Phosphorylation Of Src Family Kinases Research Articles

Plasmodium falciparum ‐induced CD36 clustering rapidly strengthens cytoadherence via p130CAS‐mediated actin cytoskeletal rearrangement

The adhesion of infected red blood cells (IRBCs) to microvascular endothelium is critical in the pathogenesis of severe malaria. Here we used atomic force and confocal microscopy to examine the adhesive forces between IRBCs and human dermal microvascular endothelial cells. Initial contact of the cells generated a mean ± sd adhesion force of 167 ± 208 pN from the formation of single or multiple bonds with CD36. The strength of adhesion increased by 5- to 6-fold within minutes of contact through a signaling pathway initiated by CD36 ligation by live IRBCs, or polystyrene beads coated with anti-CD36 or PpMC-179, a recombinant peptide representing the minimal binding domain of the parasite ligand PfEMP1 to CD36. Engagement of CD36 led to localized phosphorylation of Src family kinases and the adaptor protein p130CAS, resulting in actin recruitment and CD36 clustering by 50-60% of adherent beads. Uninfected red blood cells or IgG-coated beads had no effect. Inhibition of the increase in adhesive strength by the Src family kinase inhibitor PP1 or gene silencing of p130CAS decreased adhesion by 39 ± 12 and 48 ± 20%, respectively, at 10 dyn/cm(2) in a flow chamber assay. Modulation of adhesive strength at PfEMP1-CD36-actin cytoskeleton synapses could be a novel target for antiadhesive therapy.

Src Family Kinases Contribute to GI and Gq-Mediated Platelet Activation

Abstract 5263The Src family kinases (SFKs) play an essential role in collagen- and von Willebrand factor (vWF)-mediated platelet activation. However, the role of SFKs in G protein-coupled receptor (GPCR)-mediated platelet activation is not fully understood, and little is known about the molecular mechanisms by which SFKs are activated by GPCR stimulation. Here we demonstrate that SFKs are activated by the Gq and Gi pathways, respectively and SFKs play important roles in Gq- and Gi-dependent secretion and activation. ADP induced SFK phosphorylation in wild type and Gq−/− platelets, and ADP-induced SFK phosphorylation was inhibited by the P2Y12 antagonist AR-C69931MX or P2Y12 knockout but was not affected by the P2Y1 antagonist MRS-2179. Lyn and Fyn were co-immunoprecipitated with Gi2 in platelets, and ADP-induced SFK phosphorylation was diminished in Lyn−/− platelets. These results demonstrate that ADP induces SFK activation mainly depending on the Gi pathway activated via its receptor P2Y12. Furthermore, epinephrine also dose-dependently induced SFK phosphorylation in mouse platelets. A selective inhibitor of Src family kinase PP2 inhibited ADP-induced Akt phosphorylation, fibrinogen binding, and platelet aggregation. Thus, activation of Gi is sufficient to induce SFK activation, which plays an important role in Gi-dependent platelet activation. We further show that the thrombin receptor PAR4 peptide AYPGKF elicited SFK phosphorylation in P2Y12−/−, but not in Gq−/− platelets, and AYPGKF-induced SFK phosphorylation was inhibited by the calcium chelator dimethyl-BAPTA, suggesting that Gq-dependent SFK phosphorylation is downstream from the Ca2+ signaling. The calcium ionophore, A23187-induced TXA2 synthesis, platelet aggregation and secretion were inhibited by pre-incubation of platelets with PP2. PAR4-induced TXA2 synthesis was also abolished by PP2. Moreover, PAR4-mediated granule secretion, integrin aIIbb3 activation, and aggregation of P2Y12 deficient platelets were partially inhibited by PP2 or a PKC inhibitor Ro-31-8220, but were completely abolished by Ro-31-8220 plus PP2 or dimethyl-BAPTA, suggesting that Ca2+/SFKs and PKC represent two parallel signaling pathways mediating Gq-dependent platelet activation. In summary, SFKs can be activated by Gq/Ca2+-dependent mechanisms and by Gi-dependent mechanisms, and SFKS play important roles in Gq- and Gi-dependent platelet activation. Disclosures:No relevant conflicts of interest to declare.

Over-Expression of the Mas Receptor Decreases Arterial Thrombosis Risk in B2R KO Mice by Elevating NO and Prostacyclin and Reducing GPVI Activation

Abstract 700 Background:Bradykinin B2 receptor KO mice (B2R KO) were recognized to have delayed rose bengal and ferric chloride carotid artery thrombosis times and long tail bleeding times. In B2R KO, elevated serum angiotensin II (AngII) binds to an over-expressed angiotensin receptor 2 (AT2R) to increase plasma NO and prostacyclin (Blood 108:192, 2006). It was proposed that the combined presence of elevated AngII and AT2R are essential for the thrombosis protection phenotype. However, after losartan treatment, an angiotensin receptor 1 antagonist, thrombosis remains delayed in B2R KO despite reduction in AngII levels from 258±64 to 40±15 pg/ml. An additional pathway for thrombosis protection in B2R KO mice was sought. Methods and Results:Losartan treatment lowers ACE mRNA in mice along with lowering AngII levels. However, even in the presence of low AngII levels, plasma Ang1-7 levels, the breakdown product of AngII, in losartan-treated B2R KO are increased [21±2.2 pg/ml vs 14.4±0.7 pg/ml in wild type mice (WT)]. Upon examination, B2R KO also have elevated Mas mRNA, the receptor for angiotensin1-7, with over-expressed renal Mas protein. Treatment of B2R KO with A-779, a Mas antagonist, shortens carotid artery thrombosis time (58±4 to 38±4 min) and tail bleeding time (170±13 to 88±8 sec) and lowers plasma concentrations of nitrate (from 21.5±3.6 micromolar in untreated B2R KO to 15±5 micromolar in A-779-treated mice) and 6-keto-PGF1alpha (259±103 pg/ml in untreated B2R KO to 132±58 pg/ml). ADP- or thrombin-induced platelet (plt) activation are not abnormal in B2R KO, either by aggregation studies or by flow cytometric studies examining for fibrinogen binding or JON/A integrin epitope binding and P-Selectin expression, respectively. Although static and flow adherence to collagen was normal, B2R KO plts have defective spreading on collagen (20±0.6 microns in B2R KO versus 27±0.4 microns in WT, p<0.0001) under static conditions. Resting B2R KO plts constitutively also have increased DAF-FM fluorescence (649±41 AFU versus 405±36 AFU, p=0.0012), suggesting increased plt NO without stimulation. GSNO, an NO donor, or carbaprostacyclin, a stable derivative of prostacyclin, inhibits murine plt spreading on collagen in a concentration-dependent manner. B2R KO plts have reduced GPVI activation using CRP or convulxin even though GPVI expression on B2R KO and WT plts are similar. Further, B2R KO plts have reduced Syk phosphorylation after convulxin stimulation and reduced phosphorylation of Src family kinases when spreading on collagen. Treatment of B2R KO plts in vivo with A-779 corrects the spreading and Src family kinase phosphorylation defects upon collagen exposure, suggesting an in vivo role of the Mas receptor in the plt function defect and thrombosis delay. Conclusion:B2R KO are protected from thrombosis by over-expression of two receptors (AT2R and Mas) from the renin-angiotensin system resulting in increased NO and prostacyclin and an acquired platelet function defect. The receptors AT2R and MAS compensate for the absence of the B2R. These studies show novel mechanism(s) whereby receptors in the renin-angiotensin system influence platelet function and arterial thrombosis risk in vivo. Disclosures:No relevant conflicts of interest to declare.

Defining the Substrate Specificity Determinants Recognized by the Active Site of C-Terminal Src Kinase-Homologous Kinase (CHK) and Identification of β-Synuclein as a Potential CHK Physiological Substrate

C-Terminal Src kinase-homologous kinase (CHK) exerts its tumor suppressor function by phosphorylating the C-terminal regulatory tyrosine of the Src-family kinases (SFKs). The phosphorylation suppresses their activity and oncogenic action. In addition to phosphorylating SFKs, CHK also performs non-SFK-related functions by phosphorylating other cellular protein substrates. To define these non-SFK-related functions of CHK, we used the "kinase substrate tracking and elucidation" method to search for its potential physiological substrates in rat brain cytosol. Our search revealed β-synuclein as a potential CHK substrate, and Y127 in β-synuclein as the preferential phosphorylation site. Using peptides derived from β-synuclein and positional scanning combinatorial peptide library screening, we defined the optimal substrate phosphorylation sequence recognized by the CHK active site to be E-x-[Φ/E/D]-Y-Φ-x-Φ, where Φ and x represent hydrophobic residues and any residue, respectively. Besides β-synuclein, cellular proteins containing motifs resembling this sequence are potential CHK substrates. Intriguingly, the CHK-optimal substrate phosphorylation sequence bears little resemblance to the C-terminal tail sequence of SFKs, indicating that interactions between the CHK active site and the local determinants near the C-terminal regulatory tyrosine of SFKs play only a minor role in governing specific phosphorylation of SFKs by CHK. Our results imply that recognition of SFKs by CHK is mainly governed by interactions between motifs located distally from the active site of CHK and determinants spatially separate from the C-terminal regulatory tyrosine in SFKs. Thus, besides assisting in the identification of potential CHK physiological substrates, our findings shed new light on how CHK recognizes SFKs and other protein substrates.

The SRC-associated protein CUB Domain-Containing Protein-1 regulates adhesion and motility

Multiple SRC-family kinases (SFKs) are commonly activated in carcinoma and appear to have a role in metastasis through incompletely understood mechanisms. Recent studies have shown that CDCP1 (CUB (complement C1r/C1s, Uegf, Bmp1) Domain-Containing Protein-1) is a transmembrane protein and an SRC substrate potentially involved in metastasis. Here we show that increased SFK and CDCP1 tyrosine phosphorylation is, surprisingly, associated with a decrease in FAK phosphorylation. This appears to be true in human tumors as shown by our correlation analysis of a mass spectrometric data set of affinity-purified phosphotyrosine peptides obtained from normal and cancer lung tissue samples. Induction of tyrosine phosphorylation of CDCP1 in cell culture, including by a mAb that binds to its extracellular domain, promoted changes in SFK and FAK tyrosine phosphorylation, as well as in PKC(TM), a protein known to associate with CDCP1, and these changes are accompanied by increases in adhesion and motility. Thus, signaling events that accompany the CDCP1 tyrosine phosphorylation observed in cell lines and human lung tumors may explain how the CDCP1/SFK complex regulates motility and adhesion.

Identification of signaling pathways involved in the mechanism of action of the immunomodulatory peptide gamma-D-glutamyl-L-tryptophan.

5591 Background: Gamma-D-glutamyl-L-tryptophan (SCV-07) is being studied for attenuation of oral mucositis (OM) in head and neck cancer patients receiving chemoradiation therapy. In a pilot study SCV-07-treated subjects had delayed onset of ulcerative OM (Adkins 2009) which correlated with changes in circulating cytokines (Alterovitz 2010). In animal studies SCV-07 inhibits growth of different xenografted tumor types. SCV-07 acts via immune modulation; initial evaluation of SCV-07 signaling demonstrated inhibition of cytokine induced STAT3 transcriptional activity. In this study we explored further the molecular mechanisms by which SCV-07 interferes with mucositis pathobiology and concurrently exhibits anti-tumor activity. Methods: Tyrosine phosphorylated STAT3 (STAT3 PY) was evaluated by Western blot of Jurkat or CD45 mutant Jurkat cells with or without IL-10 or IFNα stimulation and SCV-07. Global phosphorylation profiles were analyzed using phosphoprotein detection array kits. C57/B6 mice inoculated with B16F0 tumor cells were treated with vehicle or SCV-07 and plasma cytokines analyzed by multiplex assay when tumor inhibition was evident. Results: SCV-07 significantly inhibited IL-10- and IFNα-induced STAT3 PY in Jurkat cells which was blocked by pervanadate treatment, implicating tyrosine phosphatase activity. Phosphatase CD45 was not required since STAT3 PY inhibition was seen in CD45 mutant Jurkat cells. However, SCV-07 induced activating phosphorylation of the phosphatase SHP-2 and altered phosphorylation of Src family kinases and STAT5a/b, known substrates for SHP-2. SCV-07 also decreased IFNα stimulated phosphorylation of SHP-2 associated immunoreceptors Siglec3 (CD33) and SIRPbeta1 (CD172B). SCV-07-treatment of B16 bearing mice slowed tumor growth and significantly decreased the STAT3-induced cytokines MCP-1 and IL-12p40 which are implicated in immune pathology. Conclusions: These data suggest a model where SCV-07, via SHP-2 activation and STAT3 inhibition, could shift macrophage subsets from M2 to M1 and T cells from Th2 to Th1, thereby promoting an immune regulatory environment that inhibits both mucositis and tumor progression.

Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition.

Despite the initial effectiveness of the tyrosine kinase inhibitor lapatinib against HER2 gene-amplified breast cancers, most patients eventually relapse after treatment, implying that tumors acquire mechanisms of drug resistance. To discover these mechanisms, we generated six lapatinib-resistant HER2-overexpressing human breast cancer cell lines. In cells that grew in the presence of lapatinib, HER2 autophosphorylation was undetectable whereas active PI3K-Akt and MAPK were maintained. To identify networks maintaining these signaling pathways, we profiled the tyrosine phosphoproteome of sensitive and resistant cells using an immunoaffinity-enriched mass spectrometry method. We found increased phosphorylation of Src family kinases (SFK) and putative Src substrates in several resistant cell lines. Treatment of these resistant cells with Src kinase inhibitors partially blocked PI3K-Akt signaling and restored lapatinib sensitivity. Further, SFK mRNA expression was upregulated in primary HER2+ tumors treated with lapatinib. Finally, the combination of lapatinib and the Src inhibitor AZD0530 was more effective than lapatinib alone at inhibiting pAkt and growth of established HER2-positive BT-474 xenografts in athymic mice. These data suggest that increased Src kinase activity is a mechanism of lapatinib resistance and support the combination of HER2 antagonists with Src inhibitors early in the treatment of HER2+ breast cancers in order to prevent or overcome resistance to HER2 inhibitors.

Reduced Spinal Microglial Activation and Neuropathic Pain after Nerve Injury in Mice Lacking all Three Nitric Oxide Synthases

BackgroundSeveral studies have investigated the involvement of nitric oxide (NO) in acute and chronic pain using mice lacking a single NO synthase (NOS) gene among the three isoforms: neuronal (nNOS), inducible (iNOS) and endothelial (eNOS). However, the precise role of NOS/NO in pain states remains to be determined owing to the substantial compensatory interactions among the NOS isoforms. Therefore, in this study, we used mice lacking all three NOS genes (n/i/eNOS-/-mice) and investigated the behavioral phenotypes in a series of acute and chronic pain assays.ResultsIn a model of tissue injury-induced pain, evoked by intraplantar injection of formalin, both iNOS-/-and n/i/eNOS-/-mice exhibited attenuations of pain behaviors in the second phase compared with that in wild-type mice. In a model of neuropathic pain, nerve injury-induced behavioral and cellular responses (tactile allodynia, spinal microglial activation and Src-family kinase phosphorylation) were reduced in n/i/eNOS-/-but not iNOS-/-mice. Tactile allodynia after nerve injury was improved by acute pharmacological inhibition of all NOSs and nNOS. Furthermore, in MG-5 cells (a microglial cell-line), interferon-γ enhanced NOSs and Mac-1 mRNA expression, and the Mac-1 mRNA increase was suppressed by L-NAME co-treatment. Conversely, the NO donor, sodium nitroprusside, markedly increased mRNA expression of Mac-1, interleukin-6, toll-like receptor 4 and P2X4 receptor.ConclusionsOur results provide evidence that the NOS/NO pathway contributes to behavioral pain responses evoked by tissue injury and nerve injury. In particular, nNOS may be important for spinal microglial activation and tactile allodynia after nerve injury.

Signaling mechanisms of inhibition of phospholipase D activation by CHS-111 in formyl peptide-stimulated neutrophils

