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Lactosylceramide (LacCer), a neutral glycosphingolipid, is abundantly expressed on human neutrophils, and specifically recognizes several pathogenic microorganisms. LacCer forms membrane microdomains coupled with the Src family kinase Lyn on the plasma membrane, and ligand binding to LacCer activates Lyn, resulting in neutrophil functions. In contrast, neutrophilic differentiated HL-60 cells do not have Lyn-associated LacCer-enriched microdomains and lack LacCer-mediated functions. In neutrophil plasma membranes, the very long fatty acid C24:0 and C24:1 chains are the main components of LacCer, whereas plasma membrane of D-HL-60 cells mainly includes C16-LacCer species. Here, we suggest that LacCer species containing very long fatty acid chains are indispensable for the association of Lyn with LacCer-enriched microdomains and LacCer-mediated functions., (Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Engagement of FcepsilonRI causes its phosphorylation by Lyn kinase. Two alternatively spliced variants, Lyn A and B, are expressed in mast cells, and both isoforms interact with FcepsilonRI. Unlike Lyn A, Lyn B lacks a 21-aa region in the N-terminal unique domain. In this study, we investigated the role of Lyn A and B isoforms in mast cell signaling and responses. Lyn B was found to be a poor inducer of mast cell degranulation and was less potent in both inositol 1,4,5-triphosphate production and calcium responses. Expression of Lyn B alone showed reduced phosphorylation of both phospholipase Cgamma-1 and -2 and decreased interaction of phospholipase Cgamma-1 with the phosphorylated linker for activation of T cells. Lyn B also showed increased binding of tyrosine-phosphorylated proteins, which included the negative regulatory lipid phosphatase SHIP-1. In contrast, both Lyn A and B caused similar total cellular tyrosine phosphorylation and FcepsilonRI phosphorylation and neither Lyn A nor Lyn B alone could completely restore mast cell degranulation or dampen the excessive cytokine production seen in the absence of Lyn. However, expression of both isoforms showed complementation and normalized responses. These findings demonstrate that Lyn B differs from Lyn A in its association with SHIP-1 and in the regulation of calcium responses. However, complementation of both isoforms is required in mast cell activation.

Objective: The activity of the SH2-containing-phosphatidylinositol-5'-phosphatase (SHIP, also known as SHIP1), a critical hematopoietic-restricted negative regulator of the PI3 kinase (PI3K) pathway, is regulated in large part via its protein levels. We sought to determine the mechanism(s) involved in its downregulation by BCR-ABL and by interleukin (IL)-4., Materials and Methods: We used Ba/F3(p210-tetOFF) cells to study the downregulation of SHIP by BCR-ABL and bone marrow-derived macrophages to study SHIP's downregulation by IL-4., Results: We show herein that BCR-ABL downregulates SHIP, but not SHIP2 or PTEN, and this can be blocked with the Src kinase inhibitor PP2, which inhibits the tyrosine phosphorylation of SHIP, or with the proteasomal inhibitor MG-132. We also show, using anti-SHIP immunoprecipitates, that c-Cbl and Cbl-b are associated with SHIP and that BCR-ABL induces SHIP's polyubiquitination. This ubiquitination can be blocked with PP2, consistent with the tyrosine phosphorylation of SHIP acting as a signal for its ubiquitination. In bone marrow-derived macrophages, IL-4 also leads to the proteasomal degradation of SHIP but, unlike in Ba/F3(p210-tetOFF) cells, SHIP2 is also proteasomally degraded and the degradation of both inositol phosphatases can be prevented with PP2 or MG-132., Conclusion: Our results suggest that SHIP protein levels can be reduced via BCR-ABL and/or Src family member-induced tyrosine phosphorylation of SHIP because this triggers its polyubiquitination and degradation within the proteasome., (2010 ISEH-Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

The antimicrobial peptide human beta-defensin-3 (hBD-3) is produced by epidermal keratinocytes and protects the skin from infections. This peptide induces the release of a lipid mediator, prostaglandin D(2) from dermal mast cells. Prostaglandin D(2) binds to cell-surface G protein-coupled receptors, D prostanoid receptor, and chemoattractant receptor-homologous molecule expressed on T helper cell type 2 (CRTH2). Both receptors are detected on epidermal keratinocytes. It is reported that prostaglandin D(2) is involved in cutaneous allergy, however, its role in antimicrobial defense is unknown. We examined the in vitro effects of prostaglandin D(2) on hBD-3 production in normal human keratinocytes. Prostaglandin D(2) enhanced hBD-3 secretion and mRNA expression in human keratinocytes. Prostaglandin D(2)-induced hBD-3 production was suppressed by the CRTH2 antagonist ramatroban and by antisense oligonucleotides against c-Jun and c-Fos, components of a transcription factor, activator protein-1 (AP-1). Prostaglandin D(2) enhanced the transcriptional activity and DNA binding of AP-1, expression, phosphorylation, and DNA binding of c-Fos proteins in keratinocytes. Prostaglandin D(2)-induced hBD-3 production, AP-1 activity, and c-Fos expression and phosphorylation were suppressed by U0126, PP2, and pertussis toxin, which are inhibitors of mitogen-activated protein kinase kinase (MEK), src, and G(i) proteins, respectively. The phosphorylation of extracellular signal-regulated kinase (ERK), downstream kinase of MEK, was induced by prostaglandin D(2), and suppressed by ramatroban, pertussis toxin, PP2, and U0126. These results suggest that prostaglandin D(2) induces hBD-3 production in human keratinocytes by activating AP-1 through the expression and phosphorylation of c-Fos via the CRTH2/G(i)/src/MEK/ERK pathway. Prostaglandin D(2) may promote cutaneous antimicrobial activity via hBD-3., (Copyright 2009 Elsevier Inc. All rights reserved.)

The widespread nature of protein phosphorylation/dephosphorylation underscores its key role in cell signaling metabolism, growth and differentiation. Tyrosine phosphorylation of cytoplasmic proteins is a critical event in the regulation of intracellular signaling pathways activated by external stimuli. An adequate balance in protein phosphorylation is a major factor in the regulation of osteoclast and osteoblast activities involved in bone metabolism. However, although phosphorylation is widely recognized as an important regulatory pathway in skeletal development and maintenance, the mechanisms involved are not fully understood. Among the putative protein-tyrosine kinases (ptk) and protein-tyrosine phosphatases (ptp) involved in this phenomenon there is increasing evidence that Src and low molecular weight-ptps play a central role in a range of osteoblast activities, from adhesion to differentiation. A role for Src in bone metabolism was first demonstrated in Src-deficient mice and has since been confirmed using low molecular weight Src inhibitors in animal models of osteoporosis. Several studies have shown that Src is important for cellular proliferation, adhesion and motility. In contrast, few studies have assessed the importance of the ptk/ptp balance in driving osteoblast metabolism. In this review, we summarize our current knowledge of the functional importance of the ptk/ptp balance in osteoblast metabolism, and highlight directions for future research that should improve our understanding of these critical signaling molecules., (Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.)

