Lupus-like kidney disease in mice deficient in the Src family tyrosine kinases Lyn and Fyn

Systemic lupus erythematosus (SLE) is an autoimmune disease whose cause is poorly understood. Mice rendered deficient in specific genes have served as useful animal models in deciphering the genetic control of the disease [1]. We [2] and others [3, 4] previously demonstrated that mice deficient in the Src family tyrosine kinase Lyn developed a mild lupus-like disease with high survival rates. During the course of investigating the functional interaction of Src family kinases, we generated a mouse strain deficient in both Lyn and Fyn. The double-mutant mice died at relatively young ages and developed a severe lupus-like kidney disease. Unlike the double-mutant mice, single mutants deficient in either Lyn or Fyn lived longer and had distinct subsets of the symptoms found in the former. Lyn deficiency led to high levels of autoantibody production and glomerulonephritis, as previously reported [2--4], whereas loss of Fyn contributed to proteinuria by a B and T lymphocyte-independent mechanism. Our data suggest that the severe kidney disease in the double-mutant mice results from a combination of immunological and kidney-intrinsic defects. This new animal model may be informative about the causes of human SLE.     

Src protein tyrosine kinase family and acute inflammatory responses

Acute inflammatory responses are one of the major underlying mechanisms for tissue damage of multiple diseases, such as ischemia-reperfusion injury, sepsis, and acute lung injury. By use of cellular and molecular approaches and transgenic animals, Src protein tyrosine kinase (PTK) family members have been identified to be essential for the recruitment and activation of monocytes, macrophages, neutrophils, and other immune cells. Src PTKs also play a critical role in the regulation of vascular permeability and inflammatory responses in tissue cells. Importantly, animal studies have demonstrated that small chemical inhibitors for Src PTKs attenuate tissue injury and improve survival from a variety of pathological conditions related to acute inflammatory responses. Further investigation may lead to the clinical application of these inhibitors as drugs for ischemia-reperfusion injury (such as stroke and myocardial infarction), sepsis, acute lung injury, and multiple organ dysfunction syndrome.     

Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation.     

Induction of rac-guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) following phosphorylation by the nonreceptor tyrosine kinase Src

Ras-GRF1/CDC25(Mm) has been implicated as a Ras-guanine nucleotide exchange factor (GEF) expressed in brain. Ras-GEF activity of Ras-GRF1 is augmented in response to Ca(2+) influx and G protein betagamma subunit (Gbetagamma) stimulation. Ras-GRF1 also acts as a GEF toward Rac, but not Rho and Cdc42, when activated by Gbetagamma-mediated signals. Tyrosine phosphorylation of Ras-GRF1 is critical for the induction of Rac-GEF activity as evidenced by inhibition by tyrosine kinase inhibitors. Herein, we show that the nonreceptor tyrosine kinase Src phosphorylates Ras-GRF1, thereby inducing Rac-GEF activity. Ras-GRF1 transiently expressed with v-Src was tyrosine-phosphorylated and showed significant GEF activity toward Rac, but not Rho and Cdc42, which was comparable with that induced by Gbetagamma. In contrast, Ras-GEF activity remained unchanged. The recombinant c-Src protein phosphorylated affinity-purified glutathione S-transferase-tagged Ras-GRF1 in vitro and thereby elicited Rac-GEF activity. Taken together, tyrosine phosphorylation by Src is sufficient for the induction of Rac-GEF activity of Ras-GRF1, which may imply the involvement of Src downstream of Gbetagamma to regulate Ras-GRF1.     

Modulation of TRPV1 by nonreceptor tyrosine kinase, c-Src kinase

The capsaicin receptor TRPV1 is a nonselective cation channel that is expressed in sensory neurons. In this study, we examined the role of the nonreceptor cellular tyrosine kinase c-Src kinase in the modulation of the rat TRPV1. Capsaicin-induced currents in identified colonic dorsal root ganglion neurons were blocked by the c-Src kinase inhibitor PP2 and enhanced by the tyrosine phosphatase inhibitor sodium orthovandate. PP2 also abolished currents in human embryonic kidney-293 cells transfected with rat TRPV1, whereas cotransfection of TRPV1 with v-Src resulted in fivefold increase in capsaicin-induced currents. In cells transfected with dominant-negative c-Src and TRPV1, capsaicin-induced currents were decreased by approximately fourfold. TRPV1 co-immunoprecipitated with Src kinase and was tyrosine phosphorylated. These studies demonstrate that TRPV1 is a potential target for cellular tyrosine kinase-dependent phosphorylation.     

Endosomal trafficking of Src tyrosine kinase

Endosomal trafficking is an essential cellular process involved in the transport of proteins such as integrins, hormone receptors, growth factor receptors, receptor tyrosine kinases, and lipids (e.g. sphingomyelin). Regulation of this process is highly complex and involves Arf GAPs, SNAREs, Rab proteins, Rho GTPases and the actin cytoskeleton. In this article, we focus on the intracellular targeting of the Src family of non-receptor tyrosine kinases (nRTKs), and the role of endosomes in the delivery of nRTKs to the plasma membrane. Furthermore, we discuss the role of the actin cytoskeleton in this process and consider how endosome-regulated intracellular trafficking affects cell signalling.     

Hierarchical disabled-1 tyrosine phosphorylation in Src family kinase activation and neurite formation

There are two developmentally regulated alternatively spliced forms of Disabled-1 (Dab1) in the chick retina: an early form (Dab1-E) expressed in retinal precursor cells and a late form (Dab1-L) expressed in neuronal cells. The main difference between these two isoforms is the absence of two Src family kinase (SFK) recognition sites in Dab1-E. Both forms retain two Abl/Crk/Nck recognition sites implicated in the recruitment of SH2 domain-containing signaling proteins. One of the Dab1-L-specific SFK recognition sites, at tyrosine(Y)-198, has been shown to be phosphorylated in Reelin-stimulated neurons. Here, we use Reelin-expressing primary retinal cultures to investigate the role of the four Dab1 tyrosine phosphorylation sites on overall tyrosine phosphorylation, Dab1 phosphorylation, SFK activation and neurite formation. We show that Y198 is essential but not sufficient for maximal Dab1 phosphorylation, SFK activation and neurite formation, with Y232 and Y220 playing particularly important roles in SFK activation and neuritogenesis, and Y185 having modifying effects secondary to Y232 and Y220. Our data support a role for all four Dab1 tyrosine phosphorylation sites in mediating the spectrum of activities associated with Reelin-Dab1 signaling in neurons.     

Identification of PSD-93 as a substrate for the Src family tyrosine kinase Fyn

In order to study the role of tyrosine kinase signaling in the post-synaptic density (PSD), tyrosine-phosphorylated proteins associated with the PSD-95/NMDA receptor complex were analyzed. The NMDA receptor complex from the mouse brain was successfully solubilized with deoxycholate and immunopurified with anti-PSD-95 or anti-phosphotyrosine antibody. Immunoblot analyses revealed that the predominantly tyrosine-phosphorylated proteins in the NMDA receptor complex are the NR2A/B subunits and a novel 120 kDa protein. Purification and microsequencing analysis showed that the 120 kDa protein is mouse PSD-93/Chapsyn-110. Recombinant PSD-93 was phosphorylated by Fyn in vitro, and Tyr-384 was identified as a major phosphorylation site. Tyrosine phosphorylation of PSD-93 was greatly reduced in brain tissue from Fyn-deficient mice compared with wild-type mice. Furthermore, an N-terminal palmitoylation signal of PSD-93 was found to be essential for its anchoring to the membrane, where Fyn is also localized. In COS7 cells, exogenously expressed PSD-93 was phosphorylated, dependent on its membrane localization. In addition, tyrosine-phosphorylated PSD-93 was able to bind to Csk, a negative regulator of Src family kinases, in vitro as well as in a brain lysate. These results suggest that PSD-93 serves as a membrane-anchored substrate of Fyn and plays a role in the regulation of Fyn-mediated modification of NMDA receptor function.     

Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae

Recent work has demonstrated that p56lck, a member of the Src family of protein tyrosine kinases (PTKs), is modified by palmitoylation of a cysteine residue(s) within the first 10 amino acids of the protein (in addition to amino-terminal myristoylation that is a common modification of the Src family of PTKs). This is now extended to three other members of this family by showing incorporation of [3H]palmitate into p59fyn, p55fgr, and p56hck, but not into p60src. The [3H]palmitate was released by treatment with neutral hydroxylamine, indicating a thioester linkage to the protein. Individual replacement of the two cysteine residues within the first 10 amino acids of p59fyn and p56lck with serine indicated that Cys3 was the major determinant of palmitoylation, as well as association of the PTK with glycosyl-phosphatidylinositol- anchored proteins. Introduction of Cys3 into p60src led to its palmitoylation. p59fyn but not p60src partitioned into Triton-insoluble complexes that contain caveolae, microinvaginations of the plasma membrane. Mapping of the requirement for partitioning into caveolae demonstrated that the amino-terminal sequence Met-Gly-Cys is both necessary and sufficient within the context of a Src family PTK to confer localization into caveolae. Palmitoylation of this motif in p59fyn also modestly increased its overall avidity for membranes. These results highlight the role of the amino-terminal motif Met-Gly-Cys in determining the structure and properties of members of the Src family of PTKs.     

