Fyn Tyrosine Kinase-mediated Tyrosine Phosphorylation of Roundabout (Robo), the Slit Receptor

전은숙(Eun Sook Jeon),배기원(Kee Won Bae),박환태(Hwan Tae Park) 대한해부학회 2004 Anatomy & Cell Biology Vol.37 No.3

본 연구에서는 Slit의 세포막 수용체인 Roundabout (Robo)의 tyrosine 인산화의 분자적 기전을 연구하였다. Human embryonic kidney 세포에 인위적으로 발현시킨 Robo의 tyrosine 인산화는 tyrosine phosphatase 억제제의 처리에 의해 증가 되었으며, src family kinase의 억제제인 PP2의 처리에 의해 감소하였다. 활성화된 형태의 Fyn tyrosine kinase (Fyn)를 Robo와 함께 발현시켰을 때 Robo의 tyrosine 인산화의 증가가 관찰되었으나, 비활성 형태의 Fyn은 인산화를 증가 시키지 못하였다. 또한, 인산화된 tyrosine기에 결합하는 Fyn SH2도메인이 Robo와 결합함이 관찰되었으며 이 결합은 tyrosine phosphatase 억제제의 처리에 의해 증가되었다. 따라서 이러한 결과는 Robo의 tyrosine 인산화가 Fyn에 의해 매개됨을 시사한다. In this study, the molecular mechanism of tyrosine phosphorylation of Roundabout (Robo), the transmembrane receptor for slits, was investigated. The tyrosine phosphorylation of intracellular portion of Robo was increased by the treatment of tyrosine phosphatase inhibitors in human embryonic kidney cells transfected with Robo. The Robo tyrosine phosphorylation was inhibited by the treatment of Src family kinase inhibitor, PP2. The co-transfection of constitutively active form of Fyn, not the dominant negative form of Fyn, and Robo dramatically enhanced the tyrosine phosphorylation of Robo. Furthermore, the SH2 domain of Fyn, which binds to phosphorylated tyrosine residues, interact with Robo, and the interaction was increased by the inhibition of tyrosine phosphatases. These findings indicate that the tyrosine phosphorylation of Robo is regulated by Fyn.

Tyrosine kinase Fyn regulates iNOS expression in LPS-stimulated astrocytes via modulation of ERK phosphorylation

Ko, Hyun Myung,Lee, Sung Hoon,Bang, Minji,Kim, Ki Chan,Jeon, Se Jin,Park, Yeong-Min,Han, Seol-Heui,Kim, Hahn Young,Lee, Jongmin,Shin, Chan Young Elsevier 2018 Biochemical and biophysical research communication Vol.495 No.1

<P><B>Abstract</B></P> <P>The high concentrations of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in activated glial cells in response to neuroinflammatory stimuli have neurotoxic effects on the brain. At basal levels, iNOS expression is low, and proinflammatory stimuli induce iNOS expression in astrocytes, microglia, and oligodendrocytes. Fyn, a non-receptor tyrosine kinase, regulates iNOS expression in several types of immune cells. However, its role in stimulated astrocytes is less clear. In this study, we investigated the role of Fyn in the regulation of lipopolysaccharide (LPS)-induced iNOS expression in astrocytes from mice and rats. Intracerebroventricular LPS injections in cortical regions enhanced iNOS mRNA and protein levels, which were increased in Fyn-deficient mice. Accordingly, LPS-induced nitrite production was enhanced in primary astrocytes cultured from Fyn-deficient mice or rats. Similar results were observed in cultured astrocytes after the siRNA-induced knockdown of Fyn expression. Finally, we observed increased LPS-induced extracellular signal-regulated protein kinase (ERK) activation in Fyn-deficient astrocytes. These results suggested that Fyn has a regulatory role in iNOS expression in astrocytes during neuroinflammatory responses.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Fyn regulates LPS-induced iNOS expression in the cortices. </LI> <LI> Fyn modulates iNOS expression and nitrite levels in LPS-stimulated astrocytes. </LI> <LI> Fyn regulates iNOS expression in LPS-stimulated astrocytes via ERK phosphorylation. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Fyn promotes proliferation, differentiation, survival and function of osteoclast lineage cells

