Rapid tyrosine phosphorylation of focal adhesion kinase, paxillin, and p130Cas by gastrin in human colon cancer cells

Although the expression of CCK(2) receptors is widely reported in human colorectal cancers, little is known on its role in mediating the proliferative effects of mature amidated gastrin (G17 amide) on colorectal cancers. The purpose of the present study was to determine the effects of G17 amide on tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and p130 Crk-associated substrate (p130(Cas)) in Colo 320 cells, a human colorectal cancer cell line which expresses CCK(2) receptors. By immunoprecipitation and immunoblotting, an increase in tyrosine phosphorylation of FAK (tyrosine-397), paxillin (tyrosine-31), and p130(Cas) was detected in a time- and dose-dependent manner. Overexpression of CCK(2) receptors in Colo 320 cells (Colo 320 WT) by stable transfection with the human CCK(2) receptor cDNA resulted in an increased tyrosine phosphorylation of FAK, paxillin, and p130(Cas). After incubation with 1 microM L-365,260, a specific CCK(2) receptor antagonist, this increase was completely inhibited. Our results demonstrate that in human colon cancer cells, gastrin caused a rapid tyrosine phosphorylation of FAK, paxillin, and p130(Cas) by activation of CCK(2) receptor. The phosphorylation of these proteins might be important in mediating gastrin effects on proliferation, apoptosis, and metastasis.     

Ochratoxin A affects COS cell adhesion and signaling

Ochratoxin A (OTA), a metabolite produced by strains of Aspergillus and Penicillium, has nephritogenic, carcinogenic, and teratogenic activity in animals and humans. Nanomolar concentrations of OTA promote apoptosis in a cell-type specific fashion. In this study, we have analyzed the molecular mechanism by which OTA affects COS cell adhesion and signaling resulting in an apoptotic response. OTA, at noncytotoxic doses, was able to detach collagen- and fibronectin-adherent cells from immobilized substratum. However, prior to inducing detachment of adherent cells, OTA caused apoptosis as measured by caspase-3 activation. The treatment of adherent cells by OTA caused a reduction of tyrosine phosphorylation levels of FAK and of the adapter protein paxillin. The down-regulation of FAK preceded apoptosis and cell detachment induced by OTA. The mycotoxin was also able to cause a decrease of the phosphorylation levels of the two Shc isoforms, P66 and P52, in adherent cells. Since these Shc isoforms have been implicated in the activation of protein kinase c-Src, which is required for FAK tyrosine phosphorylation, the observed dephosphorylation of FAK and of the FAK substrate paxillin by OTA could be ascribed to the early down-regulation of Shc isoforms. However, whether FAK and Shc phosphorylation contribute both to the same pathway leading to the induction of apoptosis by OTA or are involved in two parallel signaling pathways remains to be investigated.     

Lidocaine increases phosphorylation of focal adhesion kinase in rat hippocampal slices

We examined the effect of lidocaine on phosphorylation of the tyrosine kinase focal adhesion kinase (PP125FAK) in rat hippocampal slices by immunoblotting with both antiphosphotyrosine and specific anti-PP125FAK antibodies in the presence of tetrodotoxin (1 microM). Lidocaine induced a concentration-related increase in tyrosine phosphorylation of the 125-kDa band corresponding to PP125FAK phosphorylation (EC50 value=0.39+/-0.09 microM, maximal effect=169+/-28% of control, P<0.001). This effect was sensitive to neither the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine (MK 801, 10 microM) nor the inhibitor of the ryanodine receptor dantrolene (30 microM). In contrast, it was completely blocked by the protein kinase C (PKC) inhibitors chelerythrin, bisindolylmaleimide I (GF 109203X) and bisindolylmaleimide IX (RO-318220, 10 microM). We conclude that lidocaine increases phosphorylation of the tyrosine kinase PP125FAK in the rat hippocampus by a tetrotoxin (TTX)-insensitive mechanism which involves activation of PKC.     

Targeting vascular cell migration as a strategy for blocking angiogenesis: the central role of focal adhesion protein tyrosine kinase family

The formation of capillary-like structures during angiogenesis requires a series of well-orchestrated cellular events allowing endothelial cells and pericytes to migrate into the perivascular space. The proper activation of the migratory machinery in these cells is fine controlled by the presence of angiogenic challenges and by the interactions with extracellular matrix. The two members of the focal adhesion protein tyrosine kinases (FA-PTKs), FAK and PYK2, play a central role in modulating endothelial and vascular smooth muscle cells migration confirming the well consolidated observations in other migrating cell types. However accumulating data reveal that FAK and PYK2 are involved in several cell processes including cell proliferation and survival. FAK, once localized to focal adhesions, is thought to be one of the principal effectors in linking signals initiated by integrins and growth factor receptors to cytoskeleton, thus controlling migration. Although more obscure, and differently regulated, the function of PYK2 seems to be similar to that of FAK, but restricted to few cell types, including vasculature forming cells. FAK and PYK2 exert a primary role as adaptor proteins able to recruit, with high turnover, several proteins which in turn, through their docking domains and tyrosine kinase activity, determine both the turnover in focal adhesion assembly and the specificity of downstream signaling. The characterization of functional interactions of FA-PTKs may provide new potential therapeutic targets in order to control vascular pathological processes including angiogenesis.     

Transinactivation of the epidermal growth factor receptor tyrosine kinase and focal adhesion kinase phosphorylation by dietary flavonoids: effect on invasive potential of human carcinoma cells

Focal adhesion kinase (FAK), a member of a growing family of structurally distinct protein tyrosine kinases (PTK), has been linked to specific phosphorylation events, and the elevation of FAK activity in human carcinoma cells correlated with increased invasive potential. Transactivation of the epidermal growth factor receptor (EGFR) tyrosine kinase activity is proposed to stimulate cell migration and the subsequent activation of downstream signaling pathways. Quercetin (Qu) and luteolin (Lu), are potent PTK inhibitors as well as putative chemopreventive agents. The present work, we demonstrate that Qu and Lu at concentration of 20 microM transinactivated EGFR tyrosine kinase activity with marked reduction in phosphotyrosyl level of 170, 125, 65, 60 and 42 kDa cellular proteins, and induced apoptosis in MiaPaCa-2 cells. The 125 kDa protein was further identified as a FAK by immunoprecipitation and immunoblotting analyses. Tumor cells treated with Lu or Qu dampened the phosphorylation of FAK. In addition, our data clearly demonstrated that tumor cells responded to Qu and Lu by parallel reductions in the levels of phosphorylated FAK and the secreted matrix metalloproteinase (MMP) that may lead to the suppression of invasive potential and cell migration in vitro. While the molecular mechanism of FAK regulation of MMP secretion in tumor cells remains unclear, our results suggested that blockade of the EGFR-signaling pathway may contributed to the net effect. As suggested in the current study, targeting EGFR and FAK with the objective of modulating their regulatory pathways could offer prospects for the treatment of EGFR-responsive cancers in the future.     

Mechanisms of focal adhesion kinase regulation

Focal adhesion kinase (FAK) is a tyrosine kinase whose phosphorylation state and activity is tightly linked to cell adhesion to the extracellular matrix through integrin receptors. FAK's regulation by adhesion places it in a key position to be able to influence cellular events that are either dependent on cell adhesion like cell proliferation and survival, or that require modulation of cell adhesion like cell migration. FAK's involvement in cellular pathways that regulate cell growth and cell movement suggests that it may contribute to the development of cancer or other diseases. FAK's possible involvement in these pathways makes it a potential drug target. In this review we will focus on the developing view how FAK's activity and phosphorylation are regulated within the cell. Specifically, we will address the contribution of integrins and growth factor dependent pathways to FAK's activation. The role of the tyrosine kinase Src in FAK's regulation will be discussed. The contribution of various negative regulators of FAK's phosphorylation on its regulation including phosphatases and proteases will be discussed. Lastly, the emerging role of FAK's amino terminal FERM like domain in FAK's regulation will be explored. FAK's function within a cell are tightly linked to its phosphorylation state, thus understanding its normal regulation in the cell will provide important insight into drug development by highlighting novel regulatory mechanisms within FAK that potentially may be exploited.     