A selective phospholipase D (PLD) inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) inhibited the O 2 − generation and cell migration but not degranulation in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils. A novel benzyl indazole compound 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111), which inhibited O 2 − generation and cell migration, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC 50 3.9 ± 1.2 μM). CHS-111 inhibited the interaction of PLD1 with ADP-ribosylation factor (Arf) 6 and Ras homology (Rho) A, and reduced the membrane recruitment of RhoA in fMLP-stimulated cells but not in GTPγS-stimulated cell-free system. CHS-111 reduced the cellular levels of GTP-bound RhoA, membrane recruitment of Rho-associated protein kinase 1 and the downstream myosin light chain 2 phosphorylation, and attenuated the interaction between phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and Arf6, whereas it only slightly inhibited the guanine nucleotide exchange activity of human Dbs (DH/PH) protein and did not affect the arfaptin binding to Arf6. CHS-111 inhibited the interaction of RhoA with Vav, the membrane association and the phosphorylation of Vav. CHS-111 had no effect on the phosphorylation of Src family kinases (SFK) but attenuated the interaction of Vav with Lck, Hck, Fgr and Lyn. CHS-111 also inhibited the interaction of PLD1 with protein kinase C (PKC) α, βI and βII isoenzymes, and the phosphorylation of PLD1. These results indicate that inhibition of fMLP-stimulated PLD activity by CHS-111 is attributable to the blockade of RhoA activation via the interference with SFK-mediated Vav activation, attenuation of the interaction of Arf6 with PLD1 and PIP5K, and the activation of Ca 2+-dependent PKC in rat neutrophils.

Comparative Analysis of Human Src-Family Kinase Substrate Specificity in Vitro

Src family kinases (SFKs) are the earliest known family of tyrosine kinases and are widely thought to play essential roles in cellular signal transduction. Although numerous functional analyses have been performed, no study has analyzed the specificity of all SFKs on an equal platform. To gain a better understanding of SFK phosphorylation, we designed a high-throughput in vitro kinase assay on the subproteome scale using surface plasmon resonance. We reacted each of the 11 human SFKs with 519 substrate proteins, and significant phosphorylation was detected in 33.6% (1921) of the total 5709 kinase-substrate combinations. A large number of novel phosphorylations were included among them. Many substrates were shown to be phosphorylated by multiple SFKs, which might reflect functional complementarity of SFKs. Clustering analysis of phosphorylation results grouped substrates into 10 categories, while the similarity of SFK catalytic specificity exhibited no significant correlation with that of amino acid sequences. In silico predictions of SRC-specific phosphorylation sites were not consistent with experimental results, implying some unknown SRC recognition modes. In an attempt to find biologically meaningful novel substrates, phosphorylation data were integrated with annotation data. The extensive in vitro data obtained in this study would provide valuable clues for further understanding SFK-mediated signal transduction.

C-Src-mediated activation of Erk1/2 is a reaction of epithelial cells to carbon nanoparticle treatment and may be a target for a molecular preventive strategy

Owing to their specific physico/chemical properties, engineered as well as environmental nanoparticles can induce pathogenic endpoints in humans. Earlier studies demonstrated that pure carbon nanoparticles induce cell signaling events at the level of membrane receptor activation in lung epithelial cells. As a possible link between receptor activation and subsequent MAP-kinase signaling, the involvement of Src family kinases was investigated in cell lines of organs potentially exposed to environmental nanoparticles. Human cells from bronchus, intestine, and skin (keratinocytes) as well as rat lung epithelial cells showed similar time patterns for the activation of mitogen-activated protein kinases Erk1/2 as well as Src family kinases (SFK) when treated with carbon nanoparticles. Moreover, c-Src was identified as an integral part of the signaling mediating the transfer of information from membrane receptors to members of the proliferative signaling cascade in lung epithelial cells. Pretreatment of cells with the compatible solute ectoine, which is known to stabilize macromolecules, reduced the nanoparticle specific phosphorylation of SFK. Together with earlier in vivo and in vitro data, this demonstrates that compatible solutes prevent nanoparticle-induced signaling steps at the level of membrane-coupled signaling.

Involvement of Src family kinase activation in angiotensin II-induced hyperresponsiveness of rat bronchial smooth muscle

Angiotensin II (Ang II) might be an important mediator in pathogenesis of airway hyperresponsiveness (AHR) that is the asthmatic characteristic feature of asthma, although the mechanisms of AHR caused by Ang II are not yet clear. Presently, the RT-PCR analyses revealed that all the Src family kinases (SFKs), such as Fyn, Lck, Lyn, Hck, Src, Yes, Blk, Fgr and Frk, were expressed in the lungs and main bronchi of rats. The phosphorylation (activation) of SFK (Tyr416) was increased in bronchial smooth muscle (BSM) by Ang II. The Ang II-induced SFK phosphorylation was inhibited by pretreatment with SU6656, an SFK inhibitor. The concentration–contraction curves to carbachol (CCh) were shifted to the left in the presence of Ang II. The maximal contraction of CCh was also significantly increased by pretreatment with Ang II. These results indicate that Ang II causes BSM hyperresponsiveness. The Ang II-induced BSM hyperresponsiveness was significantly inhibited by SU6656, although the carbachol (CCh)-induced contraction itself was not changed by SU6656. In conclusion, Ang II induced a BSM hyperresponsiveness through activation of SFK, and might play an important role in pathophysiology of bronchial asthma.