Kim, Hyun‐,Ju,Warren, Julia T.,Kim, Shin‐,Yoon,Chappel, Jean C.,DeSelm, Carl J.,Ross, F. Patrick,Zou, Wei,Teitelbaum, Steven L. Wiley Subscription Services, Inc., A Wiley Company 2010 Journal of cellular biochemistry Vol.111 No.5

<P><B>Abstract</B></P><P>c‐Src and Lyn are the only Src family kinases (SFKs) with established activity in osteoclasts (OCs). c‐Src promotes function via cytoskeletal organization of the mature resorptive cell while Lyn is a negative regulator of osteoclastogenesis. We establish that Fyn, another SFK, also impacts the OC, but in a manner distinctly different than c‐Src and Lyn. Fyn deficiency principally alters cells throughout the osteoclastogenic process, resulting in diminished numbers of resorptive polykaryons. Arrested OC formation in the face of insufficient Fyn reflects reduced proliferation of precursors, in response to M‐CSF and retarded RANK ligand (RANKL)‐induced differentiation, attended by suppressed activation of the osteoclastogenic signaling molecules, c‐Jun, and NF‐κB. The anti‐apoptotic properties of RANKL are also compromised in cells deleted of Fyn, an event mediated by increased Bim expression and failed activation of Akt. The defective osteoclastogenesis of Fyn−/− OCs dampens bone resorption, in vitro. Finally, while Fyn deficiency does not regulate basal osteoclastogenesis, in vivo, it reduces that stimulated by RANKL by ∼2/3. Thus, Fyn is a pro‐resorptive SFK, which exerts its effects by prompting proliferation and differentiation while attenuating apoptosis of OC lineage cells. J. Cell. Biochem. 111: 1107–1113, 2010. © 2010 Wiley‐Liss, Inc.</P>

Phosphorylation at Tyr-838 in the kinase domain of EphA8 modulates Fyn binding to the Tyr-615 site by enhancing tyrosine kinase activity

Choi, Sunga,Park, Soochul 한림대학교 부설 환경ㆍ생명과학연구소 1999 일송 의학ㆍ생명과학 심포지엄 Vol.- No.1

Eph-related receptors and their ephrin lignds are highly conserved protein families which play important roles in targeting axons and migrating cells. In this study we have examined the functional roles of two major autophosphorylation sites, Tyr-615 and Tyr-838, in the EphA8 receptor. Two-dimensional phosphopeptide mapping analysis demonstrated that Tyr-615 and Tyr 838 constitute major autophosphorylation sites in EphA8. Tyr-615 was phosphorylated to the highest stoichiometry, suggesting that phosphorylation at this site may have a physiologically important role. Upon conservative mutation of Tyr 838 located int he tyrosine kinase domain, the catalytic activity of EphA8 was strikingly reduced both in vitro and in vivo, whereas a mutation at Tyr-615 in the juxtamembrane domain did not impair the tyrosine kinase activity. In vitro binding experiments revealed that phosphorylation at Tyr-615 in EphA8 emdiates the preferential binding to Fyn-SH2 domain rather than Sre and Ras GTPase-activating protein (Ras GAP-SH2 domains. Addtionally, a high level of EphA8 was detected in Fyn immunoprecipitates in intact cells, indicating that EphA8 and Fyn can physically, associate in vivo. In contrast, the association of full-length Fyn to EphA8 containing mutation at either Tyr-615 or Tyr 838 was greatly reduced. These data indicate that phosphorylation of Tyr-615 is critical for determining the association with Fyn whereas the integrity of Tyr-838 phosphorylation is required for efficient phosphorylation at Tyr-615 as well aw other major sites. Finally, it was observed that cell attachment responses are attenuated by overexpression of wild type EphA8 receptor but to much less extent by EphA8 mutants lacking phosphorylation at ixtent by EphA8 mutants lacking phosphorylation at either Tyr-615 or Tyr 838. furthermore, transient expression of kinase-inactive Fyn in EphA8-overexpressing cells blocked cell attachment responses attenuated by the EphA8 signaling. We therefore propose that Fyn kinase is one of the major downstream targets for the EphA8 signaling pathway leading to a modification of cell adhesion, and that autophosphorylation at Tyr-838 is critical for positively regulating the EphA8 signaling event.

Roles of Src-family kinase isoforms, Lyn, Fyn, Fgr, and c-Src on degranulation in RBL-2H3 mast cells

이준호,문세환,고나영,김지완,김도균,김주동,허억,최완수,Lee, Jun-Ho,Mun, Se-Hwan,Ko, Na-Young,Kim, Jie-Wan,Kim, Do-Kyun,Kim, Joo-Dong,Her, Erk,Choi, Wahn-Soo Korean Society of Life Science 2007 생명과학회지 Vol.17 No.3

흰쥐유래의 비만세포인 RBL-2H3 세포는 다양한 Src-family kinase를 발현한다. 현재까지의 연구결과에 의하면 비만세포의 초기 활성화에 Lyn kinase가 중요한 역할을 한다고 알려져 왔다. 그러나 그 세포에서 발현되는 다양한 다른 Src-family kinase의 역할은 불분명하다. 본 연구에서는 비만세포에서 다양한 Src-family kinase가 세포내 다른 곳에서 다양하게 발현되고 있다는 사실을 RT-PCR, immunoblotting 그리고 confocal microscopy 기법을 이용하여 증명하였다. 그 결과 Lyn 및 Fgr kinase는 세포막에 위치하고 c-Src 및 Yes kinase는 세포 내 과립에 존재하는 것을 알 수 있었다. 모든 Src-family kinase를 클로닝하고 과발현하여 탈과립 대한 영향을 평가하였다. 그 결과 fyn과 Fgr kinase는 비만세포에서 항원 유도의 탈과립을 증가시켰으며 반면 Lyn kinsae는 탈과립을 억제시키는 것을 확인할 수 있었다. 이러한 결과는 비만세포 초기 신호전달계에서 Fgr가 중요한 역할을 할 가능성을 제시한다. The rat RBL-2H3 mast cells contain various Src-family kinases. Previous reports with this cell line indicated that Lyn activation is an important initial signaling for the activation of the cells. However, the role and location of other Src-family kinase isoforms which are expressed in the cells are not clear. In this study, we now show that isoforms of Src-family kinases, Lyn, fyn, Fgr, c-Src, and Yes are differentially expressed and located differently in the cells as indicated by RT-PCR, immunoblotting analysis, and confocal microscopy. Lyn and Fgr were located on plasma membrane but on the other hand c-Src and Yes were located on intracellular organelle. All of Src-family kinases were cloned and overexpressed for investigating the roles of the isoforms. Overexpression of Fyn and Fgr, not Lyn and c-Src, stimulated Ag-induced degranulation in the cells. Our findings strongly suggest for the first time that each of Src-family kinase isoform can regulate differentially $Fc{\varepsilon}RI$-mediated signaling in RBL-2H3 mast cells.

Saucerneol F inhibits tumor necrosis factor- α and IL-6 production by suppressing Fyn-mediated pathways in FcεRI-mediated mast cells

( Yue Lu ),( Donggen Piao ),( Haiyan Zhang ),( Xian Li ),( Guang Hsuan Chao ),( Soon Jin Park ),( Young Chae Chang ),( Cheorl Ho Kim ),( Makoto Murakami ),( Seung Hyun Jung ),( Jung Hye Choi ),( Yong 영남대학교 약품개발연구소 2014 영남대학교 약품개발연구소 연구업적집 Vol.24 No.0

The aim of this study was to investigate the effect of saucerneol F (SF) on the productions of the pro-inflammatory cytokines, TNF-α and IL-6, in IgE/Ag-induced mouse bone marrow-derived mast cells (BMMCs). SF dose-dependently suppressed the transcriptions of these pro-inflammatory cytokines. To identify the molecular mechanisms responsible for these suppressions, we examined the effect of SF on three important transcription factors; activator protein-1 (AP-1), nuclear factor-κB (NF-κB), and STAT5. It was found that SF inhibited the nuclear translocation of the p65 subunit of NF-κB to the nucleus and its DNA-binding ability. SF also attenuated mitogen-activated protein kinase (MAPK)-mediated AP-1 activation and STAT5 activation. Biochemical analysis of FcεRI-mediated signaling pathways demonstrated that SF inhibited the phosphorylation of Fyn and multiple downstream signaling processes, including Syk, Gab2, and the Akt/IKK/IκB and MAPK pathways. Taken together, our results suggest that SF inhibits the production of pro-inflammatory cytokines by suppressing Fyn kinase-dependent signaling events.Crown Copyright ⓒ2013 Published by Elsevier Ltd. All rights reserved.

AMP-activated protein kinase negatively regulates FcεRI-mediated mast cell signaling and anaphylaxis in mice

Hwang, Seung-Lark,Li, Xian,Lu, Yue,Jin, Ye,Jeong, Yong-Tae,Kim, Yong Deuk,Lee, In-Kyu,Taketomi, Yoshitaka,Sato, Hiroyasu,Cho, You Sook,Murakami, Makoto,Chang, Hyeun Wook Elsevier 2013 The Journal of allergy and clinical immunology Vol.132 No.3

<P><B>Background</B></P> <P>Aggregation of FcεRI activates a cascade of signaling events leading to mast cell activation, followed by inhibitory signals that turn off the activating signals. However, the overall view of negative signals in mast cells is still incomplete. Although AMP-activated protein kinase (AMPK), which is generally known as a regulator of energy metabolism, is also associated with anti-inflammation, little is known about the role of AMPK in mast cells.</P> <P><B>Objectives</B></P> <P>We investigated the role of AMPK and its regulatory mechanism in mast cells.</P> <P><B>Method</B></P> <P>The roles of AMPK in FcεRI-dependent activation of bone marrow–derived mast cells (BMMCs) were evaluated by using chemical agents, small interfering RNAs (siRNAs), or adenovirus that modulated the activity or expression of AMPK signaling components. In addition, <I>AMPKα2</I> <SUP>−/−</SUP> mice were used to verify the role of AMPK in anaphylactic models.</P> <P><B>Results</B></P> <P>FcεRI signaling and associated effector functions in BMMCs were suppressed by the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) and were conversely augmented by siRNA knockdown of AMPKα2 or liver kinase B1 (LKB1), an upstream kinase of AMPK. Furthermore, <I>AMPKα2</I> deficiency led to increased FcεRI-mediated BMMC activation and anaphylaxis that were insensitive to AICAR, whereas enforced expression of AMPKα2 in <I>AMPKα2</I> <SUP>−/−</SUP> BMMCs reversed the hypersensitive FcεRI signaling to normal levels. Pharmacologic inhibition or siRNA knockdown of Fyn mimicked AMPK activation, suggesting that Fyn counterregulates the LKB1-AMPK axis. Mechanistically, Fyn controlled AMPK activity by regulating LKB1 localization.</P> <P><B>Conclusions</B></P> <P>The Fyn-regulated LKB1-AMPK axis acts as a novel inhibitory module for mast cell activation, which points to AMPK activators as therapeutic drugs for allergic diseases.</P>

Manassantin B Isolated from Saururus chinensis Inhibits Cyclooxygenase-2-Dependent Prostaglandin D2 Generation by Blocking Fyn-Mediated Nuclear Factor-kappaB and Mitogen Activated Protein Kinase Pathways in Bone Marrow Derived-Mast cells

( Yue Lu ),( Seung Lark Hwang ),( Jong Keun Son ),( Hyeun Wook Chang ) 영남대학교 약품개발연구소 2013 영남대학교 약품개발연구소 연구업적집 Vol.23 No.0

