced cytoskeletal rearrangement was retarded in PTP alpha-/- cells, with delayed filamentous actin stress fiber assembly and focal adhesion formation. This mimicked the effects of treating wild-type fibroblasts with the src family protein tyrosine kinase (Src-PTK) inhibitor PP2. These results, together with the reduced src/fyn tyrosine kinase activity in PTP alpha-/- fibroblasts (Ponniah et al., 1999; Su et al., 1999), suggest that PTP alpha functions in integrin signaling and cell migration as an Src-PTK activator. Our paper establishes that PTP alpha is required for early integrin-proximal events, acting upstream of FAK to affect the timely and efficient phosphorylation of FAK Tyr-397.     

Stanniocalcin-1 regulates re-epithelialization in human keratinocytes

Stanniocalcin-1 (STC1), a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam) formation, followed by cell migration. In this study, staurosporine (STS) treatment induced human keratinocyte (HaCaT) e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK), the surge of intracellular calcium level [Ca²⁺]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca²⁺]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca²⁺]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl) could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing.