Sphingosine kinase-dependent directional migration of leukocytes in response to phorbol ester

Syndecan-4 participates in focal adhesion by non-G protein-dependent activation of protein kinase C. Ligation of syndecan-4 with antithrombin elicits pertussis toxin-sensitive chemotaxis of leukocytes. As activation of protein kinase C stimulates release of sphingosine-1-phosphate, a chemoattracting G protein-coupled receptor agonist, we studied directional migration of leukocytes in response to phorbol myristate acetate (PMA), a direct activator of protein kinase C. Human peripheral blood neutrophils, monocytes, and lymphocytes were purified and tested for chemotactic migration in micropore filter assays in response to PMA. Dose-dependent stimulation of migration was seen only when leukocytes were exposed to concentration gradients of PMA; in the absence of such a gradient, inhibition of random migration was induced. Dimethylsphingosine inhibited PMA-induced leukocyte chemotaxis, indicating that activation of sphingosine kinase for enhanced production of sphingosine-1-phosphate mediates the chemotactic response to PMA. Pertussis toxin abrogated the chemotactic response to PMA, suggesting involvement of G protein-coupled sphingosine-1-phosphate receptor. Dimethylsphingosine also inhibited leukocyte chemotaxis toward antithrombin, indicating that similar mechanisms may be involved upon syndecan-4 ligation. Data show that protein kinase C-dependent activation of sphingosine kinase may play a central role in leukocyte chemotaxis toward non-G protein-coupled receptor agonists.     

Hydrogen peroxide induces Src family tyrosine kinase-dependent activation of Xenopus eggs

Fertilization is accompanied by a rapid and transient calcium release in eggs, which is required for the onset of zygotic developmental program or 'egg activation'. Recently, it was found that Src family tyrosine kinase (SFK)-dependent phospholipase C (PLC) activity is necessary for the calcium transience in fertilized Xenopus eggs. The present study demonstrates that hydrogen peroxide (H2O2) stimulates protein-tyrosine phosphorylation in Xenopus eggs, which occurs primarily in the egg cortex of the animal hemisphere as revealed by indirect immunofluorescence study. Egg SFK was found to be upregulated by H2O2 while the SFK-specific inhibitor PP1 effectively blocked H2O2-induced tyrosine phosphorylation. As in fertilized eggs, PLCgamma, but not Shc, was tyrosine-phosphorylated in H2O2-treated eggs. H2O2 also caused inositol 1,4,5-trisphosphate (IP3) production and sustained calcium release. After limited application of H2O2, elevated SFK activity and tyrosine phosphorylation were quickly reversed. Under such conditions, eggs showed cortical contraction and dephosphorylation of p42 MAP kinase, both of which are indicative of egg activation. These egg activation events, as well as H2O2-induced IP3 production and calcium release, were sensitive to PP1 and PLC inhibitor U-73122. Together, the present study demonstrated that H2O2 can mimic, at least in part, early events of Xenopus egg activation that require an SFK-dependent PLC pathway.     

Src和Abl酪氨酸蛋白激酶家族参与病原微生物感染的研究进展

Src和Abl家族激酶属于非受体型酪氨酸激酶(Nonreceptor tyrosine kinase,NRTK)家族重要成员,广泛存在于各种细胞中,参与细胞内信号传递并调节细胞生理过程,它们在维持细胞、组织和器官稳态功能中发挥着至关重要的作用。研究表明,Src和Abl家族激酶通过多种机制参与病原微生物的感染(如与病原微生物的脯氨酸基序-PXXP互作)。因此,从Src和Abl家族激酶角度出发探究病原微生物感染机制逐渐成为一个热点。本文就Src和Abl家族激酶的结构特点以及参与病原微生物感染的研究报道进行综述,以期为病原微生物感染的致病机制、防控和药物研发提供参考。     

Differential effects of tyrosine kinase inhibitors on volume-sensitive chloride current in human atrial myocytes: evidence for dual regulation by Src and EGFR kinases