Neurotensin causes tyrosine phosphorylation of focal adhesion kinase in lung cancer cells

The effects of neurotensin on focal adhesion kinase were investigated using lung cancer cells. Neurotensin bound with high affinity to large cell carcinoma cell line NCI-H1299 as did neurotensin-(8-13), but not neurotensin-(1-7) or levocabastine. Addition of 100 nM neurotensin to NCI-H1299 cells caused transient tyrosine phosphorylation of focal adhesion kinase which was maximal after 1-2.5 min. Also, neurotensin-(8-13), but not neurotensin-(1-8) or levocabastine, caused tyrosine phosphorylation of focal adhesion kinase after addition to NCI-H1299 cells. Focal adhesion kinase tyrosine phosphorylation caused by neurotensin was inhibited by the nonpeptide neurotensin receptor antagonist (2-(1-(7-chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carbonyl)amino)-adamantane-2-carboxylic acid) (SR48692). SR48692 inhibited the clonal growth of NCI-H1299 cells, whereas neurotensin-stimulated proliferation and levocabastine had no effect. These results indicate that lung cancer cells have functional neurotensin receptors which regulate focal adhesion kinase tyrosine phosphorylation. It remains to be determined if neurotensin receptors and focal adhesion kinase plays a role in lung cancer cellular adhesion and migration.     

2,3,7,8-tetrachlorodibenzo-p-dioxin and epidermal growth factor cooperatively suppress peroxisome proliferator-activated receptor-gamma1 stimulation and restore focal adhesion complexes during adipogenesis: selective contributions of Src, Rho, and Erk distinguish these overlapping processes in C3H10T1/2 cells

Stimulation of PPARgamma1 and adipogenesis in multipotential C3H10T1/2 cells by the combination of dexamethasone and 3-isobutyl-1-methylxanthine (DM) is suppressed by 2,3,7,8 tetrachlorodibenzodioxin (TCDD) (10 nM). This suppression requires sustained activation of extracellular signal-regulated kinase (Erk)1/2. We show that it arises from an effect of TCDD on epidermal growth factor (EGF) signaling. DM initiates an early loss of cell adhesion that is reversed by this TCDD/EGF synergy. Src kinase activity was completely essential for adhesion restoration, sustained Erk activation, and suppression of peroxisome proliferator-activated receptor (PPAR)gamma1. MEK/Erk activity did not contribute, however, to TCDD-induced adhesion. Stimulation of adhesion may therefore precede elevation of Erk. Adhesion is produced by interaction of alphabeta integrins with extracellular matrix proteins and subsequent Src-mediated phosphorylation of focal adhesion kinase (FAK, Tyr576/577) and paxillin (Tyr118). TCDD enhanced the steady state Src-mediated phosphorylation of FAK but not of paxillin. Protein tyrosine phosphatase (PTPase) inhibition by orthovanadate (OVA) showed that this Src activity is highly restricted by PTPases. Partial inhibition of PTPases by OVA mimicked TCDD in producing EGF- and Src-dependent effects on cell adhesion and PPARgamma1 suppression. TCDD may therefore induce a protein that enhances Src effectiveness at adhesion sites. Rho kinase (ROCK) inhibition blocked TCDD/EGF stimulation of clustered focal adhesion complexes without affecting either sustained Erk activation or suppression of PPARgamma1. Thus, this ROCK-mediated clustering of integrin complexes is not needed for the effects of TCDD on Erk and PPARgamma1. A minimal cholesterol depletion with beta-methylcyclodextrin attenuated TCDD effects on PPARgamma1 and Erk activation. TCDD intervention is therefore linked to extracellular proteins. It indicates that TCDD-enhanced stimulation of EGF signaling to Erk may derive from the initial alphabeta integrin complexes.     

Roles of focal adhesion proteins in skeleton and diseases

The skeletal system,which contains bones,joints,tendons,ligaments and other elements,plays a wide variety of roles in body shaping,support and movement,protection of internal organs,production of blood cells and regulation of calcium and phosphate metabolism.The prevalence of skeletal diseases and disorders,such as osteoporosis and bone fracture,osteoarthritis,rheumatoid arthritis,and intervertebral disc degeneration,increases with age,causing pain and loss of mobility and creating a huge social...     

Low-dose exposure of intestinal epithelial cells to formaldehyde results in MAP kinase activation and molecular alteration of the focal adhesion protein paxillin

We investigated the potential pathophysiological role of non-lethal formaldehyde concentrations on human intestinal epithelial HT-29 cells. Expression levels of actin, tubulin and detectable cytokeratin isoforms 5, 13, 18, 19 and 20 were not affected after 24h of exposure to 1mM formaldehyde. By contrast, cellular organization of cytoskeletal constituents was already changed after 60 min. Within 15 min, formaldehyde induced profound tyrosine phosphorylation of the focal adhesion protein paxillin and of proteins at about 120-130 kDa. Concomitantly, phosphorylation of ERK-1/2 and p38 MAP kinase occurred. Paxillin was not only tyrosine phosphorylated but underwent a sustained molecular weight shift representing serine/threonine phosphorylation that was independent of MAP kinase activity and EGF-R-mediated signalling. Our data show that exposure of intestinal epithelial cells to low-dose formaldehyde is followed by rapid and profound signalling events. The data suggest a modifier role of environmental or endogenous formaldehyde for epithelial cell functions.     

