共查询到20条相似文献,搜索用时 15 毫秒
1.
Chong YP Chan AS Chan KC Williamson NA Lerner EC Smithgall TE Bjorge JD Fujita DJ Purcell AW Scholz G Mulhern TD Cheng HC 《The Journal of biological chemistry》2006,281(44):32988-32999
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. 相似文献
2.
Nef is an HIV-1 virulence factor that promotes viral pathogenicity by altering host cell signaling pathways. Nef binds several members of the Src kinase family, and these interactions have been implicated in the pathogenesis of HIV/AIDS. However, the direct effect of Nef interaction on Src family kinase (SFK) regulation and activity has not been systematically addressed. We explored this issue using Saccharomyces cerevisiae, a well defined model system for the study of SFK regulation. Previous studies have shown that ectopic expression of c-Src arrests yeast cell growth in a kinase-dependent manner. We expressed Fgr, Fyn, Hck, Lck, Lyn, and Yes as well as c-Src in yeast and found that each kinase was active and induced growth suppression. Co-expression of the negative regulatory kinase Csk suppressed SFK activity and reversed the growth-inhibitory effect. We then co-expressed each SFK with HIV-1 Nef in the presence of Csk. Nef strongly activated Hck, Lyn, and c-Src but did not detectably affect Fgr, Fyn, Lck, or Yes. Mutagenesis of the Nef PXXP motif essential for SH3 domain binding greatly reduced the effect of Nef on Hck, Lyn, and c-Src, suggesting that Nef activates these Src family members through allosteric displacement of intramolecular SH3-linker interactions. These data show that Nef selectively activates Hck, Lyn, and c-Src among SFKs, identifying these kinases as proximal effectors of Nef signaling and potential targets for anti-HIV drug discovery. 相似文献
3.
John J. Alvarado Laurie Betts Jamie A. Moroco Thomas E. Smithgall Joanne I. Yeh 《The Journal of biological chemistry》2010,285(46):35455-35461
Most mammalian cell types depend on multiple Src family kinases (SFKs) to regulate diverse signaling pathways. Strict control of SFK activity is essential for normal cellular function, and loss of kinase regulation contributes to several forms of cancer and other diseases. Previous x-ray crystal structures of the SFKs c-Src and Hck revealed that intramolecular association of their Src homology (SH) 3 domains and SH2 kinase linker regions has a key role in down-regulation of kinase activity. However, the amino acid sequence of the Hck linker represents a suboptimal ligand for the isolated SH3 domain, suggesting that it may form the polyproline type II helical conformation required for SH3 docking only in the context of the intact structure. To test this hypothesis directly, we determined the crystal structure of a truncated Hck protein consisting of the SH2 and SH3 domains plus the linker. Despite the absence of the kinase domain, the structures and relative orientations of the SH2 and SH3 domains in this shorter protein were very similar to those observed in near full-length, down-regulated Hck. However, the SH2 kinase linker adopted a modified topology and failed to engage the SH3 domain. This new structure supports the idea that these noncatalytic regions work together as a “conformational switch” that modulates kinase activity in a manner unique to the SH3 domain and linker topologies present in the intact Hck protein. Our results also provide fresh structural insight into the facile induction of Hck activity by HIV-1 Nef and other Hck SH3 domain binding proteins and implicate the existence of innate conformational states unique to individual Src family members that “fine-tune” their sensitivities to activation by SH3-based ligands. 相似文献
4.