Influence of NKG2D/DNAM-1 interaction with tumor cell ligands on the ability of lenalidomide to enhance ADCC of antibody-coated solid tumor cells.

e21008 Background: Lenalidomide has been shown to enhance the antibody- dependent cellular cytotoxicity (ADCC) of rituximab-treated B cell chronic lymphocytic leukemia (B-CLL) and non-Hodgkin lymphoma (NHL) tumor cells. The combination appears to be especially active in patients so we sought to build on these observations by determining lenalidomide's ability to enhance ADCC of solid tumor cells. Methods: Purified NK cells were used as effector cells and tumor cell lines were used as targets after pre-treatment with cetuximab or trastuzumab. 23 tumor cell lines were used; 11 colorectal (CRC), 3 breast, 3 ovary, 2 lung, 2 head and neck and 2 bone sarcoma. Cytotoxicity was assessed using a standard lactate dehydrogenase release assay and specific lysis calculated. NK cell expression of activating receptors from 30 donors and tumor cell expression of NK cell ligands were assessed by flow cytometry. Results: Lenalidomide increased NK cell-specific lysis of cetuximab-coated CRC cells; HCT- 116, from 19% to 39% (p < 0.01), HT-29, 32% to 50% (p < 0.05) and also of trastuzumab-coated breast cancer cells; SKBR-3, 32% to 44% (p < 0.01), MCF-7, 30% to 70% (p < 0.01). Variable sensitivity of CRC cell lines was dependent on EGFR expression (range 5%-96%) but independent of either KRAS or BRAF mutational status or FCγRIIIa genotype. CRC cell line expression of NK cell ligands, PVR, MIC-A, ULBP2 and ULBP3 correlated with sensitivity to ADCC. NK cell expression of DNAM-1, NKG2D, but not NKp46, correlated with the extent of ADCC and its enhancement by lenalidomide. Antibody blocking of NKG2D, and to a lesser extent DNAM-1, inhibited CRC cell killing from 70% to 31% and 48% respectively (both p < 0.05), whereas blocking of individual cognate ligands had minimal effect. Lenalidomide enhanced NK cell production of gmcsf, IL-8, MIP-1α/βm RANTES, MCP-1 and IP-10 but reversed phosphorylation of Src family kinases induced by antibody alone. Conclusions: Lenalidomide enhances the ADCC of solid tumor cells, the extent of which is largely dependent on NKG2D/NKG2D ligand interactions, providing a rationale for exploratory clinical studies and assessment of potential biomarkers predictive lenalidomide-mediated ADCC. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Celgene Celgene

Hydrogen sulfide induces ICAM-1 expression and neutrophil adhesion to caerulein-treated pancreatic acinar cells through NF-κB and Src-family kinases pathway

We have earlier shown that mouse pancreatic acinar cells produce hydrogen sulfide (H 2S), which plays a key role in the pathogenesis of acute pancreatitis (AP). H 2S-dependent induction of inflammation is mediated by the activation of transcription factor NF-κB. We now provide evidence that activation of Src family kinases (SFKs) is crucial in signaling H 2S-induced intracellular adhesion molecule (ICAM)-1 expression via NF-κB. Stimulation of acini with H 2S resulted in a time-dependent activation of SFKs. In order to better understand this effect of H 2S, acinar cells were stimulated with caerulein after addition of H 2S donor, NaHS. Inhibition of SFKs impaired H 2S-induced NF-κB activity and ICAM-1 expression in caerulein treated acinar cells. We also observed that H 2S-induced up-regulation of ICAM-1 enhanced the adhesion of neutrophils onto acinar cells. Analysis of NF-κB pathway revealed that the effect of SFKs inhibition correlated with IκBα degradation and NF-κB DNA binding function. Interestingly, H 2S-induced association of SFKs with translocation of NF-κB, and inhibition of SFKs prevented this response, indicating that this interaction may depend on activation of SFKs. These data suggest that H 2S, by activating the phosphorylation of SFKs, may promote the transcriptional activity of NF-κB and eventually lead to an upregulation of ICAM-1 expression.