Antillatoxin (ATX) is a structurally novel lipopeptide that activates voltage-gated sodium channels (VGSC) leading to sodium influx in cerebellar granule neurons and cerebrocortical neurons 8 to 9 days in vitro (Li et al., 2001; Cao et al., 2008). However, the precise recognition site for ATX on the VGSC remains to be defined. Inasmuch as elevation of intracellular sodium ([Na(+)](i)) may increase N-methyl-d-aspartate receptor (NMDAR)-mediated Ca(2+) influx, Na(+) may function as a signaling molecule. We hypothesized that ATX may enhance neurite outgrowth in cerebrocortical neurons by elevating [Na(+)](i) and augmenting NMDAR function. ATX (30-100 nM) robustly stimulated neurite outgrowth, and this enhancement was sensitive to the VGSC antagonist, tetrodotoxin. To unambiguously demonstrate the enhancement of NMDA receptor function by ATX, we recorded single-channel currents from cell-attached patches. ATX was found to increase the open probability of NMDA receptors. Na(+)-dependent up-regulation of NMDAR function has been shown to be regulated by Src family kinase (SFK) (Yu and Salter, 1998). The Src kinase inhibitor PP2 abrogated ATX-enhanced neurite outgrowth, suggesting a SFK involvement in this response. ATX-enhanced neurite outgrowth was also inhibited by the NMDAR antagonist, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), and the calmodulin-dependent kinase kinase (CaMKK) inhibitor, 1,8-naphthoylene benzimidazole-3-carboxylic acid (STO-609), demonstrating the requirement for NMDAR activation with subsequent downstream engagement of the Ca(2+)-dependent CaMKK pathway. These results with the structurally and mechanistically novel natural product, ATX, confirm and generalize our earlier results with a neurotoxin site 5 ligand. These data suggest that VGSC activators may represent a novel pharmacological strategy to regulate neuronal plasticity through NMDAR-dependent mechanisms.

Carbonic anhydrase IX (CA IX) belongs to the physiologically important enzymes which contribute to tumor physiology. Tumor-associated expression of CA IX is induced mainly due to its strong transcriptional activation via hypoxia-inducible factor 1 (HIF-1). Therefore, CA IX can serve as a surrogate marker of hypoxia and a prognostic indicator. HIF-1 is a master transcription factor that mediates essential homeostatic responses to cellular and systemic hypoxia by activating transcription of multiple genes including those encoding glycolytic enzymes and vascular endothelial growth factor. In addition to hypoxia, HIF-1alpha expression can be up-regulated by growth factors and oncogenic signals (e.g. Src oncogene). Consequently, induction of the HIF-1alpha transcription factor up-regulates target gene expression. The results from the present study suggest that Src oncogene induces CA IX expression under normoxic, as well as hypoxic conditions. Moreover, we demonstrate that Src-mediated induction of CA IX expression is critically dependent on HIF-1alpha activity. Transcriptional activity of the CA9 promoter was significantly increased by expression of v-Src or c-Src. The effect was more prominent in normoxia, most likely because of already high level of HIF-1alpha expression in hypoxia. By co-transfection with dominant-negative HIF-1alpha we confirmed that Src-induced stimulation of CA9 transcription is mediated via HIF-1alpha. Consistent with this, Src-expressing HeLa cells displayed higher levels of HIF-1alpha protein. Finally, these results indicate a novel regulatory pathway responsible for increased CA IX expression in tumor cells and define CA IX as a new down-stream target for Src oncogene.

Matrix metalloproteinase-9 (MMP-9) is the major MMP produced by B-CLL cells and contributes to their tissue infiltration by degrading extracellular and membrane-anchored substrates. Here we describe a different function for MMP-9 in B-CLL, which involves the hemopexin domain rather than its catalytic function. Binding of soluble or immobilized (pro)MMP-9, a catalytically inactive proMMP-9 mutant, or the MMP-9 hemopexin domain to its docking receptors alpha4beta1 integrin and CD44v, induces an intracellular signaling pathway that prevents B-CLL apoptosis. This pathway is induced in all B-CLL cases, is active in B-CLL lymphoid tissues, and consists of Lyn activation, STAT3 phosphorylation, and Mcl-1 upregulation. Our results establish that MMP/receptor binding induces intracellular survival signals and highlight the role of (pro)MMP-9 in B-CLL pathogenesis., (2010 Elsevier Inc. All rights reserved.)

The FcgammaRIIB is a potent inhibitory coreceptor that blocks BCR signaling in response to immune complexes and, as such, plays a decisive role in regulating Ab responses. The recent application of high-resolution live cell imaging to B cell studies is providing new molecular details of the earliest events in the initiation BCR signaling that follow within seconds of Ag binding. In this study, we report that when colligated to the BCR through immune complexes, the FcgammaRIIB colocalizes with the BCR in microscopic clusters and blocks the earliest events that initiate BCR signaling, including the oligomerization of the BCR within these clusters, the active recruitment of BCRs to these clusters, and the resulting spreading and contraction response. Fluorescence resonance energy transfer analyses indicate that blocking these early events may not require molecular proximity of the cytoplasmic domains of the BCR and FcgammaRIIB, but relies on the rapid and sustained association of FcgammaRIIB with raft lipids in the membrane. These results may provide novel early targets for therapies aimed at regulating the FcgammaRIIB to control Ab responses in autoimmune disease.

EGF induces signal transduction between EGFR and FAK, and FAK is required for EGF-induced cell migration. It is unknown, however, what factor mediates the interaction between EGFR and FAK and leads to EGF-induced FAK phosphorylation. Here, we identify SRC-3Delta4, a splicing isoform of the SRC-3 oncogene, as a signaling adaptor that links EGFR and FAK and promotes EGF-induced phosphorylations of FAK and c-Src. We identify three PAK1-mediated phosphorylations in SRC-3Delta4 that promote the localization of SRC-3Delta4 to the plasma membrane and mediate the interactions with EGFR and FAK. Importantly, overexpression of SRC-3Delta4 promotes MDA-MB231-induced breast tumor metastasis. Our findings identify phosphorylated SRC-3Delta4 as a missing adaptor between EGFR and its downstream signaling molecule FAK to coordinately regulate EGF-induced cell migration. Our study also reveals that a nuclear receptor coactivator can act in the periphery of a cell to directly mediate activation of an enzyme.

Lyn-deficient mice develop Ab-mediated autoimmune disease resembling systemic lupus erythematosus where hyperactive B cells are major contributors to pathology. In this study, we show that an inflammatory environment is established in Lyn(-/-) mice that perturbs several immune cell compartments and drives autoimmune disease. Lyn(-/-) leukocytes, notably B cells, are able to produce IL-6, which facilitates hyperactivation of B and T cells, enhanced myelopoiesis, splenomegaly, and, ultimately, generation of pathogenic autoreactive Abs. Lyn(-/-) dendritic cells show increased maturation, but this phenotype is independent of autoimmunity as it is reiterated in B cell-deficient Lyn(-/-) mice. Genetic deletion of IL-6 on a Lyn-deficient background does not alter B cell development, plasma cell accumulation, or dendritic cell hypermaturation, suggesting that these characteristics are intrinsic to the loss of Lyn. However, hyperactivation of B and T cell compartments, extramedullary hematopoiesis, expansion of the myeloid lineage and autoimmune disease are all ameliorated in Lyn(-/-)IL-6(-/-) mice. Importantly, our studies show that although Lyn(-/-) B cells may be autoreactive, it is the IL-6-dependent inflammatory environment they engender that dictates their disease-causing potential. These findings improve our understanding of the mode of action of anti-IL-6 and B cell-directed therapies in autoimmune and inflammatory disease treatment.