Nasertorabi F Tars K Becherer K Kodandapani R Liljas L Vuori K Ely KR 《Journal of molecular recognition : JMR》2006,19(1):30-38
The docking protein p130Cas (Cas) becomes tyrosine-phosphorylated in its central substrate domain in response to extracellular stimuli such as integrin-mediated cell adhesion, and transmits signals through interactions with various intracellular signaling molecules such as the adaptor protein Crk. Src-family kinases (SFKs) bind a specific site in the carboxyl-terminal region of Cas and subsequently SFKs phosphorylate progressively the substrate domain in Cas. In this study crystallography, mutagenesis and binding assays were used to understand the molecular basis for Cas interactions with SFKs. Tyrosine phosphorylation regulates binding of Cas to SFKs, and the primary site for this phosphorylation, Y762, has been proposed. A phosphorylated peptide corresponding to Cas residues 759MEDpYDYVHL767 containing the key phosphotyrosine was crystallized in complex with the SH3-SH2 domain of the SFK Lck. The results provide the first structural data for this protein-protein interaction. The motif in Cas 762pYDYV binds to the SH2 domain in a mode that mimics high-affinity ligands, involving dual contacts of Y762 and V765 with conserved residues in SFK SH2 domains. In addition, Y764 is in position to make an electrostatic contact after phosphorylation with a conserved SFK arginine that mediates interactions with other high-affinity SH2 binders. These new molecular data suggest that Cas may regulate activity of Src as a competing ligand to displace intramolecular interactions that occur in SFKs (between the C-terminal tail and the SH2 domain) and restrain and down-regulate the kinase in an inactive form. 相似文献
5.
Artemin, one of the glial cell line-derived neurotrophic factor (GDNF) family, enhances the generation and survival of early sympathetic neurons and superior cervical ganglion (SCG) neurons. Src-family kinases (SFK) are involved in the growth and differentiation of cells, which are composed of unique Src homology 2 (SH2), Src homology 3 (SH3) and kinase domains. Various extra-cellular molecules containing growth factors and G-protein coupled receptors stimulate SFK. In this report, artemin is shown to have a significant effect on the neurite growth of dorsal root ganglia (DRG) neurons. Also, artemin triggers Src-family kinase activation and the phosphorylation of extra-cellular signal-regulated kinases (ERK) mitogen-activated protein kinase (MAPK). Artemin also regulated actin polymerization. There are several indications that another SH3-containing protein, Hck, and an SH3-containing adaptor protein, Nck1, play an important role in the organization of the actin cytoskeleton by cellular signalling. These findings suggest that the exploration of binding partners for the SH3 domain could provide an insight into regulation between the microtubule and actin networks. The binding partners for the SH3 domains of Nck, Src and Hck that we identified were Smc chromosome segregation ATPases, FOG Zn-finger protein and the FYVE zinc-binding domain, respectively. 相似文献
6.
Groveman BR Feng S Fang XQ Pflueger M Lin SX Bienkiewicz EA Yu X 《The FEBS journal》2012,279(1):20-28
Src family kinases (SFKs) play critical roles in the regulation of many cellular functions by growth factors, G-protein-coupled receptors and ligand-gated ion channels. Recent data have shown that SFKs serve as a convergent point of multiple signaling pathways regulating N-methyl-d-aspartate (NMDA) receptors in the central nervous system. Multiple SFK molecules, such as Src and Fyn, closely associate with their substrate, NMDA receptors, via indirect and direct binding mechanisms. The NMDA receptor is associated with an SFK signaling complex consisting of SFKs; the SFK-activating phosphatase, protein tyrosine phosphatase α; and the SFK-inactivating kinase, C-terminal Src kinase. Early studies have demonstrated that intramolecular interactions with the SH2 or SH3 domain lock SFKs in a closed conformation. Disruption of the interdomain interactions can induce the activation of SFKs with multiple signaling pathways involved in regulation of this process. The enzyme activity of SFKs appears 'graded', exhibiting different levels coinciding with activation states. It has also been proposed that the SH2 and SH3 domains may stimulate catalytic activity of protein tyrosine kinases, such as Abl. Recently, it has been found that the enzyme activity of neuronal Src protein is associated with its stability, and that the SH2 and SH3 domain interactions may act not only to constrain the activation of neuronal Src, but also to regulate the enzyme activity of active neuronal Src. Collectively, these findings demonstrate novel mechanisms underlying the regulation of SFKs. 相似文献
7.
Bcr-Abl is the constitutively active protein-tyrosine kinase expressed as a result of the Philadelphia translocation in chronic myelogenous leukemia. Bcr-Abl is coupled to many of the same signaling pathways normally regulated by hematopoietic cytokines. Recent work shows that Hck, a member of the Src tyrosine kinase family with myeloid-restricted expression, associates with and is activated by Bcr-Abl. Here we investigated the mechanism of Hck interaction with Bcr-Abl and the requirement for Hck activation in Bcr-Abl transformation signaling. Binding studies demonstrated that the Hck SH3 and SH2 domains are sufficient for interaction with Bcr-Abl in vitro. Hck binding localizes to the Abl SH2, SH3, and kinase domains as well as the distal portion of the C-terminal tail. To address the requirement for endogenous Src family kinase activation in Bcr-Abl signaling, a kinase-defective mutant of Hck was stably expressed in the cytokine-dependent myeloid leukemia cell line DAGM. Kinase-defective Hck dramatically suppressed Bcr-Abl-induced outgrowth of these cells in the absence of cytokine compared with a control cell line expressing beta-galactosidase. In contrast, kinase-defective Hck did not affect cell proliferation in response to interleukin-3, suggesting that the effect is specific for Bcr-Abl. These data show that Hck interacts with Bcr-Abl through a complex mechanism involving kinase-dependent and -independent components and that interaction with Hck or other Src family members is essential for transformation signaling by Bcr-Abl. 相似文献
8.
The role of Src-family protein tyrosine kinases (SFKs) in egg activation has been established, in large part, by the observation that GST fusion proteins encoding the SH2 domain of Src or Fyn suppress the sperm-induced calcium transient and cause polyspermy in marine invertebrate eggs. These fusion proteins are thought to act as dominant-negative inhibitors of SFK function; however, the mechanism by which they work is not known. The objective of the present study was to test the hypothesis that fusion proteins containing the above SH2 domains prevent activation of SFKs in response to fertilization. A single cell assay was developed that allows estimation of SFK activity in eggs injected with the GST-Fyn-SH2 fusion protein. The results demonstrate that the GST-Fyn-SH2 fusion protein prevents fertilization induced stimulation of SFK activity at concentrations that also suppress the sperm-induced calcium transient in zebrafish eggs. 相似文献
9.
10.
Chong YP Mulhern TD Zhu HJ Fujita DJ Bjorge JD Tantiongco JP Sotirellis N Lio DS Scholz G Cheng HC 《The Journal of biological chemistry》2004,279(20):20752-20766
Although C-terminal Src kinase (CSK)-homologous kinase (CHK) is generally believed to inactivate Src-family tyrosine kinases (SFKs) by phosphorylating their consensus C-terminal regulatory tyrosine (Tyr(T)), exactly how CHK inactivates SFKs is not fully understood. Herein, we report that in addition to phosphorylating Tyr(T), CHK can inhibit SFKs by a novel non-catalytic mechanism. First, CHK directly binds to the SFK members Hck, Lyn, and Src to form stable protein complexes. The complex formation is mediated by a non-catalytic Tyr(T)-independent mechanism because it occurs even in the absence of ATP or when Tyr(T) of Hck is replaced by phenylalanine. Second, the non-catalytic CHK-SFK interaction alone is sufficient to inactivate SFKs by inhibiting the catalytic activity of autophosphorylated SFKs. Third, CHK and Src co-localize to specific plasma membrane microdomains of rat brain cells, suggesting that CHK is in close proximity to Src such that it can effectively inactivate Src in vivo. Fourth, native CHK.Src complex exists in rat brain, and recombinant CHK.Hck complex exists in transfected HEK293T cells, implying that CHK forms stable complexes with SFKs in vivo. Taken together, our findings suggest that CHK inactivates SFKs (i) by phosphorylating their Tyr(T) and (ii) by this novel Tyr(T)-independent mechanism involving direct binding of CHK to SFKs. It has been documented that autophosphorylated SFKs can still be active, in some cases even when their Tyr(T) is phosphorylated. Thus, the ability of the Tyr(T)-independent mechanism to suppress the activity of both non-phosphorylated and autophosphorylated SFKs represents a fail-safe measure employed by CHK to down-regulate SFK signaling under all circumstances. 相似文献
11.