CD72 Negatively Regulates KIT-Mediated Responses in Human Mast Cells

KIT activation, through binding of its ligand, stem cell factor, is crucial for normal mast cell growth, differentiation, and survival. Furthermore, KIT may also contribute to mast cell homing and cytokine generation. Activating mutations in KIT lead to the dysregulated mast cell growth associated with the myeloproliferative disorder, mastocytosis. We investigated the potential of downregulating such responses through mast cell inhibitory receptor activation. In this study, we report that the B cell-associated ITIM-containing inhibitory receptor, CD72, is expressed in human mast cells. Ligation of CD72 with the agonistic Ab, BU40, or with recombinant human CD100 (rCD100), its natural ligand, induced the phosphorylation of CD72 with a resulting increase in its association with the tyrosine phosphatase SH2 domain-containing phosphatase-1. This, in turn, resulted in an inhibition of KIT-induced phosphorylation of Src family kinases and extracellular-regulated kinases (ERK1/2). As a consequence of these effects, KIT-mediated mast cell proliferation, chemotaxis, and chemokine production were significantly reduced by BU40 and rCD100. Furthermore, BU40 and rCD100 also downregulated the growth of the HMC1.2 human mast cell line. Thus, targeting CD72 may provide a novel approach to the suppression of mast cell disease such as mastocytosis.

RPTPα and PTPε signaling via Fyn/Yes and RhoA is essential for zebrafish convergence and extension cell movements during gastrulation

Convergence and extension (C&E) cell movements are essential to shape the body axis during vertebrate gastrulation. We have used the zebrafish to assess the role of the receptor protein-tyrosine phosphatases, RPTPα and PTPε, in gastrulation cell movements. Both RPTPα and PTPε knockdown and ptpra−/− embryos show defects in C&E movements. A method was developed to track gastrulation cell movements using confocal microscopy in a quantitative manner and ptpra−/− embryos displayed reduced convergence as well as extension speeds. RPTPα and PTPε knockdowns cooperated with knockdown of a well known factor in C&E cell movement, non-canonical Wnt11. RPTPα and PTPε dephosphorylate and activate Src family kinases in various cell types in vitro and in vivo. We found that Src family kinase phosphorylation was enhanced in ptpra−/− embryos, consistent with reduced Src family kinase activity. Importantly, both ptpra−/− and RPTPα and PTPε knockdown induced C&E defects were rescued by active Fyn and Yes. Moreover, active RhoA rescued the RPTPα and PTPε knockdown and ptpra−/− induced gastrulation cell movement defects as well. Our results demonstrate that RPTPα and PTPε are essential for C&E movements in a signaling pathway parallel to non-canonical Wnts and upstream of Fyn, Yes and RhoA.

Activities of SYK and PLCγ2 Predict Apoptotic Response of CLL Cells to SRC Tyrosine Kinase Inhibitor Dasatinib

B-cell receptor signaling plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). However, blocking B-cell receptor signaling with dasatinib, an inhibitor of SRC kinase, produced variable results in preclinical and clinical studies. We aim to define the molecular mechanisms underlying the differential dasatinib sensitivity and to uncover more effective therapeutic targets in CLL. Fresh CLL B cells were treated with dasatinib, and cell viability was followed. The CLL cases were then divided into good and poor responders. The cellular response was correlated with the activities of B-cell receptor signaling molecules, as well as with molecular and cytogenetic prognostic factors. Among 50 CLL cases, dasatinib treatment reduced cell viability by 2% to 90%, with an average reduction of 47% on day 4 of culture. The drug induced CLL cell death through the intrinsic apoptotic pathway mediated by reactive oxygen species. Unexpectedly, phosphorylation of SRC family kinases was inhibited by dasatinib in good, as well as poor, responders. As opposed to SRC family kinases, activities of two downstream molecules, SYK and phospholipase Cgamma2, correlate well with the apoptotic response of CLL cells to dasatinib. Thus, SYK inhibition predicts cellular response to dasatinib. SYK, together with phospholipase Cgamma2, may serve as potential biomarkers to predict dasatinib therapeutic response in patients. From the pathogenic perspective, our study suggests the existence of alternative mechanisms or pathways that activate SYK, independent of SRC kinase activities. The study further implicates that SYK might serve as a more effective therapeutic target in CLL treatment.

