Lyn Knockdown Research Articles

A novel lncRNA associated with the prognosis of patients with colorectal cancer resists apoptosis through the LYN/BCL-2 pathway

PurposeWe found a novel lncRNA named lncAC138150.2 related to the overall survival and staging of patients with colorectal cancer (CRC) by bioinformatic analysis using data from the Cancer Genome Atlas (TCGA), and the study aimed to elucidate the function of lncAC138150.2 and underlying mechanisms. MethodsTarget molecules were knocked down by transfection with antisense oligonucleotides (ASOs), siRNAs, or lentiviruses and overexpressed by transfection with plasmids. The function of lncAC138150.2 was determined using histological, cytological, and molecular biology methods. The underlying mechanism of lncAC138150.2 function was investigated using RNA-seq, bioinformatics analysis, and molecular biology methods. ResultsThe expression of lncAC138150.2 was increased in colorectal tissues compared with paired normal tissues. The lncAC138150.2 knockdown increased apoptosis but did not change the cell proliferation, cell cycle distribution, or cell migration ability of CRC cells, while lncAC138150.2 overexpression decreased CRC apoptosis. lncAC138150.2 was mainly located in the cell nucleus, and each lncAC138150.2 transcript knockdown increased CRC apoptosis. BCL-2 pathway was significantly altered in apoptosis induced by lncAC138150.2 knockdown, which was alleviated by BAX knockdown. The expression of LYN was significantly decreased with lncAC138150.2 knockdown, LYN knockdown increased CRC apoptosis, and its overexpression completely alleviated CRC apoptosis induced by lncAC138150.2 knockdown. ConclusionlncAC138150.2 significantly inhibited CRC apoptosis and affected the prognosis of patients with CRC, through the LYN/BCL-2 pathway.

Pan-Src kinase inhibitor treatment attenuates diabetic kidney injury via inhibition of Fyn kinase-mediated endoplasmic reticulum stress

Src family kinases (SFKs) have been implicated in the pathogenesis of kidney fibrosis. However, the specific mechanism by which SFKs contribute to the progression of diabetic kidney disease (DKD) remains unclear. Our preliminary transcriptome analysis suggested that SFK expression was increased in diabetic kidneys and that the expression of Fyn (a member of the SFKs), along with genes related to unfolded protein responses from the endoplasmic reticulum (ER) stress signaling pathway, was upregulated in the tubules of human diabetic kidneys. Thus, we examined whether SFK-induced ER stress is associated with DKD progression. Mouse proximal tubular (mProx24) cells were transfected with Fyn or Lyn siRNA and exposed to high glucose and palmitate (HG-Pal). Streptozotocin-induced diabetic rats were treated with KF-1607, a novel pan-Src kinase inhibitor (SKI) with low toxicity. The effect of KF-1607 was compared to that of losartan, a standard treatment for patients with DKD. Among the SFK family members, the Fyn and Lyn kinases were upregulated under diabetic stress. HG-Pal induced p70S6 kinase and JNK/CHOP signaling and promoted tubular injury. Fyn knockdown but not Lyn knockdown inhibited this detrimental signaling pathway. In addition, diabetic rats treated with KF-1607 showed improved kidney function and decreased ER stress, inflammation, and fibrosis compared with those treated with losartan. Collectively, these findings indicate that Fyn kinase is a specific member of the SFKs implicated in ER stress activation leading to proximal tubular injury in the diabetic milieu and that pan-SKI treatment attenuates kidney injury in diabetic rats. These data highlight Fyn kinase as a viable target for the development of therapeutic agents for DKD.

The interactions of CAP and LYN with the insulin signaling transducer CBL play an important role in polycystic ovary syndrome

BackgroundPolycystic ovary syndrome (PCOS) is a hormonal disorder characterized by hyperandrogenism, ovulatory dysfunction, and insulin resistance. Evidence suggests that aberrations in insulin signaling-associated pathways may underlie PCOS pathogenesis. Our aim was to investigate the molecular mechanisms underlying PCOS and associated insulin resistance using in silico analyses, in vitro cell models, and in vivo murine models. MethodsR-based bioinformatics analysis was performed on granulosa cell microarray data from three human cohorts: healthy control, PCOS patients without insulin resistance, and PCOS patients with insulin resistance. Transgenic human granulosa cell models were utilized for in vitro studies. Transgenic murine models of dehydroepiandrosterone (DHEA)-induced PCOS were utilized for in vivo studies. ResultsSorbin and SH3 Domain Containing 1 (SORBS1), the parent gene of the insulin receptor-associated Casitas B-lineage lymphoma protein (CBL)-associated protein (CAP), is a key downregulated gene in PCOS patients with insulin resistance. CAP binding to CBL reduced CBLY731 phosphorylation, CBL-phosphoinositide 3-kinase (PI3K) p85α interactivity, protein kinase B (Akt)S473 phosphorylation, and NFκB-induced inflammatory marker expression but enhanced CRKII-mediated membrane GLUT4 translocation in granulosa cells. In contrast, the tyrosine kinase Lck/Yes-Related Novel Protein (LYN) is upregulated in PCOS patients with insulin resistance. LYN binding to CBL enhanced CBLY731 phosphorylation, CBL-PI3K p85α interactivity, AktS473 phosphorylation, and NFκB-induced inflammatory marker expression but did not impact membrane GLUT4 translocation. In PCOS mice, Cap overexpression, Cap transactivation by metformin, or enhancing Cbl-CrkII binding improved insulin sensitivity and ovarian dysfunction (i.e., estrous cycle disruption, cyst-like follicle formation, and sex hormone dysregulation). In contrast, Lyn knockdown, Lyn inhibition by PP2, or CBL-PI3K p85α blockade improved only ovarian dysfunction. Cbl3YF phosphomutant overexpression (which enhances Cbl-CrkII binding but blocks Cbl-PI3K p85α binding) ameliorated both ovarian dysfunction and insulin resistance. ConclusionsThe interactions of CAP and LYN with CBL, and the resulting effects on CBL phosphorylation and activity, may play an important role in PCOS pathogenesis. Targeting these players may be a viable therapeutic strategy for PCOS.

Oncogenic role of LYN in human gastric cancer via the Wnt/β-catenin and AKT/mTOR pathways.

LYN kinase (LYN) is a member of the Src tyrosine kinase family, which plays an important role in multiple tumor-related functions. The current study demonstrated that LYN functions as a pro-oncogene in AGS gastric cancer cells. It was found that LYN expression levels were significantly raised in gastric cancer tissue and were significantly associated with the pathological grades of patients with gastric cancer. This was accomplished by knocking down LYN in AGS cells using short hairpin RNA (shRNA) plasmid transfection, with reverse transcription-quantitative PCR detection verifying the effectiveness of RNA interference. It was found that the cell proliferation and colony formation abilities of AGS cells were significantly inhibited, using CCK-8 and clone formation assays, respectively. Furthermore, LYN knockdown was found to induce apoptosis and inhibit both migration and invasion in AGS cells, using flow cytometry and Transwell assays, respectively. A mechanical investigation further suggested that LYN knockdown resulted in the activation of the mitochondrial apoptotic pathway. Likewise, the Wnt/β-catenin pathway was inactivated by LYN knockdown, including decreased levels of Wnt3a, β-catenin, snail family transcriptional repressor (Snail)1 and Snail2. Epithelial-mesenchymal transition mesenchymal markers (including N-cadherin and vimentin) were also found to be downregulated, and E-cadherin was upregulated in LYN-silenced AGS cells. Finally, the AKT/mTOR pathway was found to be downregulated by LYN knockdown in AGS cells, including decreased levels of phosphorylated (p)-AKT (Ser473), p-mTOR (Ser2448), and the down-stream effector p70S6 kinase (p70S6K). Furthermore, the AKT pathway activator, insulin like growth factor-1 (IGF-1), was found to reverse the inhibitory effects of LYN knockdown on the proliferation, migration and invasion of AGS cells. In conclusion, the current study demonstrated that LYN plays an oncogenic role in the proliferation, survival and movement of human gastric cancer cells by activating the mitochondrial apoptotic pathway, and downregulating the Wnt/β-catenin and AKT/mTOR pathways. The current research provides a comprehensive insight into the regulation of LYN in gastric cancer and may help with the development of new tumor treatment strategies.

Roscovitine enhances All-trans retinoic acid (ATRA)-induced leukemia cell differentiation: Novel effects on signaling molecules for a putative Cdk2 inhibitor

All-trans retinoic acid (ATRA)-based differentiation therapy has been unsuccessful in treating t(15;17) negative acute myeloid leukemia (AML) patients, motivating interest in combination therapies using ATRA plus other agents. Using the t (15, 17) negative HL-60 human myeloblastic leukemia model, we find that the cyclin-dependent kinase (CDK) inhibitor, roscovitine, augments signaling by an ATRA-induced macromolecular signalsome that propels differentiation and enhances ATRA-induced differentiation. Roscovitine co-treatment enhanced ATRA-induced expression of pS259- pS289/296/301- pS621-c-Raf, pS217/221-Mek, Src Family Kinases (SFKs) Lyn and Fgr and SFK Y416 phosphorylation, adaptor proteins c-Cbl and SLP-76, Vav, and acetylated 14–3-3 in the signalsome. Roscovitine enhanced ATRA-induced c-Raf interaction with Lyn, Vav, and c-Cbl. Consistent with signalsome hyper-activation, roscovitine co-treatment enhanced ATRA-induced G1/0 arrest and expression of differentiation markers, CD11b, ROS and p47 Phox. Because roscovitine regulated Lyn expression, activation and partnering, a stably transfected Lyn knockdown was generated from wt-parental cells to investigate its function in ATRA-induced differentiation. Lyn-knockdown enhanced ATRA-induced up-regulation of key signalsome molecules, c-Raf, pS259-c-Raf, pS289/296/301-c-Raf, Vav1, SLP-76, and Fgr, but with essentially total loss of pY416-SFK. Compared to ATRA-treated wt-parental cells, differentiation markers p47 phox, CD11b, G1/G0 arrest and ROS production were enhanced in ATRA-treated Lyn-knockdown stable transfectants, and addition of roscovitine further enhanced these ATRA-inducible markers. The Lyn-knockdown cells expressed slightly higher c-Raf, pS259-c-Raf, pS289/296/301-c-Raf, and SLP-76 than wt-parental cells, and this was associated with enhanced ATRA-induced upregulation of Fgr and cell differentiation, consistent with heightened signaling, suggesting that enhanced Fgr may have compensated for loss of Lyn to enhance differentiation in the Lyn-knockdown cells.

Lyn Kinase Promotes the Proliferation of Malignant Melanoma Cells through Inhibition of Apoptosis and Autophagy via the PI3K/Akt Signaling Pathway.

Melanoma is a malignant tumor of cutaneous melanocytes that is characterized by high grade malignancy, rapid progression and high mortality. Thus far, its specific etiological mechanism has been unclear. In this study, we discovered that Lyn kinase expression was up-regulated in melanoma tissues and cells. The function of Lyn was determined by knocking down its expression with a lentivirus containing Lyn shRNA and upregulating its expression with pcDNA3.1-Lyn in the melanoma cell lines M14 and A375. The results showed that Lyn knockdown could significantly inhibit the proliferation, migration and invasiveness through its inhibition of apoptosis and autophagy via the PI3K/Akt pathway in melanoma cell lines. This was further confirmed by treatment with PI3K inhibitor BEZ235. Up-regulation of Lyn promoted the expression of p-Akt and Cyclin D1. Additionally, we investigated the effects of Lyn inhibitor Bafetinib on melanoma cells and the results were consistent with Lyn knockdown. Collectively, our results indicated that Lyn plays a carcinogenic role in multiple cellular functions during melanoma development through regulating apoptosis and autophagy via the PI3K/Akt pathway and may be a valuable potential target for the clinical treatment of melanoma.

LYN, a key mediator in estrogen-dependent suppression of osteoclast differentiation, survival, and function

Estrogen insufficiency at menopause cause accelerated bone loss due to unwarranted differentiation and function of osteoclasts. Unraveling the underlying mechanism/s may identify mediators of estrogen action which can be targeted for improved management of osteoporosis. Towards this, we analyzed the effect of 17β-estradiol on the proteomes of differentiating human osteoclasts. The major proteomic changes observed included upregulation of LYN by estrogen. We, therefore, investigated the effect of estrogen on osteoclast differentiation, survival, and function in control and LYN knockdown conditions. In control condition, estrogen treatment increased the apoptosis rate and suppressed the calcium signaling by reducing the intracellular Ca2+ levels as well as expression and activation of NFATc1 and c-Src during differentiation, resulting in reduced osteoclastogenesis. These osteoclasts were smaller in size with reduced extent of multinuclearity and produced significantly low levels of bone resorbing enzymes. They also exhibited disrupted sealing zone formation with low podosome density, impaired cell polarization and reduced resorption of dentine slices. Interestingly, in LYN knockdown condition, estrogen failed to induce apoptosis and inhibit activation of NFATc1 and c-Src. Compared to effect of estrogen on osteoclast in control condition, LYN knockdown osteoclasts did not show reduction in production of bone resorbing enzymes and had defined sealing zone formation with high podosome density with no impairment in cell polarization. They resorbed significant area on dentine slices. Thus, the inhibitory action of estrogen on osteoclast was severely restrained in LYN knockdown condition, demonstrating the importance of LYN as a key mediator of the effect of estrogen on osteoclastogenesis.

Lyn regulates mucus secretion and MUC5AC via the STAT6 signaling pathway during allergic airway inflammation

Hypersecretion of mucus is an important component of airway remodeling and contributes to the mucus plugs and airflow obstruction associated with severe asthma phenotypes. Lyn has been shown to down-regulate allergen-induced airway inflammation. However, the role of Lyn in mucin gene expression remains unresolved. In this study, we first demonstrate that Lyn overexpression decreased the mucus hypersecretion and levels of the muc5ac transcript in mice exposed to ovalbumin (OVA). Lyn overexpression also decreased the infiltration of inflammatory cells and the levels of IL-13 and IL-4 in OVA-challenged airways. Whereas Lyn knockdown increased the IL-4 or IL-13-induced MUC5AC transcript and protein levels in the human bronchial epithelial cell line, 16HBE, Lyn overexpression decreased IL-4- or IL-13-induced MUC5AC transcript and protein levels. Overexpression of Lyn also decreased the expression and phosphorylation of STAT6 in OVA-exposed mice, whereas Lyn knockdown increased STAT6 and MUC5AC levels in 16HBE cells. Finally, chromatin immunoprecipitation analysis confirmed that Lyn overexpression decreased the binding of STAT6 to the promoter region of Muc5ac in mice exposed to OVA. Collectively, these findings demonstrated that Lyn overexpression ameliorated airway mucus hypersecretion by down-regulating STAT6 and its binding to the MUC5AC promoter.

Src family kinases differentially influence glioma growth and motility

Src-family kinase (SFK) signaling impacts multiple tumor-related properties, particularly in the context of the brain tumor glioblastoma. Consequently, the pan-SFK inhibitor dasatinib has emerged as a therapeutic strategy, despite physiologic limitations to its effectiveness in the brain. We investigated the importance of individual SFKs (Src, Fyn, Yes, and Lyn) to glioma tumor biology by knocking down individual SFK expression both in culture (LN229, SF767, GBM8) and orthotopic xenograft (GBM8) contexts. We evaluated the effects of these knockdowns on tumor cell proliferation, migration, and motility-related signaling in culture, as well as overall survival in the orthotopic xenograft model. The four SFKs differed significantly in their importance to these properties. In culture, Src, Fyn, and Yes knockdown generally reduced growth and migration and altered motility-related phosphorylation patterns while Lyn knockdown did so to a lesser extent. However the details of these effects varied significantly depending on the cell line: in no case were conclusions about the role of a particular SFK applicable to all of the measures or all of the cell types examined. In the orthotopic xenograft model, mice implanted with non-target or Src or Fyn knockdown cells showed no differences in survival. In contrast, mice implanted with Yes knockdown cells had longer survival, associated with reduced tumor cell proliferation. Those implanted with Lyn knockdown cells had shorter survival, associated with higher overall tumor burden. Together, our results suggest that Yes signaling directly affects tumor cell biology in a pro-tumorigenic manner, while Lyn signaling affects interactions between tumor cells and the microenvironment in an anti-tumor manner. In the context of therapeutic targeting of SFKs, these results suggest that pan-SFK inhibitors may not produce the intended therapeutic benefit when Lyn is present.

Abstract 2507: Mechanistic and functional characterization of the atypical kinase SgK269/PEAK1 in breast cancer.

Abstract The human genome encodes 518 protein kinases which can be mined for potential cancer drug targets and biomarkers. Previously, we performed SILAC labelling in combination with mass spectrometry-based phosphoproteomics to identify seven proteins exhibiting decreased tyrosine phosphorylation upon Lyn knockdown in the BT549 and MDA-MB-231 basal breast cancer cell lines. From these we selected the atypical kinase SgK269 (also known as PEAK1) for further study. SgK269 is classified as a pseudokinase since it contains critical substitutions in one of the three conserved motifs important for kinase activity. The primary objective of this study was to functionally characterize SgK269 and to identify its signaling role through proteomic and phosphoproteomic profiling in mammary epithelial cells. Initial pull-down experiments using SgK269 recombinant protein were performed to identify binding partners of SgK269 in MDA-MB-231 cells. Functional annotation of the identified binding partners subsequently indicated that SgK269 may be involved in MAPK signaling, regulation of the actin cytoskeleton and focal adhesions, and ubiquitin-mediated proteolysis. To further interrogate the role of SgK269 in breast cancer, SgK269 was overexpressed in the human mammary epithelial cell line MCF-10A. We also expressed a mutant form of SgK269 where the Lyn phosphorylation site, Y635, was altered to phenylalanine (F). Overexpression of SgK269 resulted in a change in cellular morphology. In monolayer, cells were elongated resembling a mesenchymal phenotype and exhibited a decreased expression of E-cadherin and an increase in N-cadherin, indicative of epithelial-to-mesenchymal transition (EMT). When grown in Matrigel, the SgK269-overexpressing cells formed large, aberrant acini, while the SgK269 Y635F mutant was unable to enhance acinar size. In terms of signaling, overexpression of SgK269 increased Erk phosphorylation in three-dimensional culture, an effect associated with SgK269/Grb2 binding and enhanced interaction between the EGFR and Grb2. Since SgK269 showed strong binding with Stat3 in pull-down studies, we also characterized Stat3 signalling in this model. SgK269 overexpression led to enhanced Stat3 tyrosine phosphorylation and transcriptional activity whilst the Y635F mutation abolished this effect. The global tyrosine phosphorylation signature associated with SgK269 overexpression in MCF-10A cells is currently under investigation. SgK269 is a poorly characterized kinase that may contribute to the progression of breast and other cancers. This study provides novel and highly significant insights into the signaling mechanism and function of SgK269 that highlights potential strategies for the improved treatment of basal breast cancers, as well as other cancers where SgK269 is overexpressed. Citation Format: Ling Liu, Howard Chan, David Croucher, Falco Hochgräfe, Luxi Zhang, Carole Tactacan, Roger Daly. Mechanistic and functional characterization of the atypical kinase SgK269/PEAK1 in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2507. doi:10.1158/1538-7445.AM2013-2507 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Src inhibitors, PP2 and dasatinib, increase retinoic acid-induced association of Lyn and c-Raf (S259) and enhance MAPK-dependent differentiation of myeloid leukemia cells

All-trans-retinoic-acid (ATRA)-induced differentiation of human myeloid leukemia cells is characterized by persistent MAPK signaling. Fragmentary data suggests Src family kinase (SFK) inhibitors enhance differentiation and thus have potential therapeutic value. The present study shows that SFK inhibitors PP2 and dasatinib enhance aspects of MAPK signaling and regulate a panel of differentiation markers including CD11b and p47phox. HL-60 and NB4 myeloid leukemia cells show accelerated ATRA-induced G1/0 arrest/differentiation with inhibitor co-treatment. We also identified components of a Lyn- and c-Raf-containing MAPK signaling complex augmented by the inhibitors. PP2 and dasatinib increased ATRA-induced expression of Lyn and c-Raf (total and c-RafpS259) and their interaction. The Lyn-associated serine/threonine kinase CK2 also complexed with c-Raf and c-RafpS259, and the KSR1 scaffold protein bound c-Raf, Lyn, and ERK. c-Raf/ERK association was increased by the inhibitors, which is significant since ERK may cause c-Raf C-terminal domain (CTD) phosphorylation in a putative feedback mechanism. Consistent with this, inhibitor treatment caused more CTD phosphorylation. Lyn knockdown decreased c-Raf CTD and S259 phosphorylation. This is the first evidence suggesting SFK inhibitors enhance ATRA-induced differentiation through a possible feedback loop involving KSR1-scaffolded c-Raf and ERK complexed with Lyn and CK2.

Abstract 3131: A novel tyrosine kinase signaling pathway in human basal breast cancer

Abstract Basal-type breast cancers account for up to 25 % of breast cancer cases, depending on the patient population. These cancers usually lack estrogen, progesterone and erbB2 receptors and are therefore resistant to endocrine or trastuzumab therapy, and markers that stratify this subset according to clinical behaviour have yet to be identified. Consequently, basal breast cancers present a major treatment dilemma. In order to identify potential therapeutic targets and prognostic markers for this disease subgroup, we are currently undertaking a detailed characterization of tyrosine kinase signaling networks in basal breast cancer cells. Mass spectrometry (MS)-based global profiling of the tyrosine phosphoproteome of basal breast cancer cells identified a prominent Src family kinase (SFK) signaling network that featured high expression of the SFK Lyn (Hochgräfe et al, Cancer Res 2010 doi: 10.1158/0008-5472.CAN-10-0911). In order to identify Lyn substrates, we used stable isotope labelling with amino acids in cell culture (SILAC) in combination with immunoaffinity-based phosphotyrosine enrichment and LC-MS/MS to profile cellular tyrosine phosphorylation following transient siRNA-mediated Lyn knockdown in MDA-MB-231 and BT-549 basal breast cancer cells. This identified several Lyn substrates characteristic of both cell lines, which included annexin A2 (Y42), 40S ribosomal protein S10 (Y12) and SgK269 (Y635). The latter is a relatively understudied kinase that contains several tyrosine phosphorylation sites that are likely to act as recruitment sites for SH2/PTB domain-containing signalling proteins, and a C-terminal kinase-like domain that lacks the consensus DFG motif usually regarded as essential for kinase activity. Protein expression of SgK269 was markedly higher in basal, versus luminal, breast cancer cells, while mRNA transcript levels in the two cell types were similar, indicating post-transcriptional regulation of SgK269 expression. Phosphorylation of SgK269 on Y635 could be detected readily in basal breast cancer cells. Overexpression of SgK269 in MCF-10A immortalized mammary epithelial cells led to a more elongated morphology in monolayer and perturbed acinar morphogenesis in Matrigel, resulting in large, multilobular acini that failed to undergo proliferative suppression in long-term culture. SgK269-overexpressing cells also exhibited enhanced tyrosine phosphorylation of Stat3. In addition, we undertook stable shRNA-mediated knockdown of SgK269 in BT-549 cells. This reduced soft agar growth and Stat3 activation. These studies identify a novel oncogenic signalling pathway in basal breast cancer involving the kinases Lyn and SgK269, which may represent a target for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3131. doi:10.1158/1538-7445.AM2011-3131