Structural basis of the auto-inhibition mechanism of nonreceptor tyrosine kinase PTK6

Protein tyrosine kinase 6 (PTK6) is composed of SH3, SH2, and Kinase domains, with a linker region (Linker) between the SH2 and Kinase domains. Here, we report the structural basis of the SH3-Linker interaction that results in auto-inhibition of PTK6. The solution structures of the SH3 domain and SH3/Linker complex were determined by NMR spectroscopy. The structure of the SH3 domain forms a conventional β-barrel with two β-sheets comprised of five β-strands. However, the molecular topology and charge distribution of PTK6-SH3 slightly differs from that of the other SH3 domains. The structure of the N-terminal Linker within the complex showed that the proline-rich region (P175-P187) of the Linker forms a compact hairpin structure through hydrophobic interactions. The structure of the SH3/Linker complex revealed intra-molecular interaction between the amino acid pairs R22/E190, W44/W184, N65/P177, and Y66/P179. Mutations in PTK6 at R22, W44, N65, and Y66 residues in the SH3 domain increased catalytic activity compared with wild-type protein, implying that specific interactions between hydrophobic residues in the proline-rich linker region and hydrophobic residues in the SH3 domain are mainly responsible for down-regulating the catalytic activity of PTK6.     

Regulation of the nonreceptor tyrosine kinase Brk by autophosphorylation and by autoinhibition

Brk (breast tumor kinase) is a nonreceptor tyrosine kinase that is most closely related to the Frk family of kinases, and more distantly to Src family kinases. Brk was originally identified in a screen for tyrosine kinases that are overexpressed in human metastatic breast tumors. To shed light on the activity and regulation of Brk and related tyrosine kinases, we expressed and purified Brk using the Sf9/baculovirus system. We characterized the substrate specificity of Brk using synthetic peptides, and we show that the kinetic parameters K(m) and k(cat) both play a role in specificity. We carried out mass spectrometry experiments to show that Brk autophosphorylates within the predicted kinase activation loop and at additional sites in the N terminus. Autophosphorylation increases enzyme activity of wild-type Brk but not of a Y342A mutant form of Brk. We also carried out experiments to address the possible involvement of the Src homology (SH) 2 and SH3 domains of Brk in enzyme regulation. Mutation of a C-terminal tyrosine (Tyr-447) increases enzyme activity and SH2 domain accessibility, consistent with a role for this residue in autoinhibition. A proline-rich peptide activates Brk, suggesting that the SH3 domain is also involved in maintaining an inactive form of Brk. These biochemical results for Brk may aid in the understanding of other tyrosine kinases in the Frk family.     

Src family kinase involvement in muscarinic receptor-induced tyrosine phosphorylation in differentiated SH-SY5Y cells

Muscarinic receptor-mediated changes in protein tyrosine phosphorylation were examined in differentiated human neuroblastoma SH-SY5Y cells. Treatment of differentiated cells with 1 mM carbachol caused rapid increases in the tyrosine phosphorylation of focal adhesion kinase (FAK), Cas, and paxillin. The src family kinase-selective inhibitor PP1 reduced carbachol-stimulated tyrosine phosphorylation of FAK, Cas, and paxillin by 50 to 75%. In contrast, carbachol-stimulated activation of ERK1/2 was unaffected by PP1. Src family kinase activation by carbachol was further demonstrated by increased carbachol-induced tyrosine phosphorylation of the src-substrate, p120, and tyrosine phosphorylation of the src family kinase activation-associated autophosphorylation site. Site-specific FAK phosphotyrosine antibodies were used to determine that the carbachol-stimulated increase in the autophosphorylation of FAK was unaffected by pretreatment with PP1, whereas the carbachol-stimulated increase in the src family kinase-mediated phosphotyrosine of FAK was completely blocked by pretreatment with PP1. In SH-SY5Y cell lines stably overexpressing Fyn, the phosphotyrosine immunoreactivity of FAK was 625% that of control cells. Thus, muscarinic receptors activate protein tyrosine phosphorylation in differentiated cells, and the tyrosine phosphorylation of FAK, Cas, and paxillin, but not ERK1/2, is mediated by a src family tyrosine kinase activated in response to stimulation of muscarinic receptors.     

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。     

Characterization of the expression of the ALK receptor tyrosine kinase in mice

Anaplastic Lymphoma Kinase (ALK), a receptor tyrosine kinase, was first described as the fusion product causing a subtype of non-Hodgkin's lymphoma. To date Alk has been reported to be mainly expressed in CNS and other parts of the brain. Here we describe an extensive characterization of the mRNA and protein expression of ALK during mouse development. We show that mRNA and ALK protein show overlapping expressing patterns in specific regions of the central and the peripheral nervous systems. Furthermore, ALK is also expressed in the eye, nasal epithelium, olfactory nerve, tongue, skin, tissue surrounding the esophagus, stomach and midgut but not the hindgut. Expression of ALK is also found in testis and ovary.     