To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (I(Cl.vol)) in human atrial myocytes and to identify the PTKs involved, we studied the effects of broad-spectrum and selective PTK inhibitors and the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (VO(4)(-3)). I(Cl.vol) evoked by hyposmotic bath solution (0.6-times isosmotic, 0.6T) was enhanced by genistein, a broad-spectrum PTK inhibitor, in a concentration-dependent manner (EC(50) = 22.4 microM); 100 microM genistein stimulated I(Cl.vol) by 122.4 +/- 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 150 microM) and tamoxifen (20 microM), blockers of I(Cl.vol). Moreover, the current augmented by genistein was volume dependent; it was abolished by hyperosmotic shrinkage in 1.4T, and genistein did not activate Cl(-) current in 1T. In contrast to the stimulatory effects of genistein, 100 microM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited I(Cl.vol) by 38.2 +/- 4.9% and 40.9 +/- 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter I(Cl.vol). In addition, the PTP inhibitor VO(4)(-3) (1 mM) reduced I(Cl.vol) by 53.5 +/- 4.5% (IC(50) = 249.6 microM). Pretreatment with VO(4)(-3) antagonized genistein-induced augmentation and A23- or A25-induced suppression of I(Cl.vol). Furthermore, the selective Src-family PTK inhibitor PP2 (5 microM) stimulated I(Cl.vol), mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 microM) reduced I(Cl.vol), mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO(4)(-3). The results suggest that I(Cl.vol) is regulated in part by the balance between PTK and PTP activity. Regulation is complex, however. Src and EGFR kinases, distinct soluble and receptor-mediated PTK families, have opposing effects on I(Cl.vol), and multiple target proteins are likely to be involved.     

Structural versatility that serves the function of the HRD motif in the catalytic loop of protein tyrosine kinase,Src

Site‐directed mutagenesis is a traditional approach for structure–function analysis of protein tyrosine kinases, and it requires the generation, expression, purification, and analysis of each mutant enzyme. In this study, we report a versatile high throughput bacterial screening system that can identify functional kinase mutants by immunological detection of tyrosine phosphorylation. Two key features of this screening system are noteworthy. First, instead of blotting bacterial colonies directly from Agar plates to nitrocellulose membrane, the colonies were cultured in 96‐well plates, and then spotted in duplicate onto the membrane with appropriate controls. This made the screening much more reliable compared with direct colony blotting transfer. A second feature is the parallel use of a protein tyrosine phosphatase (PTP)‐expressing host and a non‐PTP‐expressing host. Because high activity Src mutants are toxic to the host, the PTP system allowed the identification of Src mutants with high activity, while the non‐PTP system identified Src mutants with low activity. This approach was applied to Src mutant libraries randomized in the highly conserved HRD motif in the catalytic loop, and revealed that structurally diverse residues can replace the His and Arg residues, while the Asp residue is irreplaceable for catalytic activity.     

Transmission of growth cone traction force through apCAM-cytoskeletal linkages is regulated by Src family tyrosine kinase activity.

Tyrosine kinase activity is known to be important in neuronal growth cone guidance. However, underlying cellular mechanisms are largely unclear. Here, we report how Src family tyrosine kinase activity controls apCAM-mediated growth cone steering by regulating the transmission of traction forces through receptor-cytoskeletal linkages. Increased levels of tyrosine phosphorylation were detected at sites where beads coated with apCAM ligands were physically restrained to induce growth cone steering, but not at unrestrained bead binding sites. Interestingly, the rate and level of phosphotyrosine buildup near restrained beads were decreased by the myosin inhibitor 2,3-butanedione-2-monoxime, suggesting that tension promotes tyrosine kinase activation. While not affecting retrograde F-actin flow rates, genistein and the Src family selective tyrosine kinase inhibitors PP1 and PP2 strongly reduced the growth cone's ability to apply traction forces through apCAM-cytoskeletal linkages, assessed using the restrained bead interaction assay. Furthermore, increased levels of an activated Src family kinase were detected at restrained bead sites during growth cone steering events. Our results suggest a mechanism by which growth cones select pathways by sampling both the molecular nature of the substrate and its ability to withstand the application of traction forces.     