The present review focuses primarily on the studies we made in recent years to improve the understanding of the molecular mechanisms of PGF2alpha-induced hypertrophy of Vascular Smooth Muscle Cells (VSMC). In this review, we will summarize the recent findings in the context of the PGF2alpha signaling pathway in three parts: PGF2alpha binding to its receptor, transactivation of EGF receptor, two independent signaling transduction pathways increasing the expression of NOX1 gene.

Although the role of the actin cytoskeleton has become increasingly elucidated, the role of actin polymerization in inflammatory processes remains poorly understood. Here, we examine the role of the actin cytoskeleton during LPS-mediated inflammatory events in RAW264.7 cells and peritoneal macrophages. We observed that actin cytoskeleton disruption by cytochalasin B and siRNA to cytoplasmic actin strongly down-regulated LPS-mediated inflammatory responses such as NO production, PGE(2) release, and TNF-alpha secretion. Actin cytoskeleton disruption by cytochalasin B down-regulated a series of signaling cascades including PI3K, Akt, and IKK, but not MAPKs, necessary for NF-kappaB activation without down-regulating total forms of the proteins as assessed by measuring their phosphorylation levels. In particular, cytochalasin B significantly inhibited LPS-induced both phosphorylation and kinase activity of Src without altering total level, implying that Src may be a potential pharmacological target of actin cytoskeleton rearrangement. Moreover, the direct association of Src with actin was actin polymerization-dependent according to immunoprecipitation analysis performed with a GFP-actin wild type and HA-tagged Src. Therefore, our data suggest that actin cytoskeleton rearrangement may be a key event during the regulation of inflammatory responses that modulates the activity of Src and its downstream signaling molecules.

The two main tyrosine kinases (TK) in the brain are p60Src and p59Fyn, expressed as specific isoforms (p60SrcNI, p60SrcNI+NII and p59fynB). They play a pivotal role in some major processes such as neuronal growth and myelinisation. Another member of this TK family was then reported in brain, the p56lck. Its name Lck (lymphocyte cell kinase) indicates its cellular specificity observed initially, so its presence in the brain was intriguing. But no further studies were performed to understand its role in brain until recent clinical studies on Alzheimer patients' brains. One study reveals a decreased p56lck level in the brains of these patients while another study shows an association between one peculiar SNP (single nucleotide polymorphism) of the lck gene and some cases of the disease. These new data prompt us to reinvestigate the original biochemical data and to confront them with the present knowledge. This analysis suggests some hypothesis concerning both the Lck protein expressed in the brain (rather an isoform than the lymphocyte protein itself) and its role (to maintain the neuronal survival presumably by protecting them from inflammation, the main pathway that leads to neuron degeneracy)., (Copyright 2009 Académie des sciences. Published by Elsevier SAS. All rights reserved.)

Aims: Increased levels of resistin, a peptide secreted by adipocytes and inflammatory cells, circulate in patients with insulin resistance and early type 2 diabetes, a high-risk population for the development of a diffuse and extensive pattern of arteriosclerosis. Recent data suggest that resistin may activate vascular cells such as smooth muscle cells and endothelial cells, but hitherto nothing is known about the role of resistin in CD4-positive lymphocytes. Therefore, the present study examined the effect of resistin on CD4-positive lymphocyte migration, an important process in early atherogenesis., Methods and Results: Resistin stimulated CD4-positive cell chemotaxis in a concentration-dependent manner with a maximal induction of 2.25 +/- 0.54 at 100 ng/mL (P < 0.05, n = 7). This process involves pertussis toxin-sensitive G-proteins as well as activation of Src- and phosphoinositide 3-kinase (PI 3-K). Biochemical analysis showed that resistin induces phosphorylation of Src and PI 3-K activation in human CD4-positive cells. In addition, resistin activates RhoA, Rac-1, and Cdc42 in these cells as shown by affinity precipitation experiments. Finally, resistin-induced phosphorylation of myosin light chain was inhibited by Src short interference RNA transfection, underscoring the importance of the upstream signalling molecule Src in resistin-induced migration., Conclusion: These data support an active role of resistin in CD4-positive lymphocyte chemotaxis and elucidate molecular mechanisms in resistin-induced cell migration.

Heparin-binding epidermal growth factor EGF-like growth factor (HB-EGF) is a member of the epidermal growth factor family that is expressed in many cell types. We have previously reported the effects of HB-EGF on intestinal epithelial cells and endothelial cells after exposure to ischemia/reperfusion in vivo or anoxia/reoxygenation injury in vitro. However, the effect of HB-EGF on neuronal cells is largely unexplored. In this study, we examined the effect of HB-EGF on neurite outgrowth in pheochromocytoma (PC12) cells as well as the neuroprotective effect of HB-EGF on injured PC12 cells exposed to oxygen and glucose deprivation (OGD), which mimics ischemic conditions. We found that HB-EGF significantly promotes PC12 cell neurite outgrowth and that this effect was blocked by EGF receptor (EGFR) inhibition or mitogen-activated protein kinase (MAPK) inhibition, but not by tyrosine kinase inhibition. In the face of OGD injury, HB-EGF preserves cell viability and decreases apoptosis and LDH release in PC12 cells. HB-EGF-mediated cytoprotection was abolished by EGFR inhibition and MAPK inhibition. We conclude that HB-EGF, through its interaction with the EGF receptor, activates the MAPK signaling pathway in PC12 cells under basal or injury conditions, leading to enhanced neurite outgrowth and neuroprotection against ischemic injury., (Copyright © 2010 S. Karger AG, Basel.)

We have shown the increase of Src(Tyr416) phosphorylation in rat colonic mucosa at early stages of 6% iodoacetamide-induced ulcerative colitis (UC), while the level of Src protein expression was not changed. Pretreatment of rats with Src inhibitor PP1 (0.2 mg/100 g, subcutaneously) decreased the colonic vascular permeability (VP) (P < or = 0.001) and pSrc(Tyr416) level during iodoacetamide-UC. Iodoacetamide-induced autophosphorylation and upregulation of VEGFR-2 was associated with Src activation in colonic mucosa of rats. Sequentially, protein-protein interaction between beta-arrestine2 and VE-cadherine was enhanced, that might be a reason of colonic endothelium barrier disruption. We concluded that Src plays a key role in the mechanisms of increasing the colonic VP during experimental UC.