C-terminal Src kinase-homologous kinase (CHK), a unique inhibitor inactivating multiple active conformations of Src family tyrosine kinases

The Src family of protein kinases (SFKs) mediates mitogenic signal transduction, and constitutive SFK activation is associated with tumorigenesis. To prevent constitutive SFK activation, the catalytic activity of SFKs in normal mammalian cells is suppressed mainly by two inhibitors called C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK), which inactivate SFKs by phosphorylating a consensus tyrosine near the C terminus of SFKs (Y(T)). The phosphorylated Y(T) intramolecularly binds to the SH2 domain of SFKs. This interaction, known as pY(T)/SH2 interaction, together with binding between the SH2 kinase linker and the SH3 domain of SFKs (linker/SH3 interaction) stabilizes SFKs in a "closed" inactive conformation. We previously discovered an alternative mechanism CHK employs to inhibit SFKs. This mechanism, referred to as the non-catalytic inhibitory mechanism, involves tight binding of CHK to SFKs; the binding alone is sufficient to inhibit SFKs. Herein, we constructed multiple active conformations of an SFK member, Hck, by systematically disrupting the two inhibitory interactions. We found that CHK employs the non-catalytic mechanism to inactivate these active conformations of Hck. However, CHK does not bind Hck when it adopts the inactive conformation in which both inhibitory interactions are intact. These data indicate that binding of CHK to SFKs via the non-catalytic mechanism is governed by the conformations of SFKs. Although CSK is also an inhibitor of SFKs, it does not inhibit SFKs by a similar non-catalytic mechanism. Thus, the non-catalytic inhibitory mechanism is a unique property of CHK that allows it to down-regulate multiple active conformations of SFKs.     

Cytokine-induced arginase activity in pulmonary endothelial cells is dependent on Src family tyrosine kinase activity

We hypothesized that the Src family tyrosine kinases (STKs) are involved in the upregulation of arginase and inducible nitric oxide synthase (iNOS) expression in response to inflammatory stimuli in pulmonary endothelial cells. Treatment of bovine pulmonary arterial endothelial cells (bPAEC) with lipopolysaccharide and tumor necrosis factor-alpha (L/T) resulted in increased urea and nitric oxide (NO) production, and this increase in urea and NO production was inhibited by the STK inhibitor PP1 (10 microM). The STK inhibitors PP2 (10 microM) and herbimycin A (10 microM) also prevented the L/T-induced expression of both arginase II and iNOS mRNA in bPAEC. Together, the data demonstrate a central role of STK in the upregulation of both arginase II and iNOS in bPAEC in response to L/T treatment. To identify the specific kinase(s) required for the induction of urea and NO production, we studied human pulmonary microvascular endothelial cells (hPMVEC) so that short interfering RNA (siRNA) techniques could be employed. We found that hPMVEC express Fyn, Yes, c-Src, Lyn, and Blk and that the protein expression of Fyn, Yes, c-Src, and Lyn could be inhibited with specific siRNA. The siRNA targeting Fyn prevented the cytokine-induced increase in urea and NO production, whereas siRNAs specifically targeting Yes, c-Src, and Lyn had no appreciable effect on cytokine-induced urea and NO production. These findings support our hypothesis that inflammatory stimuli lead to increased urea and NO production through a STK-mediated pathway. Furthermore, these results indicate that the STK Fyn plays a critical role in this process.     

Phosphorylation of cyclin dependent kinase 4 on tyrosine 17 is mediated by Src family kinases

Cyclin dependent kinase 4 is a key regulator of the cell cycle and its activity is frequently deregulated in cancer. The activity of cyclin dependent kinase 4 is controlled by multiple mechanisms, including phosphorylation of tyrosine 17. This site is equivalent to tyrosine 15 of cyclin dependent kinase 1, which undergoes inhibitory phosphorylation by WEE1 and MYT1; however, the kinases that phosphorylate cyclin dependent kinase 4 on tyrosine 17 are still unknown. In the present study, we generated a phosphospecific antibody to the tyrosine 17-phosphorylated form of cyclin dependent kinase 4, and showed that this site is phosphorylated to a low level in asynchronously proliferating HCT116 cells. We purified tyrosine 17 kinases from HeLa cells and found that the Src family non-receptor tyrosine kinase C-YES contributes a large fraction of the tyrosine 17 kinase activity in HeLa lysates. C-YES also phosphorylated cyclin dependent kinase 4 when transfected into HCT116 cells, and treatment of cells with Src family kinase inhibitors blocked the tyrosine 17 phosphorylation of cyclin dependent kinase 4. Taken together, the results obtained in the present study provide the first evidence that Src family kinases, but not WEE1 or MYT1, phosphorylate cyclin dependent kinase 4 on tyrosine 17, and help to resolve how the phosphorylation of this site is regulated.