Src-Tyrosine kinases are major agents in mitochondrial tyrosine phosphorylation

Mitochondrial tyrosine phosphorylation is emerging as an important mechanism in regulating mitochondrial function. This article, aimed at identifying which kinases are the major agents in mitochondrial tyrosine phosphorylation, shows that this role should be attributed to Src family members. Indeed, various members of this family, for example, Fgr, Fyn, Lyn, c-Src, are constitutively present in the internal structure of mitochondria as well as Csk, a key enzyme in the regulation of the activity of this family. By means of different approaches, biochemical fractioning, Western blotting and immunogold analysis "in situ" of phosphotyrosine signaling, evidence is reported on the existence of a signal transduction pathway from plasma membrane to mitochondria, resulting in increasing Src-dependent mitochondrial tyrosine phosphorylation. The activation of Src kinases at mitochondrial level is associated with the proliferative status where several mitochondrial proteins are specifically tyrosine-phosphorylated.     

Involvement of Src family tyrosine kinase in apoptosis of human neutrophils induced by protozoan parasite Entamoeba histolytica

Tyrosine kinases are one of the most important regulators for intracellular signal transduction related to inflammatory responses. However, there are no reports describing the effects of tyrosine kinases on neutrophil apoptosis induced by Entamoeba histolytica. In this study, isolated human neutrophils from peripheral blood were incubated with live trophozoites in the presence or absence of tyrosine kinase inhibitors. Entamoeba-induced receptor shedding of CD16 and PS externalization in neutrophils were inhibited by pre-incubation of neutrophils with the broad-spectrum tyrosine kinase inhibitor genistein or the Src family kinase inhibitor PP2. Entamoeba-induced ROS production was also inhibited by genistein or PP2. Moreover, genistein and PP2 blocked the phosphorylation of ERK and p38 MAPK in neutrophils induced by E. histolytica. These results suggest that Src tyrosine kinases may participate in the signaling event for ROS-dependent activation of MAPKs during neutrophil apoptosis induced by E. histolytica.     

Novel N9-arenethenyl purines as potent dual Src/Abl tyrosine kinase inhibitors

Novel N⁹-arenethenyl purines, optimized potent dual Src/Abl tyrosine kinase inhibitors, are described. The key structural feature is a trans vinyl linkage at N⁹ on the purine core which projects hydrophobic substituents into the selectivity pocket at the rear of the ATP site. Their synthesis was achieved through a Horner–Wadsworth–Emmons reaction of N⁹-phosphorylmethylpurines and substituted benzaldehydes or Heck reactions between 9-vinyl purines and aryl halides. Most compounds are potent inhibitors of both Src and Abl kinase, and several possess good oral bioavailability.     

Src 在胃癌中的作用

Src是非受体型酪氨酸激酶家族成员之一,通过广泛的信号转导通路参与HP感染、胃癌细胞粘附、受体调节、增殖和血管形成等。近年来研究表明,Src在胃癌组织中异常表达或活性增高,与胃癌的发生发展密切相关。因此,研究Src和胃癌的关系有着重要意义。目前,针对Src的靶向抑制剂已经进入临床试验。大量研究发现,Src抑制剂包括Dasatinib、AZD0530、Sunitinib等对胃癌有抑制作用,这将为胃癌的治疗开辟新的治疗途径。     

Raf-1 kinase activation is uncoupled from downstream MEK/ERK pathway in cells treated with Src tyrosine kinase inhibitor PP2

Recently we demonstrated that PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, markedly enhanced Ras-independent activation of Raf-1 by the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H(2)O(2)). We report here that Raf-1 knockdown cells were significantly more sensitive to treatment of PP2 than control cells. This PP2-induced growth inhibition was found to be linked to decreased ERK and p38 activity. Interestingly, the growth of Sprouty knockdown cells appeared to be inhibited at earlier time points of PP2 treatment when compared with control cells. Unexpectedly, siRNA-mediated knockdown of Spry2, which is known to modulate the Ras/Raf/MAPK signal through feedback regulation, resulted in decreased Raf-1 kinase activity. PP2 had limited effect on the ability of PMA/H(2)O(2) to induce significant phosphorylation of MEK/ERK proteins in both Spry2 knockdown and control cells, indicating that PP2-mediated activation of Raf-1 did not potentiate signaling through the downstream MEK/ERK pathway. Taken together our results suggest that Raf-1 signaling may be bypassed in PP2-treated cells by uncoupling from downstream MEK/ERK pathway.     

Src protein and tyrosine-phosphorylated protein profiles in marrow stroma during osteogenic stimulation

Src protein is essential for the regulation of bone turnover primarily via bone resorption because it is required in osteoclast differentiation and function. We followed temporal changes of Src protein abundance in marrow stromal cells induced to mineralize by dexamethasone (DEX), growth in cold temperature, or both. Given the tyrosine kinase function of Src and its numerous substrates, profiles of phosphotyrosine-containing proteins were followed as well. On day 11 of stimulation, specific alkaline phosphatase (ALP) activity at 30°C decreased under DEX relative to 37°C cultures, in accord with increased cell counts. Mineralization per well under DEX increased by 25% at 37°C, whereas at 30°C it increased by more than threefold regardless of the DEX stimulation. At 30°C, on a per cell basis mineralization increased 2.5 and 3 times with and without DEX, respectively. Cultures at 37°C showed a general drop per cell of many phosphotyrosine-containing proteins on day 3 relative to days 1 and 2 in both DEX-stimulated and nonstimulated cultures; several proteins did recover (recuperate) thereafter. On days 1 and 2, the phosphotyrosine signal was higher in several proteins under DEX stimulation; this trend became inverted after day 3. The changes in abundance per cell of Src protein (pp60src) followed a similar trend, and in addition a truncated Src molecule, p54/52src, was detected as a putative cleavage product presumably representing its carboxy terminus. The pp60src was most abundant, relative to its truncated product, in day 7 nonstimulated cultures, whereas under DEX stimulation the truncated species pp54/52src showed the highest relative abundance on days 7. At 30°C, DEX stimulation accentuated the increase in Src protein on day 3, showed no change on day 7, and returned to increase Src protein on day 10. Potassium ionophorvalinomycin, considered to select against mineralizing osteoprogenitors at 30°C, showed on day 10 in the absence of DEX a relative increase in truncated Src protein compared to both DEX-stimulated and nonstimulated cultures in the absence of valinomycin. On day 7 of DEX stimulation, the presence of valinomycin resulted in low p54/52src. Among phosphotyrosine-containing proteins, a 32–34 kDa band, as yet unidentified, showed the most concordant changes with mineralization induction. P32–34 decreased by DEX on days 2 and 8 and increased by low temperature alone or combined with DEX on day 3. On day 7, p32–34 did not change under DEX, but valinomycin selected cells with less phoshpotyrosine-containing p32–34. Taken together, high Src abundance at the start of osteogenic induction followed by a decrease 1 week later is probably related to energy metabolism-dependent induction of mineralization. This is in temporal accord with the increase in Src truncation and fluctuation in mitochondrial membrane potential (which affects mineralization). The reported binding of amino-terminal Src oligopeptide to p32 ADP/ATP carrier in the mitochondrial inner membrane raises the question of its possible involvement in mitochondria-regulated mineralization. J. Cell. Biochem. 69:316–325, 1998. © 1998 Wiley-Liss, Inc.     