Acquisition of independence from anchorage to the extracellular matrix is a critical event for onset and progression of solid cancers. To identify and characterize new genes conferring anchorage independence, we transduced MCF10A human normal breast cells with a retroviral cDNA expression library and selected them by growth in suspension. Microarray analysis targeted on library-derived transcripts revealed robust and reproducible enrichment, after selection, of cDNAs encoding the scaffolding adaptor Gab2. Gab2 was confirmed to strongly promote anchorage-independent growth when overexpressed. Interestingly, downregulation by RNA interference of endogenous Gab2 in neoplastic cells did not affect their adherent growth, but abrogated their growth in soft agar. Gab2-driven anchorage independence was found to specifically involve activation of the Src-Stat3 signaling axis. A transcriptional 'signature' of 205 genes was obtained from GAB2-transduced, anchorage-independent MCF10A cells, and found to contain two main functional modules, controlling proliferation and cell adhesion/migration/invasion, respectively. Extensive validation on breast cancer data sets showed that the GAB2 signature provides a robust prognostic classifier for breast cancer metastatic relapse, largely independent from existing clinical and genomic indicators and from estrogen receptor status. This work highlights a pivotal role for GAB2 and its transcriptional targets in anchorage-independent growth and breast cancer metastatic progression.

Despite the recent advances in the diagnosis of bladder cancer, recurrence after surgical intervention for muscle invasive disease is still problematic as nearly half of the patients harbor occult distant metastases and this, in turn, is associated with poor 5-year survival rate. We have recently identified Rho family GDP dissociation inhibitor 2 (RhoGDI2) protein as functional metastasis suppressor and a prognostic marker in patients after cystectomy. In identifying the mechanisms underlying metastasis suppression by RhoGDI2, we found this protein to be associated with the c-Src kinase in human tumors, where the expression of both is diminished as a function of stage. Interestingly, c-Src bound to and phosphorylated RhoGDI2 resulting in enhanced metastasis suppressive potency. In this review, we will discuss the established roles of c-Src and RhoGDI2 in bladder cancer and speculate on their therapeutic relevance.

Purpose: To determine the beta1/beta3 integrin-mediated pathways that regulate cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM., Methods: Expression of CD47 (an alphavbeta3 integrin coreceptor/thrombospondin-1 receptor) and integrins alphavbeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 integrin ligand) in the absence or presence of the beta3 integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2., Results: HTM cells expressed CD47 and integrins alphavbeta3 and beta1. beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it., Conclusions: Distinct beta1 and alphavbeta3 integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both integrin pathways were Src dependent.

Therapeutic strategies such as using channel blockers and reducing culture temperature have been used to rescue some long QT-associated voltage-gated potassium Kv trafficking defective mutant channels. A hyperpolarization-activated cyclic nucleotide-gated HCN4 pacemaker channel mutant (D553N) has been recently found in a patient associated with cardiac arrhythmias including long QT. D553N showed the defective trafficking to the cell surface, leading to little ionic current expression (loss-of-function). We show in this report that enhanced tyrosine phosphorylation mediated by Src, Fyn, and Yes kinases was able to restore the surface expression of D553N for normal current expression. Src or Yes, but not Fyn, significantly increased the current density and surface expression of D553N. Fyn accelerated the activation kinetics of the rescued D553N. Co-expression of D553N with Yes exhibited the slowest activation kinetics of D553N. Src, Fyn, and Yes significantly enhanced the tyrosine phosphorylation of D553N. A combination of Src, Fyn, and Yes rescued the current expression and the gating of D553N comparable with those of wild-type HCN4. In conclusion, we demonstrate a novel mechanism using three endogenous Src kinases to rescue a trafficking defective HCN4 mutant channel (D553N) by enhancing the tyrosine phosphorylation of the mutant channel protein.

Epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase involved in the etiology of several human cancers. Cetuximab is an EGFR-blocking antibody that has been approved for the treatment of patients with head and neck squamous cell carcinoma and metastatic colorectal cancer. Previous reports have shown that EGFR translocation to the nucleus is associated with cell proliferation. Here we investigated mechanisms of acquired resistance to cetuximab using a model derived from the non-small cell lung cancer line H226. We demonstrated that cetuximab-resistant cells overexpress HER family ligands including epidermal growth factor (EGF), amphiregulin, heparin-binding EGF and beta-cellulin. Overexpression of these ligands is associated with the nuclear translocation of the EGFR and this process was mediated by the Src family kinases (SFK). Treatment of cetuximab-resistant cells with the SFK inhibitor, dasatinib, resulted in loss of nuclear EGFR, increased membrane expression of the EGFR and resensitization to cetuximab. In addition, expression of a nuclear localization sequence-tagged EGFR in cetuximab-sensitive cells increased resistance to cetuximab both in vitro and in mouse xenografts. Collectively, these data suggest that nuclear expression of EGFR may be an important molecular determinant of resistance to cetuximab therapy and provides a rationale for investigating nuclear EGFR as a biomarker for cetuximab response. Further, these data suggest a rationale for the design of clinical trials that examine the value of treating patients with cetuximab-resistant tumors with inhibitors of SFKs in combination with cetuximab.

We have previously demonstrated the presence of active epidermal growth factor receptor (EGFR) and its involvement in sperm capacitation and the acrosome reaction; however, the mechanism of EGFR activation was not clear. We show here that the sperm EGFR can be transactivated by angiotensin II or by lysophosphatydic acid, two ligands which activate specific G-protein-coupled receptors (GPCR), or by directly activating protein kinase A using 8Br-cAMP. This transactivation occurs in noncapacitated sperm and is mediated by PKA, SRC and a metalloproteinase. We also show that the EGFR is activated in sperm incubated under in vitro capacitation conditions, without any added ligand, but not in bicarbonate-deficient medium or when PKA is blocked. Despite the fact that EGFR is activated in capacitated sperm, this state is not sufficient to induce the acrosome reaction. We conclude that the EGFR is stimulated during capacitation via PKA activation, while further activation of the EGFR in capacitated sperm is required in order to induce the acrosome reaction. The acrosome reaction can be induced by GPCR via the transactivation of the EGFR by a signaling pathway involving PKA, SRC and metalloproteinase and the EGFR down-stream effectors PI3K, PLC and PKC.