Activity of SYK and PLCγ2 Predict Apoptotic Response of Chronic Lymphocytic Leukemia Cells to SRC Tyrosine Kinase Inhibitor Dasatinib.

Abstract 1249Poster Board I-271 PurposeB-cell receptor (BCR) signaling plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). However, blocking BCR signaling with dasatinib, an inhibitor of SRC kinase, produced variable results in pre-clinical and clinical studies. We aim to define the molecular mechanisms underlying the differential dasatinib sensitivity and to uncover more effective therapeutic targets in CLL. Experimental DesignFresh CLL B cells were treated with dasatinib and cell viability was followed. The CLL cases were then divided into good and poor responders. The cellular response was correlated with the activities of BCR signaling molecules as well as with molecular and cytogenetic prognostic factors. Results and ConclusionsAmong 44 CLL cases, dasatinib treatment reduced cell viability by 2-90%, with an average reduction of 48% on day four of culture. The drug induced CLL cell death via the intrinsic apoptotic pathway mediated by reactive oxygen species. Unexpectedly, phosphorylation of SRC family kinases was inhibited by dasatinib in good as well as poor responders. As opposed to SRC family kinases, activities of two downstream molecules, SYK and PLCγ2, correlate well with the apoptotic response of CLL cells to dasatinib. Thus, SYK inhibition predicts cellular response to dasatinib. SYK, together with PLCγ2, may serve as potential biomarkers to predict dasatinib therapeutic response in patients. From the pathogenic perspective, our study suggests the existence of alternative mechanisms or pathways that activate SYK independent of SRC kinase activities. The study further implicates that SYK might serve as a more effective therapeutic target in CLL treatment. DisclosuresLee:Bristol-Myers Squibb Research & Development: Employment. Coleman:Bristol-Myers Squibb Research & Development: Consultancy.

Therapeutic implications of Src independent calcium mobilization in diffuse large B-cell lymphoma

We report that 38% of primary large B-cell lymphoma (DLBCL) tested expressed active Src family kinases, which are targeted by dasatinib. The expression of active Src family of kinases (SFK) in primary DLBCL tumors correlated with unfavorable prognostic markers such as Ki67 and Mum1. Using four DLBCL cell lines we found that: (1) sensitivity to dasatinib (but not imatinib) varied 400-fold; (2) dasatinib resistance was associated with distinct signaling profiles downstream of BCR activation. In particular, although Src family kinase phosphorylation was inhibited by 100–150 nM dasatinib in all cell lines, this failed to inhibit BCR-mediated Blnk phosphorylation, calcium signaling and proliferation in a dasatinib resistant cell line.

Dectin-1 is Inducible and Plays an Essential Role for Mycobacteria-Induced Innate Immune Responses in Airway Epithelial Cells

Airway epithelial cells are the first cells to be challenged upon contact with mycobacteria. In response, they express pattern-recognition receptors that play fundamental roles as sentinels and mediators of pulmonary innate immunity. The c-type lectin Dectin-1 is expressed predominantly on the surface of myeloid lineage cells. In this study, we examined the induction, regulation, and functions of Dectin-1 in pulmonary epithelial cells. Mycobacterium tuberculosis (Mtb) actively induced the expression of Dectin-1 mRNA and protein in A549 cells in a toll-like receptor (TLR) 2-dependent manner. In addition, Mtb-mediated generation of reactive oxygen species and Dectin-1 induction were mutually dependent. Moreover, Mtb actively induced the phosphorylation of Src family kinases at Tyr416 via TLR2. Selective inhibition of Src markedly attenuated the induction of Mtb-dependent Dectin-1 expression, indicating that Src kinases are crucial regulators of Dectin-1-dependent signaling. Mtb internalization was partially blocked by silencing Dectin-1 expression, inhibiting Src kinases, or pretreating with antioxidants. Finally, Dectin-1 was required for pro-inflammatory cytokine release and antimicrobial effects on intracellular mycobacterial growth in A549 cells. Collectively, our findings demonstrate the novel induction of Dectin-1 in type II airway epithelial cells and its critical role in the innate immune response against Mtb in non-phagocytic cells.