Novel regulation of Na, K-ATPase by Src tyrosine kinases in cortical neurons

The Na+, K+-ATPase or Na+, K+-pump plays a critical role in ion homeostasis and many cellular events. The Na+, K+-pump activity is regulated by serine/threonine phosphorylation, the role of tyrosine kinases in the regulation, however, is obscure. We now present novel evidence showing that tyrosine phosphorylation activates the Na+, K+-pump in cortical neurons. The electrogenic activity of the Na+, K+-pump was measured using whole-cell voltage clamp. A tonic activity was revealed by an inward current induced by the specific inhibitor ouabain or strophanthidin; an outward current due to activation of the pump was triggered by raising extracellular K+. The inward and outward currents were attenuated by the tyrosine kinase inhibitor genistein, herbimycin A, or lavendustin A, while blocking tyrosine phosphatases increased the pump current. Down-regulation of the pump current was also seen with the Src inhibitor PP1 and intracellularly applied anti-Lyn or anti-Yes antibody. Consistently, intracellular application of Lyn kinase up-regulated the pump current. Immunoprecipitation and western blotting showed tyrosine phosphorylation and a direct interaction between Lyn and the alpha3 subunit of the Na+, K+-pump. The tyrosine phosphorylation of the alpha3 subunit was reduced by serum deprivation. These data suggest that the Na+, K+-ATPase activity in central neurons is regulated by specific Src tyrosine kinases via a protein-protein mechanism and may play a role in apoptosis.     

A new strategy to produce active human Src from bacteria for biochemical study of its regulation

Enzymological studies of Src protein tyrosine kinase have been hindered by the lack of a suitable bacterial expression system. Poor expression of active Src appears to be due to toxicity associated with its kinase activity. To overcome this problem, we fused Src to a protein tyrosine phosphatase with an affinity tag and an appropriate thrombin cleavage site. Upon affinity purification of the fusion protein, Src was released by thrombin digestion and further purified by FPLC. This strategy has been used to produce several Src mutants that display catalytic and regulatory properties similar to those from eukaryotic expression systems. Characterization of the Src mutants confirmed that inactivation of Src by Csk through tail tyrosine phosphorylation required the Src SH3 domain.     

Click chemistry inspired one-pot synthesis of 1,4-disubstituted 1,2,3-triazoles and their Src kinase inhibitory activity

Two classes of 1,4-disubstituted 1,2,3-triazoles were synthesized using one-pot reaction of α-tosyloxy ketones/α-halo ketones, sodium azide, and terminal alkynes in the presence of aq PEG (1:1, v/v) using the click chemistry approach and evaluated for Src kinase inhibitory activity. Structure-activity relationship analysis demonstrated that insertion of C₆H₅- and 4-CH₃C₆H₄- at position 4 for both classes and less bulkier aromatic group at position 1 in class 1 contribute critically to the modest Src inhibition activity (IC₅₀ = 32-43 μM) of 1,4-disubstituted 1,2,3-triazoles.     

Salivary phospholipid secretion in response to beta-adrenergic stimulation is mediated by Src kinase-dependent epidermal growth factor receptor transactivation

Communication between receptor tyrosine kinase and G protein-coupled receptor (GPCR)-mediated signaling is recognized as a common integrator linking diverse aspects of intracellular signaling systems. Here, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation of salivary phospholipid release occurs with the involvement of epidermal growth factor receptor (EGFR). Using sublingual gland acinar cells, we show that prosecretory effect of isoproterenol on phospholipid release was subjected to suppression by EGFR kinase inhibitor, PD153035, and wortmannin, an inhibitor of PI3K, but not by PD98059, an inhibitor of extracellular signal regulated kinase (ERK). Furthermore, wortmannin, but not the ERK inhibitor, caused the reduction in the acinar cell secretory responses to beta-adrenergic agonist-generated cAMP as well as adenyl cyclase activator, forskolin. The acinar cell phospholipid secretory responses to isoproterenol, moreover, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation. Taken together, our data are the first to demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of salivary phospholipid secretion in response to beta-adrenergic GPCR activation.     