Background and Objectives: Outside-in integrin alphaIIbbeta3 signaling involves a series of tyrosine kinase reactions that culminate in platelet spreading on fibrinogen. The aim of this study was to identify novel tyrosine phosphorylated signaling proteins downstream of alphaIIbbeta3, and explore their role in platelet signaling., Methods and Results: Utilizing proteomics to search for novel platelet proteins that contribute to outside-in signaling by the integrin alphaIIbbeta3, we identified 27 proteins, 17 of which were not previously shown to be part of a tyrosine phosphorylation-based signaling complex downstream of alphaIIbbeta3. The proteins identified include the novel immunoreceptors G6f and G6b-B, and two members of the Dok family of adapters, Dok-1 and Dok-3, which underwent increased tyrosine phosphorylation following platelet spreading on fibrinogen. Dok-3 was also inducibly phosphorylated in response to the GPVI-specific agonist collagen-related peptide (CRP) and the PAR-1 and -4 agonist thrombin, independently of the integrin alphaIIbbeta3. Tyrosine phosphorylation of Dok-1 and Dok-3 was primarily Src kinase-independent downstream of the integrin, whereas it was Src kinase-dependent downstream of GPVI. Moreover, both proteins inducibly interacted with Grb-2 and SHIP-1 in fibrinogen-spread platelets., Conclusions: This study provides new insights into the molecular mechanism regulating alphaIIbbeta3-mediated platelet spreading on fibrinogen. The novel platelet adapter Dok-3 and the structurally related Dok-1 are tyrosine phosphorylated in an Src kinase-independent manner downstream of alphaIIbbeta3 in human platelets, leading to an interaction with Grb2 and SHIP-1.

The intracellular signalling cascade(s) mediating epidermal growth factor (EGF)-induced cyclooxygenase-2 (COX-2) expression is poorly defined in oral carcinomas. Investigation of two different oral squamous cell carcinoma (OSCC) cell lines with high EGF-induced COX-2 expression revealed, however, that this expression was dependent on two mitogen-activated protein kinase (MAPK) pathways [extracellular signal-regulated kinase 1/2 (ERK1/2) and p38] because combined inhibition of these pathways was needed to abolish EGF-induced COX-2 expression. Surprisingly, inhibition of phosphoinositide-3 kinase (PI3K) increased EGF-induced COX-2 expression in the basaloid OSCC cell line (C12), suggesting a PI3K-controlled, inhibitory COX-2-regulating pathway. Neither the transcription factor nuclear factor-kappaB (NF-kappaB), nor Src, was involved in EGF-induced COX-2 expression. The results suggest that EGF-induced COX-2 expression is regulated by several pathways, and emphasizes that individual tumors use different strategies for intracellular signalling.

Protein kinase C-delta (PKC-delta) is expressed in platelets and activated downstream of protease-activated receptors (PARs) and glycoprotein VI (GPVI) receptors. We have previously shown that PKC-delta positively regulates PAR-mediated dense granule secretion, whereas it negatively regulates GPVI-mediated dense granule secretion. We further investigated the mechanism of such differential regulation of dense granule release by PKC-delta in platelets. SH2 domain-containing inositol phosphatase-1 (SHIP-1) is phosphorylated on Y1020, a marker for its activation, upon stimulation of human platelets with PAR agonists SFLLRN and AYPGKF or GPVI agonist convulxin. GPVI-mediated SHIP-1 phosphorylation occurred rapidly at 15 seconds, whereas PAR-mediated phosphorylation was delayed, occurring at 1 minute. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKC-delta on stimulation of platelets with a GPVI agonist, but not with a PAR agonist. In PKC-delta-null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited. Furthermore, in Lyn null murine platelets, GPVI-mediated phosphorylations on Y-1020 of SHIP-1 and Y311 of PKC-delta were inhibited. In murine platelets lacking Lyn or SHIP-1, GPVI-mediated dense granule secretions are potentiated, whereas PAR-mediated dense granule secretions are inhibited. Therefore, we conclude that Lyn-mediated phosphorylations of PKC-delta and SHIP-1 and their associations negatively regulate GPVI-mediated dense granule secretion in platelets.

The nonreceptor isoform of tyrosine phosphatase epsilon (cyt-PTPe) supports osteoclast adhesion and activity in vivo, leading to increased bone mass in female mice lacking PTPe (EKO mice). The structure and organization of the podosomal adhesion structures of EKO osteoclasts are abnormal; the molecular mechanism behind this is unknown. We show here that EKO podosomes are disorganized, unusually stable, and reorganize poorly in response to physical contact. Phosphorylation and activities of Src, Pyk2, and Rac are decreased and Rho activity is increased in EKO osteoclasts, suggesting that integrin signaling is defective in these cells. Integrin activation regulates cyt-PTPe by inducing Src-dependent phosphorylation of cyt-PTPe at Y638. This phosphorylation event is crucial because wild-type-but not Y638F-cyt-PTPe binds and further activates Src and restores normal stability to podosomes in EKO osteoclasts. Increasing Src activity or inhibiting Rho or its downstream effector Rho kinase in EKO osteoclasts rescues their podosomal stability phenotype, indicating that cyt-PTPe affects podosome stability by functioning upstream of these molecules. We conclude that cyt-PTPe participates in a feedback loop that ensures proper Src activation downstream of integrins, thus linking integrin signaling with Src activation and accurate organization and stability of podosomes in osteoclasts.

Background and Purpose: The present study was carried out to examine the role of protein kinases in the development of acute tolerance to the effects of ethanol on spinal N-methyl-D-aspartate (NMDA) receptor-mediated pressor responses during prolonged ethanol exposure., Experimental Approach: Blood pressure responses induced by intrathecal injection of NMDA were recorded. The levels of several phosphorylated residues on NMDA receptor NR1 (GluN1) (NR1) and NMDA receptor NR2B (GluN2B) (NR2B) subunits were determined by immunohistochemistry and Western blot analysis., Key Results: Ethanol inhibited spinal NMDA-induced pressor responses at 10 min, but the inhibition was significantly reduced at 40 min following continuous infusion. This effect was dose-dependently blocked by chelerythrine [a protein kinase C (PKC) inhibitor, 1-1000 pmol] or PP2 (a Src family tyrosine kinase inhibitor, 1-100 pmol) administered intrathecally 10 min following ethanol infusion. A significant increase in the immunoreactivity of phosphoserine 896 of NR1 subunits (pNR1-Ser896) and phosphotyrosine 1336 of NR2B subunits (pNR2B-Tyr1336) was found in neurons of intermediolateral cell column during the development of tolerance. Levels of pNR1-Ser896 and pNR2B-Tyr1336 were also significantly increased in lateral horn regions of the spinal cord slices incubated with ethanol for 40 min in vitro. The increases in pNR1-Ser896 and pNR2B-Tyr1336 levels were inhibited by post-treatment with chelerythrine and PP2, respectively, both in the in vivo and in vitro studies., Conclusions and Implications: The results suggest that activation of PKC and Src tyrosine kinase during prolonged ethanol exposure leading to increases in the levels of pNR1-Ser896 and pNR2B-Tyr1336 may contribute to acute tolerance to inhibition by ethanol of NMDA receptor function.

HMGB1 is a nuclear protein that signals tissue damage, as it is released by cells dying traumatically or secreted by activated innate immunity cells. Extracellular HMGB1 elicits the migration to the site of tissue damage of several cell types, including inflammatory cells and stem cells. The identity of the signaling pathways activated by extracellular HMGB1 is not known completely: We reported previously that ERK and NF-kappaB pathways are involved, and we report here that Src is also activated. The ablation of Src or inhibition with the kinase inhibitor PP2 blocks migration toward HMGB1. Src associates to and mediates the phosphorylation of FAK and the formation of focal adhesions.