The authors investigated the effect of manassantin B (Man B) isolated from Saururus chinensis (S.chinensis) on cyclooxygenase-2 (COX-2)-dependent prostaglandin D2 (PGD2) generation in mouse bone marrow derived-mast cells (BMMCs). Man B inhibited the generation of PGD2 dose-dependently by inhibiting COX-2 expression in immunoglobulin E (IgE)/Ag-stimulated BMMCs. To elucidate the mechanism responsible for the inhibition of COX-2 expression by Man B, the effects of Man B on the activation of nuclear factor-kappaB (NF-κB), a transcription factor essential and mitogen-activated protein kinases (MAPKs) for COX-2 induction, were examined. Man B attenuated the nuclear translocation of NF-κB p65 and its DNA-binding activity by inhibiting inhibitors of kappa Bα (IκBα) degradation and concomitantly suppressing IκB kinase (IKK) phosphorylation. In addition, Man Bsuppressed phosphorylation of MAPKs including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK) and p38. It was also found that Man B suppressed Fyn kinase activation and consequent downstream signaling processes, including those involving Syk, Gab2, and Akt. Taken together, the present results suggest that Man B suppresses COX-2 dependent PGD2 generation by primarily inhibiting Fyn kinase in FcεRI-mediated mast cells.

AMP-activated protein kinase negatively regulates Fc□RI-mediated mast cell signaling and anaphylaxis in mice

( Seung Lark Hwang ),( Xian Li ),( Yue Lu ),( Ye Jin ),( Yong Tae Jeong ),( Yong Deuk Kim ),( In Kyu Lee ),( Yoshitaka Taketomi ),( Hiroyasu Sato ),( You Sook Cho ),( Makoto Murakami ),( Hyeun Wook Ch 영남대학교 약품개발연구소 2014 영남대학교 약품개발연구소 연구업적집 Vol.24 No.0

Background: Aggregation of FcεRI activates a cascade of signaling events leading to mast cell activation, followed by inhibitory signals that turn off the activating signals. However, the overall view of negative signals in mast cells is still incomplete. Although AMP-activated protein kinase (AMPK), which is generally known as a regulator of energy metabolism, is also associated with anti-inflammation, little is known about the role of AMPK in mast cells. Objectives: We investigated the role of AMPK and its regulatory mechanism in mast cells. Method: The roles of AMPK in FcεRI-dependent activation of bone marrow-derived mast cells (BMMCs) were evaluated by using chemical agents, small interfering RNAs (siRNAs), or adenovirus that modulated the activity or expression of AMPK signaling components. In addition, AMPKα2(-/-) mice were used to verify the role of AMPK in anaphylactic models. Results: FcεRI signaling and associated effector functions in BMMCs were suppressed by the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) and were conversely augmented by siRNA knockdown of AMPKα2 or liver kinase B1 (LKB1), an upstream kinase of AMPK. Furthermore, AMPKα2 deficiency led to increased FcεRI-mediated BMMC activation and anaphylaxis that were insensitive to AICAR, whereas enforced expression of AMPKα2 in AMPKα2(-/-) BMMCs reversed the hypersensitive FcεRI signaling to normal levels. Pharmacologic inhibition or siRNA knockdown of Fyn mimicked AMPK activation, suggesting that Fyn counterregulates the LKB1-AMPK axis. Mechanistically, Fyn controlled AMPK activity by regulating LKB1 localization. Conclusions: The Fyn-regulated LKB1-AMPK axis acts as a novel inhibitory module for mast cell activation, which points to AMPK activators as therapeutic drugs for allergic diseases. (J Allergy Chin Immunol 2013;132: 729-36)

Assessment of Relationship between Fyn-related Kinase Gene Polymorphisms and Overweight/Obesity in Korean Population

정미영,김범식,김윤정,고인송,정주호 대한약리학회 2008 The Korean Journal of Physiology & Pharmacology Vol.12 No.2