Src蛋白激酶活性的调节机制

Src蛋白激酶在人类多种肿瘤细胞中被激活并在肿瘤发生、发展过程中起重要作用.Src活性的调节主要包括共价修饰、异构调节,但基因突变和其他一些方式也可以调节其活性.Src共价修饰主要是磷酸化,Tyr530、Tyr419、Thr34、Thr46、Ser72、Tyr138和Tyr213等都是Src的磷酸化位点,其中Tyr530位点和Tyr419位点是Src最重要的磷酸化位点.异构调节包括SH3、SH2等区域结合的调节,分别涉及黏着斑激酶(focal adhesion kinase,FAK)、孕酮受体(progesterone receptor,PR)、雌激素受体(estrogen receptor,ER)、雄激素受体(androgen receptor,AR)、P130Cas、血小板源生长因子(the platelet-derived growth factor,PDGF)、血小板衍生生长因子受体(platelet-derived growth factor receptor,PDGFR)、表皮生长因子受体(epidermal growth factor receptor,EGFR,HER1/erb B1)、人类表皮生长因子受体2(human epidermal growth factor receptor-2,ERBB2/HER2/NEU)、胰岛素样生长因子1受体(insulin-like growth factor-1 receptor,IGF-1R)、纤维母细胞生长因子受体1(fibroblast growth factor receptor,FGFR1)、肝细胞生长因子受体(hepatocyte growth factor receptor c-Met)、人类1型T细胞白血病病毒编码的辅助蛋白p13、HIV-1毒力因子Nef和Sin.本文就Src蛋白激酶的调节机制作一简要综述.     

Heme controls the regulation of protein tyrosine kinases Jak2 and Src

Protein tyrosine kinases play key roles in many molecular and cellular processes in diverse living organisms. Their proper functioning is crucial for the normal growth, development, and health in humans, whereas their dysfunction can cause serious diseases, including various cancers. As such, intense studies have been performed to understand the molecular mechanisms by which the activities of protein tyrosine kinases are regulated in mammalian cells. Particularly, small molecules that can modulate the activity of tyrosine kinases are of great importance for discovering therapeutic drug candidates for numerous diseases. Notably, heme cannot only serve as a prosthetic group for hemoglobins and enzymes, but it also is a small signaling molecule that can control the activity of diverse signaling and regulatory proteins. Using a computational search, we found that a group of non-membrane spanning tyrosine kinases contains one or more CP motifs that can potentially bind to heme and mediate heme regulation. We then used experimental approaches to determine whether heme can affect the activity of any of these tyrosine kinases. We found that heme indeed affects the phosphorylation of key tyrosine residues in Jak2 and Src, and is therefore able to modulate Jak2 and Src activity. Further experiments showed that Jak2 and Src bind to heme and that the presence of heme alters the sensitivity of Jak2 and Src to trypsin digestion. These results suggest that heme actively interacts with Jak2 and Src and alters their conformation.     

Regulation of the Src Family Kinases by Csk

The non-receptor tyrosine kinase Csk serves as an indispensable negative regulator of the Src family tyrosine kinases (SFKs) by specifically phosphorylating the negative regulatory site of SFKs, thereby suppressing their oncogenic potential. Csk is primarily regulated through its SH2 domain, which is required for membrane translocation of Csk via binding to scaffold proteins such as Cbp/PAG1. The binding of scaffolds to the SH2 domain can also upregulate Csk kinase activity. These regulatory features have been elucidated by analyses of Csk structure at the atomic levels. Although Csk itself may not be mutated in human cancers, perturbation of the regulatory system consisting of Csk, Cbp/PAG1, or other scaffolds, and certain tyrosine phosphatases may explain the upregulation of SFKs frequently observed in human cancers. This review focuses on the molecular bases for the function, structure, and regulation of Csk as a unique regulatory tyrosine kinase for SFKs.