Background & Aims: Protein tyrosine kinase 6 (PTK6) is expressed in epithelial linings of the gastrointestinal tract. PTK6 sensitizes the nontransformed Rat1a fibroblast cell line to apoptotic stimuli. The aim of this study was to determine if PTK6 regulates apoptosis in vivo after DNA damage in the small intestine., Methods: Wild-type and Ptk6(-/-) mice were subjected to gamma-irradiation; intestinal tissues were collected, protein was isolated, and samples were fixed for immunohistochemical analyses at 0, 6, and 72 hours after the mice were irradiated. Expression of PTK6 was examined in the small intestine before and after irradiation. Apoptosis and proliferation were compared between wild-type and Ptk6(-/-) mice. Expression and activation of prosurvival signaling proteins were assessed., Results: Irradiation induced PTK6 in crypt epithelial cells of the small intestine in wild-type mice. Induction of PTK6 corresponded with DNA damage-induced apoptosis in the wild-type small intestine. Following irradiation, the apoptotic response was impaired in the intestinal crypts of Ptk6(-/-) mice. Increased activation of AKT and extracellular signal-regulated kinase (ERK)1/2 and increased inhibitory phosphorylation of the proapoptotic protein BAD were detected in Ptk6(-/-) mice after irradiation. In response to the induction of apoptosis, compensatory proliferation increased in the small intestines of wild-type mice but not in Ptk6(-/-) mice at 6 hours after irradiation., Conclusions: PTK6 is a stress-induced kinase that promotes apoptosis by inhibiting prosurvival signaling. After DNA damage, induction of PTK6 is required for efficient apoptosis and inhibition of AKT and ERK1/2.

Once cleaved by caspases, the Lyn tyrosine kinase (LynDeltaN) is relocalized from the plasma membrane to the cytoplasm of apoptotic cells, but the function of such a cleavage is incompletely understood. We evaluated the effect of LynDeltaN overexpression on imatinib sensitivity of the chronic myelogenous leukemia (CML) cell line K562. Therefore, we generated stable cells that express plasmids encoding LynDeltaN or its catalytically inactive counterpart LynDeltaNKD. We established that Lyn is cleaved in imatinib-treated parental K562 cells in a caspase-dependent manner. Lyn cleavage also occurred following BCR-ABL silencing by specific short hairpin RNA (sh-RNA). Imatinib-induced apoptosis was abrogated in LynDeltaN-overexpressing cells, but not in cells overexpressing its inactive counterpart. Conversely, the overexpression of LynDeltaN failed to affect the differentiation of K562 cells. Importantly, the protective effect of LynDeltaN was suppressed by two inhibitors of Lyn activity. LynDeltaN also inhibits imatinib-mediated caspase-3 activation in the small proportion of nilotinib-resistant K562 cells overexpressing Lyn that can engage an apoptotic program upon imatinib stimulation. Finally, Lyn knockdown by sh-RNA altered neither imatinib-mediated apoptosis nor differentiation. Taken together, our data show that the caspase-cleaved form of Lyn exerts a negative feedback on imatinib-mediated CML cell apoptosis that is entirely dependent on its kinase activity and likely on the BCR-ABL pathway.

Antiviral immune responses are initiated through Toll-like receptors (TLRs) and RIG-I (retinoic acid-inducible gene-I)-like RNA helicases that recognize nucleic acids from distinct viruses. In this study, we show that the tyrosine kinase c-Src participates in antiviral responses induced by the cytoplasmic RNA helicase RIG-I. Sendai virus (SV), which is recognized by RIG-I, induced c-Src phosphorylation. Functional impairment of c-Src through chemical inhibition or transient expression of a c-Src kinase-inactive mutant attenuated production of endogenous antiviral proteins after SV infection or after expression of RIG-I or its adapter protein MAVS. Importantly, SV-stimulated synthesis of antiviral proteins was significantly impaired in cells treated with c-Src small interfering RNA and in cells from c-Src-deficient mice. In addition, we found that c-Src interacted with components of the RIG-I pathway: RIG-I, MAVS, and TRAF3 (tumor necrosis factor receptor-associated factor-3). The interaction between c-Src and TRAF3 was found to occur within the RING domain of TRAF3. Taken together, our results suggest that c-Src enhances RIG-I-mediated signaling, acting at the level of TRAF3.

Mast cells and T cells produce reactive oxygen species (ROS) after stimulation with the high-affinity IgE receptor (Fc epsilon RI) and T cell receptor. A growing body of evidence suggests the existence of ROS-regulated intracellular and/or plasma membrane Ca(2+) channels in these cells but their molecular entities remain to be identified. Here, we report that store-operated Ca(2+) channel (SOC) activity is regulated by superoxide (O(2)(*-)) and hydrogen peroxide (H(2)O(2)) in mast cells. MnTBaP (Mn(III)tetrakis(4-benzoic acid)porphyrin) and ebselen (2-phenyl-1,2-benziso-selenazol-3(2H)-one) selectively blocked the generation of O(2)(*-) and H(2)O(2), respectively, in antigen-stimulated cells. The H(2)O(2) generation was dependent on the Src family kinase (SFK) and phosphatidylinositol-3-kinase (PI3K) activities but independent of extracellular Ca(2+), and the Fc epsilon RI beta-chain immunoreceptor tyrosine-based activation motif played an essential role. On the other hand, O(2)(*-) generation was strictly dependent on extracellular Ca(2+), but negatively regulated by the SFK and PI3K activities. Inhibition of O(2)(*-) generation resulted in increased H(2)O(2) generation and reduced SOC activity, although it had a minimal effect on endoplasmic reticulum Ca(2+) store depletion. On the contrary, inhibition of H(2)O(2) generation resulted in increased intracellular O(2)(*-) generation and augmented SOC activity. The findings suggest that O(2)(*-) and H(2)O(2), which are generated by separate signaling pathways/sources, reciprocally regulate SOC activity in mast cells. Such generations of multiple oxidant species and their distinct roles in the regulation of SOC activity may facilitate the fine tuning of Ca(2+) signaling in mast cells.

In the last few years dysregulated expression of the cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) has been associated with several cancers and this cell surface molecule has been recognized both as a tumor marker and as a potential target to disrupt progression of cancer. Here we summarize what is known about CDCP1 including its structural features, expression in normal and cancerous tissues, and the in vitro experiments and studies in animal models that have provided the key insights into its potential role in tumor formation and metastasis in humans. We conclude by highlighting opportunities and challenges in targeting CDCP1 in cancer.