Formaldehyde-induced paxillin–tyrosine phosphorylation and paxillin and P53 downexpression in Hela cells

Formaldehyde (FA) is an environmental pollutant and an endogenous product believed to be involved in tumorigenesis. However, the underlying mechanism of observed FA effects has not been clearly defined. Paxillin is a focal adhesion protein that may play an important role in several signaling pathways. Many paxillin-interacting proteins are involved in the regulation of actin cytoskeleton organization, which is necessary for cell motility events associated with diverse biological responses, such as embryonic development, wound repair and tumor metastasis. P53 is important in multicellular organisms, where it regulates the cell cycle and thus functions as a tumor suppressor that is involved in preventing cancer. In this study, we investigated the effects of FA on paxillin–tyrosine phosphorylation and P53 expression in Hela cells by Western blot and immunofluorescence. Western blot analysis revealed that nonlethal concentrations of FA (0.5, 1.0 and 2.0?mM, with the exposure time for 0.5, 1.0 and 2.0?h, respectively) had downregulated paxillin and wild-type p53 genes expression while upregulated paxillin–tyrosine phosphorylation significantly. At the same time, phosphotyrosine at the focal adhesion sites detected by immunofluorescence assay obviously increased in Hela cells incubated with 2.0?mM FA for 2?h. The results suggested that paxillin and p53 genes expression may be involved in FA-related adverse effects and the mechanism may be involved in paxillin–tyrosine phosphorylation.     

Focal adhesion kinase as a cancer therapy target

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that resides at the sites of focal adhesions. The 125 kDa FAK protein is encoded by the FAK gene located on human chromosome 8q24. Structurally, FAK consists of an amino-terminal regulatory FERM domain, a central catalytic kinase domain, and a carboxy-terminal focal adhesion targeting domain. FAK has been shown to be an important mediator of cell adhesion, growth, proliferation, survival, angiogenesis and migration, all of which are often disrupted in cancer cells. Normal tissues have low expression of FAK, while primary and metastatic tumors significantly overexpress this protein. This review summarizes expression of FAK by immunohistochemical staining in different tumor types and presents several FAK inhibition therapy approaches.     

Protein kinase B/AKT and focal adhesion kinase: two close signaling partners in cancer

AKT (or protein kinase B) and focal adhesion kinase (FAK) are two important kinases that regulate various cellular functions. Each is overexpressed and/or aberrantly activated in diverse cancers. Several small molecular inhibitors targeting either AKT or FAK are in development or in clinical trials. It is well established that FAK is an upstream regulator of AKT signaling pathway in various cancer cell lines and in xenograft tumor models. However, very recent reports from our laboratory and others demonstrate that AKT can also directly regulate FAK through direct association and serine phosphorylation. This indicates that AKT and FAK may be dual therapeutic targets for pharmacologic intervention in the treatment of primary and metastatic cancer. FAK-AKT interaction is particularly critical for metastatic adhesion. We review recent developments in AKT and FAK signaling in cancer with the particular emphasis on the novel signaling pathways in which FAK is downstream of AKT. We also provide an update on inhibitors targeting AKT or FAK currently in clinical trials.     

Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays critical roles in integrin-mediated signal transductions and also participates in signaling by other cell surface receptors. In integrin-mediated cell adhesion, FAK is activated via disruption of an auto-inhibitory intra-molecular interaction between its amino terminal FERM domain and the central kinase domain. The activated FAK forms a complex with Src family kinases, which initiates multiple downstream signaling pathways through phosphorylation of other proteins to regulate different cellular functions. Multiple downstream signaling pathways are identified to mediate FAK regulation of migration of various normal and cancer cells. Extensive studies in cultured cells as well as conditional FAK knockout mouse models indicated a critical role of FAK in angiogenesis during embryonic development and cancer progression. More recent studies also revealed kinase-independent functions for FAK in endothelial cells and fibroblasts. Consistent with its roles in cell migration and angiogenesis, increased expression and/or activation of FAK are found in a variety of human cancers. Therefore, small molecular inhibitors for FAK kinase activity as well as future development of novel therapies targeting the potentially kinase-independent functions of FAK are promising treatments for metastatic cancer as well as other diseases.     