The fyn-related kinase (FRK) belongs to the tyrosine kinase family of protein kinases. Recent studies have shown that Frk affects pancreatic beta cell number during embryogenesis and promotes beta cell cytotoxic signals in response to streptozotocin. To investigate the genetic association between FRK polymorphisms and the risk of obesity in Korean population, single nucleotide polymorphisms (SNPs) in the FRK gene region were selected and analyzed. The body mass index (BMI) was calculated, and biochemical data (systolic blood pressure, diastolic blood pressure, hemoglobin A1C, triglyceride, total cholesterol, high density lipoprotein, and low density lipoprotein) of blood sample from each subject were also measured. One hundred fifty five healthy control and 204 overweight/obesity subjects were recruited. Genotype frequencies of six SNPs [rs6568920 (+8391G>A), rs3756772 (+56780A>G), rs3798234 (+75687C>T), rs9384970 (+68506G>A), rs1933739 (+72978G>A), and rs9400883 (+ 75809A>G)] in the FRK gene were determined by Affymetrix Targeted Genotyping Chip data. According to the classification of Korean Society for the Study of Obesity, control (BMI 18 to <23) and overweight/obesity (BMI≥23) subjects were recruited. For the analysis of genetic data, EM algorithm, SNPStats, Haploview, HapAnalyzer, SNPAnalyzer, and Helixtree programs were used. Multiple logistic regression analysis (codominant, dominant, and recessive models) was performed. Age and gender as covariates were adjusted. For biochemical data, Student's t test was used. The mean value of BMI in the control and overweigh/obesity groups was 21.1±1.2 (mean±SD) and 25.6±2.0, respectively. All biochemical data of the overweight/obesity group were statistically significance, compared with the control group. Among six SNPs, two linkage disequilibrium (LD) blocks were discovered. One block consisted of rs1933739 and rs9400883, and the other comprised rs3756772 and rs3798234. One SNP (rs9384970, +68506G>A) showed an association with overweight/obesity in the codominant model (p=0.03). Interestingly, the AA genotype distribution in the overweight/obesity group (n=7, 3.5%) was higher than those in the control group (n=1, 0.6%), which is not found in either Japanese or Chinese subjects. Therefore, the AA genotype of rs9384970 may be a risk factor for development of obesity in Korean population. The results suggest that FRK may be associated with overweight/obesity in Korean population. The fyn-related kinase (FRK) belongs to the tyrosine kinase family of protein kinases. Recent studies have shown that Frk affects pancreatic beta cell number during embryogenesis and promotes beta cell cytotoxic signals in response to streptozotocin. To investigate the genetic association between FRK polymorphisms and the risk of obesity in Korean population, single nucleotide polymorphisms (SNPs) in the FRK gene region were selected and analyzed. The body mass index (BMI) was calculated, and biochemical data (systolic blood pressure, diastolic blood pressure, hemoglobin A1C, triglyceride, total cholesterol, high density lipoprotein, and low density lipoprotein) of blood sample from each subject were also measured. One hundred fifty five healthy control and 204 overweight/obesity subjects were recruited. Genotype frequencies of six SNPs [rs6568920 (+8391G>A), rs3756772 (+56780A>G), rs3798234 (+75687C>T), rs9384970 (+68506G>A), rs1933739 (+72978G>A), and rs9400883 (+ 75809A>G)] in the FRK gene were determined by Affymetrix Targeted Genotyping Chip data. According to the classification of Korean Society for the Study of Obesity, control (BMI 18 to <23) and overweight/obesity (BMI≥23) subjects were recruited. For the analysis of genetic data, EM algorithm, SNPStats, Haploview, HapAnalyzer, SNPAnalyzer, and Helixtree programs were used. Multiple logistic regression analysis (codominant, dominant, and recessive models) was performed. Age and gender as covariates were adjusted. For biochemical data, Student's t test was used. The mean value of BMI in the control and overweigh/obesity groups was 21.1±1.2 (mean±SD) and 25.6±2.0, respectively. All biochemical data of the overweight/obesity group were statistically significance, compared with the control group. Among six SNPs, two linkage disequilibrium (LD) blocks were discovered. One block consisted of rs1933739 and rs9400883, and the other comprised rs3756772 and rs3798234. One SNP (rs9384970, +68506G>A) showed an association with overweight/obesity in the codominant model (p=0.03). Interestingly, the AA genotype distribution in the overweight/obesity group (n=7, 3.5%) was higher than those in the control group (n=1, 0.6%), which is not found in either Japanese or Chinese subjects. Therefore, the AA genotype of rs9384970 may be a risk factor for development of obesity in Korean population. The results suggest that FRK may be associated with overweight/obesity in Korean population.