Integrins are important mediators of cell adhesion and migration, which in turn are essential for diverse biological functions, including wound healing and cancer metastasis. The integrin alpha9beta1 is expressed on numerous mammalian tissues and can mediate accelerated cell migration. As the molecular signaling mechanisms that transduce this effect are poorly defined, we investigated the pathways by which activated integrin alpha9beta1 signals migration. We found for the first time that specific ligation of integrin alpha9beta1 rapidly activates Src tyrosine kinase, with concomitant tyrosine phosphorylation of p130Cas and activation of Rac-1. Furthermore, activation of integrin alpha9beta1 also enhanced NO production through activation of inducible nitric oxide synthase (iNOS). Inhibition of Src tyrosine kinase or NOS decreased integrin-alpha9beta1-dependent cell migration. Src appeared to function most proximal in the signaling cascade, in a FAK-independent manner to facilitate iNOS activation and NO-dependent cell migration. The cytoplasmic domain of integrin alpha9 was crucial for integrin-alpha9beta1-induced Src activation, subsequent signaling events and cell migration. When taken together, our results describe a novel and unique mechanism of coordinated interactions of the integrin alpha9 cytoplasmic domain, Src tyrosine kinase and iNOS to transduce integrin-alpha9beta1-mediated cell migration.

Nerve growth factor (NGF)-mediated activation of mitogen-activated protein kinases (MAPK) is critical for differentiation and apoptosis of PC12 cells. Since NGF employs stress-activated c-Jun N-terminal kinase (JNK) to regulate both programmed cell death and neurite outgrowth of PC12 cells, we examined NGF-regulated JNK activity and the role of G(i/o) proteins. Induction of JNK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). To discern the participation of various signaling intermediates, PC12 cells were treated with specific inhibitors prior to NGF challenge. NGF-elevated JNK activity was abolished by inhibitors of JNK, p38 MAPK, Src, JAK3 and MEK1/2. NGF-dependent JNK phosphorylation became insensitive to PTX treatment upon transient expressions of Galpha(z) or the PTX-resistant mutants of Galpha(i1-3) and Galpha(oA). Collectively, these studies indicate that NGF-dependent JNK activity may be mediated via G(i1-3) proteins, JAK3, Src, p38 MAPK and the MEK/ERK cascade.

Sonic hedgehog (Shh) plays essential roles in developmental events such as cell fate specification and axon guidance. Shh induces cell fate specification through canonical Shh signaling, mediated by transcription. However, the mechanism by which Shh guides axons is unknown. To study this, we developed an in vitro assay for axon guidance, in which neurons can be imaged while responding to a defined gradient of a chemical cue. Axons of dissociated commissural neurons placed in a Shh gradient turned rapidly toward increasing concentrations of Shh. Consistent with this rapid response, we showed that attraction by Shh does not require transcription. Instead, Shh stimulates the activity of Src family kinase (SFK) members in a Smoothened-dependent manner. Moreover, SFK activity is required for Shh-mediated guidance of commissural axons, but not for induction of Gli transcriptional reporter activity. Together, these results indicate that Shh acts via a rapidly acting, noncanonical signaling pathway to guide axons.

This review provides an overview of the molecular mechanisms of K transport in the mammalian connecting tubule (CNT) and cortical collecting duct (CCD), both nephron segments responsible for the regulation of renal K secretion. Aldosterone and dietary K intake are two of the most important factors regulating K secretion in the CNT and CCD. Recently, angiotensin II (AngII) has also been shown to play a role in the regulation of K secretion. In addition, genetic and molecular biological approaches have further identified new mechanisms by which aldosterone and dietary K intake regulate K transport. Thus, the interaction between serum-glucocorticoid-induced kinase 1 (SGK1) and with-no-lysine kinase 4 (WNK4) plays a significant role in mediating the effect of aldosterone on ROMK (Kir1.1), an important apical K channel modulating K secretion. Recent evidence suggests that WNK1, mitogen-activated protein kinases such as P38, ERK, and Src family protein tyrosine kinase are involved in mediating the effect of low K intake on apical K secretory channels.

To better understand whether autoimmunity in Lyn-deficient mice arises from compromised central or peripheral B cell tolerance, we examined BCR signaling properties of wild-type and Lyn-deficient B cells at different stages of development. Wild-type mature follicular B cells were less sensitive to BCR stimulation than were immature transitional stage 1 B cells with regard to BCR-induced calcium elevation and ERK MAPK activation. In the absence of Lyn, mature B cell signaling was greatly enhanced, whereas immature B cell signaling was minimally affected. Correspondingly, Lyn deficiency substantially enhanced the sensitivity of mature B cells to activation via the BCR, but minimally affected events associated with tolerance induction at the immature stage. The effects of CD22 deficiency on BCR signaling were very similar in B cells at different stages of maturation. These results indicate that the Lyn-CD22-Src homology region 2 domain-containing phosphatase-1 inhibitory pathway largely becomes operational as B cell mature, and sets a threshold for activation that appears to be critical for the maintenance of tolerance in the B cell compartment.

Substance P is known to play a key role in the pathogenesis of acute pancreatitis. Src family kinases (SFKs) are known to be involved in cytokine signaling. However, the involvement of SFKs in substance P-induced chemokine production and its role in acute pancreatitis have not been investigated yet. To that end, we have used primary preparations of mouse pancreatic acinar cells as our model to show that substance P/neurokinin 1 receptor (NK1R) induced activation of SFKs. SFKs mediated the activation of mitogen-activated protein kinases [extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK)], transcription factors [signal transducer and activator of transcription (STAT) 3, nuclear factor (NF) kappaB, activator protein-1 (AP-1)], and production of chemokines in pancreatic acinar cells. We further tested the significance of the SFK signaling pathway in acute pancreatitis. Our results show, for the first time, that treatment of mice with the potent and selective SFK inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-D] pyrimidine], but not its negative inhibitor PP3 (4-amino-7-phenylpyrazol [3,4-D] pyrimidine), reduced the severity of pancreatitis. This was proven by significant attenuation of hyperamylasemia, pancreatic myeloperoxidase activity, chemokines, and water content. Histological evidence of diminished pancreatic injury also confirmed the protective effect of the inhibition of SFKs. Moreover, treatment with the substance P receptor antagonist CP96345 [(2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)-methyl)-1-azabicyclo(2.2.2.)-octan-3-amine] attenuated acute pancreatitis-induced activation of SFKs, ERK, JNK, STAT3, NFkappaB, and AP-1. The proposed signaling pathway through which substance P mediates acute pancreatitis is through substance P/NK1R-SFKs-(ERK, JNK)-(STAT3, NFkappaB, AP-1) chemokines. In light of our study, we propose that drugs targeting the substance P-mediated signaling pathways could prove beneficial in improving treatment efficacy in acute pancreatitis.

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.

Non-small cell lung cancer (NSCLC)-associated epidermal growth factor receptor (EGFR) mutants are constitutively active and induce ligand-independent transformation in non-malignant cell lines. We investigated the possibility that the ability of mutant EGFRs to transform cells reflects a constitutive cooperativity with Src using a system in which the overexpression of mutant, but not wild-type, EGFR induced anchorage-independent cell growth. Src was constitutively activated and showed enhanced interaction with mutant EGFRs, suggesting that constitutive EGFR-Src cooperativity may contribute to mutant EGFR-mediated oncogenesis. Indeed, the mutant EGFR-mediated cell transformation was inhibited by Src- as well as EGFR-directed inhibitors. Importantly, a tyrosine to phenylalanine mutation of the major Src phosphorylation site on EGFR, Y845, reduced the constitutive phosphorylation of NSCLC-EGFR mutants, as well as that of STAT3, Akt, Erk and Src, and reduced the mutant EGFR-Src association as well as proliferation, migration and anchorage-independent growth. Reduced anchorage-independent growth and migration were also observed when dominant-negative-Src was expressed in mutant EGFR-expressing cells. Overall, our findings show that mutant EGFR-Src interaction and cooperativity play critical roles in constitutive engagement of the downstream signaling pathways that allow NSCLC-associated EGFR mutants to mediate oncogenesis, and support the rationale to target Src-dependent signaling pathways in mutant EGFR-mediated malignancies.