Focal adhesion kinase controls prostate cancer progression via intrinsic kinase and scaffolding functions

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which mediates integrin signaling from the sites of connection to the extracellular membrane known as focal adhesions. FAK mediates essential cellular processes including growth, proliferation, adhesion, migration, and survival through its functions as a molecular scaffold and as a kinase. FAK is frequently overexpressed and overactive in prostate cancer, which represents the second leading cause of cancer deaths in American men. Through the activation of major oncogenic pathways, FAK promotes the growth, survival, migration, metastasis, and androgen-independence of prostate tumors in vitro and in vivo. A full examination of FAK kinase function has never been completed despite many studies suggesting its importance and the development of kinase-specific therapeutic inhibitors. An expanded understanding of FAK kinase function is required to understand the role of FAK in prostate cancer progression, thereby aiding future development of novel inhibitory drugs and screening procedures.     

The microtubule binding drug laulimalide inhibits vascular endothelial growth factor-induced human endothelial cell migration and is synergistic when combined with docetaxel (taxotere)

Laulimalide, a natural product from marine sponges, is a microtubule-stabilizing agent that binds to tubulin at a site distinct from that of the taxoids. In the present study, we found that laulimalide inhibited human umbilical vein endothelial cell (HUVEC) tubule formation and vascular endothelial growth factor (VEGF)-induced HUVEC migration, key components of the angiogenic process. These occurred at concentrations substantially lower than that which inhibited HUVEC proliferation. When combined, laulimalide and docetaxel (Taxotere) synergistically inhibited migration and tubule formation, but their combined effect on proliferation was antagonistic. Possible mechanism(s) by which laulimalide inhibited VEGF-induced HUVEC migration were explored. Similar to docetaxel, laulimalide had no effect on the VEGF-induced tyrosine phosphorylation of the VEGF receptor Flk-1/KDR (VEGFR-2). Low concentrations of laulimalide substantially blocked subsequent VEGFR-2 downstream events, as did docetaxel, including the phosphorylation of the Tyr397 and Tyr407 residues of focal adhesion kinase (FAK), the association of VEGFR-2 with FAK and Hsp90, and the Tyr31 phosphorylation of paxillin. Laulimalide inhibited integrin activation; however, compared with docetaxel, it had a weaker inhibitory effect on the VEGF-induced association of VEGFR-2 with the alpha5beta1 integrin. Compared with docetaxel, laulimalide more potently caused a reduction in the constitutive levels (i.e., in the absence of VEGF) of phosphorylated paxillin and more potently inhibited the association of RhoA with the alpha5beta1 integrin. In conclusion, although both docetaxel and laulimalide inhibited integrin-associated signaling pathways that mediated VEGF-induced cell migration, their actions on the signaling cascade seemed not to be identical. These complementary actions could account for their synergistic effects on HUVEC.     

Pelargonidin attenuates PDGF-BB-induced aortic smooth muscle cell proliferation and migration by direct inhibition of focal adhesion kinase

Pelargonidin is a natural red pigment found in fruits and vegetables, and has been reported to exhibit various effects potentially beneficial for human health. However, the possible preventive effects of pelargonidin toward atherosclerosis and mechanisms involved have not been investigated to date. Here, we compared the effects of pelargonidin and its glucoside-conjugated form, pelargonidin-3-glucoside (P3G), on proliferation and migration induced by platelet-derived growth factor (PDGF)-BB in human aortic smooth muscle cells (HASMCs). Pelargonidin, but not P3G, exhibited strong inhibitory effects against PDGF-BB-induced HASMC proliferation and migration, while suppressing PDGF-BB-induced ex vivo rat aortic ring sprouting. Immunoblot analysis revealed that pelargonidin inhibited PDGF-BB-induced phosphorylation of focal adhesion kinase (FAK) as well as F-actin reduction, whereas Src, mitogen-activated protein kinases (MAPKs) and Akt phosphorylation status were not altered. We also observed that the anti-proliferative and migratory effects of both pelargonidin and P3G corresponded with the extent of FAK inhibition. Both in vitro and ex vivo pull-down assays revealed that pelargonidin binds directly with FAK in an adenosine triphosphate-competitive manner, suggesting that FAK could be a molecular target of pelargonidin. Interestingly, pelargonidin did not exhibit inhibitory effects on the proliferation, migration or FAK phosphorylation of human umbilical vein endothelial cells (HUVECs). Taken together, our results suggest that pelargonidin exhibits potential preventive effects toward atherosclerosis through the attenuation of HASMC proliferation and migration, as well as aortic sprouting via the direct inhibition of FAK activity.     