Syk and Lyn mediate distinct Syk phosphorylation events in FcɛRI-signal transduction: Implications for regulation of IgE-mediated degranulation

Spleen tyrosine kinase (Syk) is a key regulatory factor in the IgE-mediated allergic signal transduction pathway in mast cells and basophils. Syk is phosphorylated on a number of tyrosines following the binding of IgE/allergen complexes to FcɛRI receptors leading to initiation of inflammatory signaling via downstream enzymes and scaffolding proteins. We examined the kinases responsible for the phosphorylation of key Syk tyrosines in rat RBL-2H3 basophilic cells and bone marrow-derived mast cells (BMMCs). The phosphorylation of Syk tyrosine 346 was completely blocked by the novel Src family kinase inhibitor BIRA766, suggesting this tyrosine is a pure substrate for Src family kinases. This was supported by the findings that kinase-dead (KD) Syk was efficiently phosphorylated on this tyrosine and that a specific Syk inhibitor BAY61-3606 was without effect. The phosphorylation of other Syk tyrosines 317, 342, 519 and 520 was reduced by Syk and Src family inhibitors, suggesting a role for auto- and trans-phosphorylation. Lyn was the predominant Src family kinase expressed and activated in RBL-2H3 cells, meanwhile Lyn knockdown with a specific siRNA interfered with the phosphorylation of all Syk tyrosines and the Syk substrates SLP-76 and LAT. Pharmacological inhibition of Syk completely blocked the degranulation of RBL-2H3 and BMMCs. However, Lyn knockdown sensitized RBL-2H3 cells to FcɛRI-induced degranulation. We showed that whilst interference with Lyn expression disrupts FcɛRI proximal signaling via Syk and its direct substrates including SLP-76 and LAT, distal activation of downstream proteins including Erk is enhanced. This study identifies the responsible kinases for the phosphorylation of key Syk tyrosines and the propagation of FcɛRI receptor mediated signal transduction in allergic responses.

Inhibition of imatinib-mediated apoptosis by the caspase-cleaved form of the tyrosine kinase Lyn in chronic myelogenous leukemia cells

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.

A critical role for Lyn in acute myeloid leukemia

Receptor or nonreceptor tyrosine kinases (TKs) are known to play an important role in leukemogenesis. Here we studied the level of protein tyrosine phosphorylations in a series of fresh AML samples and evaluated the effect of TK inhibitors. Compared with normal hematopoietic progenitors, a high level of tyrosine phosphorylation was detected in most acute myeloid leukemia (AML) samples. The Src family kinases (SFKs) appeared constitutively activated in most cases, including in the CD34(+)CD38(-)CD123(+) compartment as revealed by the level of phosphorylated tyrosine 416. Lyn was the major SFK family member expressed in an active form in AML cells where it was abnormally distributed throughout the plasma membrane and the cytosol as opposed to normal hematopoietic progenitors. The SFK inhibitor, PP2, strongly reduced the global level of tyrosine phosphorylations, inhibited cell proliferation, and induced apoptosis in patient samples without affecting normal granulomonocytic colony forming units. Moreover, silencing Lyn expression by small interfering RNA in primary AML cells strongly inhibited proliferation. Interestingly, a link between Lyn and the mTOR pathway was observed as PP2 and a Lyn knockdown both affected the phosphorylation of mTOR targets without inhibiting Akt phosphorylation. Lyn should be considered as a novel pharmacologic target for AML therapy.

Lyn-coupled LacCer-enriched lipid rafts are required for CD11b/CD18-mediated neutrophil phagocytosis of nonopsonized microorganisms

The integrin CD11b/CD18 plays a central role in neutrophil phagocytosis. Although CD11b/CD18 binds a wide range of ligands, including C3bi and beta-glucan, and transmits outside-in signaling, the mechanism of this signaling responsible for phagocytosis remains obscure. Here, we report that lactosylceramide (LacCer)-enriched lipid rafts are required for CD11b/CD18-mediated phagocytosis of nonopsonized zymosans (NOZs) by human neutrophils. Anti-CD11b and anti-LacCer antibodies inhibited the binding of NOZs to neutrophils and the phagocytosis of NOZs. During phagocytosis of NOZ, CD11b and LacCer were accumulated and colocalized in the actin-enriched phagocytic cup regions. Immunoprecipitation experiments suggested that CD11b/CD18 was mobilized into the LacCer-enriched lipid rafts during phagocytosis of NOZs. DMSO-treated, neutrophil-like HL-60 cells (D-HL-60 cells) lacking Lyn-coupled, LacCer-mediated signaling showed little phagocytosis of NOZs. However, loading of D-HL-60 cells with C24 fatty acid chain-containing LacCer (C24-LacCer) reconstructed functional Lyn-associated, LacCer-enriched lipid rafts, and restored D-HL-60 cell NOZ phagocytic activity, which was inhibited by anti-LacCer and anti-CD11b antibodies. Lyn knockdown by small interfering RNA blocked the effect of C24:1-LacCer loading on D-HL-60 cell phagocytosis of NOZs. CD11b/CD18 activation experiments indicated phosphorylation of LacCer-associated Lyn by activation of CD11b. Taken together, these observations suggest that CD11b activation causes translocation of CD11b/CD18 into Lyn-coupled, LacCer-enriched lipid rafts, allowing neutrophils to phagocytose NOZs via CD11b/CD18.

Involvement of very long fatty acid-containing lactosylceramide in lactosylceramide-mediated superoxide generation and migration in neutrophils

The neutral glycosphingolipid lactosylceramide (LacCer) forms lipid rafts (membrane microdomains) coupled with the Src family kinase Lyn on the plasma membranes of human neutrophils; ligand binding to LacCer activates Lyn, resulting in neutrophil functions, such as superoxide generation and migration (Iwabuchi and Nagaoka, Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils, Blood 100, 1454-1464, 2002 and Sato et al. Induction of human neutrophil chemotaxis by Candida albicans-derived beta-1,6-long glycoside side-chain-branched beta glycan, J. Leukoc. Biol. 84, 204-211, 2006). Neutrophilic differentiated HL-60 cells (D-HL-60 cells) express almost the same amount of LacCer as neutrophils. However, D-HL-60 cells do not have Lyn-associated LacCer-enriched lipid rafts and lack LacCer-mediated superoxide-generating and migrating abilities. Here, we examined the roles of LacCer molecular species of different fatty acid compositions in these processes. Liquid chromatography-mass spectrometry analyses revealed that the very long fatty acid C24:0 and C24:1 chains were the main components of LacCer (31.6% on the total fatty acid content) in the detergent-resistant membrane fraction (DRM) from neutrophil plasma membranes. In contrast, plasma membrane DRM of D-HL-60 cells included over 70% C16:0-LacCer, but only 13.6% C24-LacCer species. D-HL-60 cells loaded with C24:0 or C24:1-LacCer acquired LacCer-mediated migrating and superoxide-generating abilities, and allowed Lyn coimmunoprecipitation by anti-LacCer antibody. Lyn knockdown by siRNA completely abolished the effect of C24:1-LacCer loading on LacCer-mediated migration of D-HL-60 cells. Immunoelectron microscopy revealed that LacCer clusters were closely associated with Lyn molecules in neutrophils and C24:1-LacCer-loaded D-HL-60 cells, but not in D-HL-60 cells or C16:0-LacCer-loaded cells. Taken together, these observations suggest that LacCer species with very long fatty acids are specifically necessary for Lyn-coupled LacCer-enriched lipid raft-mediated neutrophil superoxide generation and migration.