Expression of the PTEN tumor suppressor is frequently lost in breast cancer in the absence of mutation or promoter methylation through as yet undetermined mechanisms. In this study, we demonstrate that the Rak tyrosine kinase physically interacts with PTEN and phosphorylates PTEN on Tyr336. Knockdown of Rak enhanced the binding of PTEN to its E3 ligase NEDD4-1 and promoted PTEN polyubiquitination, leading to PTEN protein degradation. Notably, ectopic expression of Rak effectively suppressed breast cancer cell proliferation, invasion, and colony formation in vitro and tumor growth in vivo. Furthermore, Rak knockdown was sufficient to transform normal mammary epithelial cells. Therefore, Rak acts as a bona fide tumor suppressor gene through the mechanism of regulating PTEN protein stability and function.

This review addresses the localized regulation of voltage-gated ion channels by phosphorylation. Comprehensive data on channel regulation by associated protein kinases, phosphatases, and related regulatory proteins are mainly available for voltage-gated Ca2+ channels, which form the main focus of this review. Other voltage-gated ion channels and especially Kv7.1-3 (KCNQ1-3), the large- and small-conductance Ca2+-activated K+ channels BK and SK2, and the inward-rectifying K+ channels Kir3 have also been studied to quite some extent and will be included. Regulation of the L-type Ca2+ channel Cav1.2 by PKA has been studied most thoroughly as it underlies the cardiac fight-or-flight response. A prototypical Cav1.2 signaling complex containing the beta2 adrenergic receptor, the heterotrimeric G protein Gs, adenylyl cyclase, and PKA has been identified that supports highly localized via cAMP. The type 2 ryanodine receptor as well as AMPA- and NMDA-type glutamate receptors are in close proximity to Cav1.2 in cardiomyocytes and neurons, respectively, yet independently anchor PKA, CaMKII, and the serine/threonine phosphatases PP1, PP2A, and PP2B, as is discussed in detail. Descriptions of the structural and functional aspects of the interactions of PKA, PKC, CaMKII, Src, and various phosphatases with Cav1.2 will include comparisons with analogous interactions with other channels such as the ryanodine receptor or ionotropic glutamate receptors. Regulation of Na+ and K+ channel phosphorylation complexes will be discussed in separate papers. This review is thus intended for readers interested in ion channel regulation or in localization of kinases, phosphatases, and their upstream regulators.

Akt is a central regulator of apoptosis, cell growth and survival. Growth factors and some G-protein-coupled receptors (GPCR) regulate Akt. Whereas growth-factor activation of Akt has been extensively studied, the regulation of Akt by GPCR's, especially gastrointestinal hormones/neurotransmitters, remains unclear. To address this area, in this study the effects of GI growth factors and hormones/neurotransmitters were investigated in rat pancreatic acinar cells which are high responsive to these agents. Pancreatic acini expressed Akt and 5 of 7 known pancreatic growth-factors stimulate Akt phosphorylation (T308, S473) and translocation. These effects are mediated by p85 phosphorylation and activation of PI3K. GI hormones increasing intracellular cAMP had similar effects. However, GI-hormones/neurotransmitters [CCK, bombesin, carbachol] activating phospholipase C (PLC) inhibited basal and growth-factor-stimulated Akt activation. Detailed studies with CCK, which has both physiological and pathophysiological effects on pancreatic acinar cells at different concentrations, demonstrated CCK has a biphasic effect: at low concentrations (pM) stimulating Akt by a Src-dependent mechanism and at higher concentrations (nM) inhibited basal and stimulated Akt translocation, phosphorylation and activation, by de-phosphorylating p85 resulting in decreasing PI3K activity. This effect required activation of both limbs of the PLC-pathway and a protein tyrosine phosphatase, but was not mediated by p44/42 MAPK, Src or activation of a serine phosphatase. Akt inhibition by CCK was also found in vivo and in Panc-1 cancer cells where it inhibited serum-mediated rescue from apoptosis. These results demonstrate that GI growth factors as well as gastrointestinal hormones/neurotransmitters with different cellular basis of action can all regulate Akt phosphorylation in pancreatic acinar cells. This regulation is complex with phospholipase C agents such as CCK, because both stimulatory and inhibitory effects can be seen, which are mediated by different mechanisms.

Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG) also known as Csk-binding protein was first fully characterized in 2000. It was initially recognized as a ubiquitously expressed adaptor protein recruiting cytoplasmic C-terminal Src-kinase to the close proximity of plasma membrane-anchored Src-kinases thereby allowing Csk to impose its inhibitory potential on these kinases. A role of PAG was initially seen in negative regulation of immune reactions. Since the year 2000 other Csk-dependent and independent interactions have been discovered and some of them showed anti-oncogenic effects in experiment. According to current opinions, these findings place PAG in a position of tumour suppressor candidate.

Reverse signaling via glycosylphosphatidylinositol (GPI)-linked Ephrins may help control cell proliferation and outgrowth within the nervous system, but the mechanisms underlying this process remain poorly understood. In the embryonic enteric nervous system (ENS) of the moth Manduca sexta, migratory neurons forming the enteric plexus (EP cells) express a single Ephrin ligand (GPI-linked MsEphrin), whereas adjacent midline cells that are inhibitory to migration express the cognate receptor (MsEph). Knocking down MsEph receptor expression in cultured embryos with antisense morpholino oligonucleotides allowed the EP cells to cross the midline inappropriately, consistent with the model that reverse signaling via MsEphrin mediates a repulsive response in the ENS. Src family kinases have been implicated in reverse signaling by type-A Ephrins in other contexts, and MsEphrin colocalizes with activated forms of endogenous Src in the leading processes of the EP cells. Pharmacological inhibition of Src within the developing ENS induced aberrant midline crossovers, similar to the effect of blocking MsEphrin reverse signaling. Hyperstimulating MsEphrin reverse signaling with MsEph-Fc fusion proteins induced the rapid activation of endogenous Src specifically within the EP cells, as assayed by Western blots of single embryonic gut explants and by whole-mount immunostaining of cultured embryos. In longer cultures, treatment with MsEph-Fc caused a global inhibition of EP cell migration and outgrowth, an effect that was prevented by inhibiting Src activation. These results support the model that MsEphrin reverse signaling induces the Src-dependent retraction of EP cell processes away from the enteric midline, thereby helping to confine the neurons to their appropriate pathways.