The focal adhesion kinase inhibitor PF-562,271 impairs primary CD4+ T cell activation

The focal adhesion kinase inhibitor, PF-562,271, is currently in clinical development for cancer, however it is not known how PF-562,271 affects T cell function. Here, we demonstrate inhibitory effects of PF-562,271 on the activation of primary human and mouse T cells. PF-562,271 inhibits T cell receptor signaling-induced T cell adhesion to intercellular adhesion molecule-1 and T cell interactions with antigen-presenting cells. An additional focal adhesion kinase inhibitor, PF-573,228, and genetic depletion of focal adhesion kinase also impair T cell conjugation with antigen-presenting cells. PF-562,271 blocks phosphorylation of the signaling molecules zeta chain associate protein of 70 kDa, linker of activated T cells, and extracellular signal-regulated kinase, and impairs T cell proliferation. The effects observed on T cell proliferation cannot solely be attributed to focal adhesion kinase inhibition, as genetic depletion did not alter proliferation. The effect of PF-562,271 on T cell proliferation is not rescued when proximal T cell receptor signaling is bypassed by stimulation with phorbol-12-myristate-13-acetate and ionomycin. Taken together, our findings demonstrate that focal adhesion kinase regulates integrin-mediated T cell adhesion following T cell receptor activation. Moreover, our findings suggest that PF-562,271 may have immunomodulatory effects that could impact its therapeutic applications.     

Involvement of Rho signaling in PAR2-mediated regulation of neutrophil adhesion to lung epithelial cells

Protease-activated receptor 2 (PAR2) has been implicated in the pathogenesis of airway inflammation. We report that epithelial PAR2 stimulation with trypsin (0.05-1 U/ml) or an agonist peptide (SLIGKV-NH2, 1-100 microM) for 0.5-3 h dose- and time-dependently enhanced neutrophil adhesion to alveolar type II epithelial cells (A549 cells) and that this stimulation also induced the formation of epithelial actin filaments. Both responses in neutrophil adhesion and epithelial actin reorganization were reduced by a Rho inhibitor, mevastatin and by a Rho-associated kinase (ROCK) inhibitor, Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide). Neutrophil adherence was also inhibited by an inhibitor of actin polymerization, cytochalasin D and a tyrosine kinase inhibitor, genistein. Further, the PAR2-mediated tyrosine phosphorylation of focal adhesion kinase (FAK), a major cytoskeleton protein, was detected, and this response was inhibited by mevastatin or Y-27632. These results suggest that PAR2 stimulation of alveolar epithelial cells enhances neutrophil adhesion presumably at least in part through Rho/ROCK signal-mediated actin cytoskeleton reorganization associated with the tyrosine phosphorylation of FAK.     

The novel synthetic ether lipid inositol-C2-PAF inhibits phosphorylation of the tyrosine kinases Src and FAK independent of integrin activation in transformed skin cells

New alkyl-phospholipids that are structurally derived from platelet-activating factor are promising candidates for anticancer treatment. The mechanism of action of derivatives of the platelet-activating factor is distinctly different from that of known DNA- or tubulin-targeting anticancer agents because they are incorporated into cell membranes, where they accumulate and interfere with a wide variety of key enzymes. We recently presented evidence of a novel group of alkyl-phospholipids, glycosidated phospholipids that efficiently inhibit cell proliferation. One member of this group, inositol-C2-PAF (Ino-C2-PAF), displays high efficacy and low cytotoxicity in HaCaT-cells, an immortalized non-tumorigenic skin keratinocyte cell line.Here, we show that Ino-C2-PAF also inhibits the motility of the skin-derived transformed cell lines HaCaT and squamous cell carcinoma (SCC)-25. This decrease in motility is accompanied by an altered F-actin cytoskeleton, increased clustering of integrins, and increased cell–matrix adhesion. Despite enhanced integrin clustering and matrix adhesion, we observed less phosphorylation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and Src, key regulators of cellular motility, at focal adhesion sites. Transient transfection of constitutively active variants of FAK and Src could at least in part bybass this inhibitory effect of Ino-C2-PAF. This fact indicates that Ino-C2-PAF interferes with the fine-tuned balance between adhesion and migration. Ino-C2-PAF at least partially uncouples integrin-mediated attachment from subsequent integrin-dependent signaling steps, which inhibits migration in transformed keratinocyte cell lines.