Ogawa A Takayama Y Sakai H Chong KT Takeuchi S Nakagawa A Nada S Okada M Tsukihara T 《The Journal of biological chemistry》2002,277(17):14351-14354
The carboxyl-terminal Src kinase (Csk) is an indispensable negative regulator for the Src family tyrosine kinases (SFKs) that play pivotal roles in various cell signalings. To understand the molecular basis of the Csk-mediated regulation of SFKs, we elucidated the crystal structure of full-length Csk. The Csk crystal consists of six molecules classified as active or inactive states according to the coordinations of catalytic residues. Csk assembles the SH2 and SH3 domains differently from inactive SFKs, and their binding pockets are oriented outward enabling the intermolecular interaction. In active molecules, the SH2-kinase and SH2-SH3 linkers are tightly stuck to the N-lobe of the kinase domain to stabilize the active conformation, and there is a direct linkage between the SH2 and the kinase domains. In inactive molecules, the SH2 domains are rotated destroying the linkage to the kinase domain. Cross-correlation matrices for the active molecules reveal that the SH2 domain and the N-lobe of the kinase domain move as a unit. These observations suggest that Csk can be regulated through coupling of the SH2 and kinase domains and that Csk provides a novel built-in activation mechanism for cytoplasmic tyrosine kinases. 相似文献
12.
Previous studies demonstrated that intra-domain interactions between Src family kinases (SFKs), stabilized by binding of the phosphorylated C-terminus to the SH2 domain and/or binding of the SH2 kinase linker to the SH3 domain, lock the molecules in a closed conformation, disrupt the kinase active site, and inactivate SFKs. Here we report that the up-regulation of N-methyl-D-aspartate receptors (NMDARs) induced by expression of constitutively active neuronal Src (n-Src), in which the C-terminus tyrosine is mutated to phenylalanine (n-Src/Y535F), is significantly reduced by dysfunctions of the SH2 and/or SH3 domains of the protein. Furthermore, we found that dysfunctions of SH2 and/or SH3 domains reduce auto-phosphorylation of the kinase activation loop, depress kinase activity, and decrease NMDAR phosphorylation. The SH2 domain plays a greater regulatory role than the SH3 domain. Our data also show that n-Src binds directly to the C-terminus of the NMDAR NR2A subunit in vitro, with a K(D) of 108.2 ± 13.3 nM. This binding is not Src kinase activity-dependent, and dysfunctions of the SH2 and/or SH3 domains do not significantly affect the binding. These data indicate that the SH2 and SH3 domains may function to promote the catalytic activity of active n-Src, which is important in the regulation of NMDAR functions. 相似文献
13.
Graham S. Baldwin Daisy Sio-Seng Lio Audrey Ferrand Bruno Catimel B. Philip Shehan Raymond S. Norton Heung-Chin Cheng 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(3):487-496
The Src-family tyrosine kinases (SFKs) are oncogenic enzymes that contribute to the initiation and progression of many types of cancer. In normal cells, SFKs are kept in an inactive state mainly by phosphorylation of a consensus regulatory tyrosine near the C-terminus (Tyr530 in the SFK c-Src). As recent data indicate that tyrosine modification enhances binding of metal ions, the hypothesis that SFKs might be regulated by metal ions was investigated. The c-Src C-terminal peptide bound two Fe3 + ions with affinities at pH 4.0 of 33 and 252 μM, and phosphorylation increased the affinities at least 10-fold to 1.4 and 23 μM, as measured by absorbance spectroscopy. The corresponding phosphorylated peptide from the SFK Lyn bound two Fe3 + ions with much higher affinities (1.2 pM and 160 nM) than the Src C-terminal peptide. Furthermore, when Lyn or Hck kinases, which had been stabilised in the inactive state by phosphorylation of the C-terminal regulatory tyrosine, were incubated with Fe3 + ions, a significant enhancement of kinase activity was observed. In contrast Lyn or Hck kinases in the unphosphorylated active state were significantly inhibited by Fe3 + ions. These results suggest that Fe3 + ions can regulate SFK activity by binding to the phosphorylated C-terminal regulatory tyrosine. 相似文献
14.
Jamie A. Moroco Jodi K. Craigo Roxana E. Iacob Thomas E. Wales John R. Engen Thomas E. Smithgall 《PloS one》2014,9(8)
Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo. 相似文献
15.
To examine the interactions between Src homology,domains and the tyrosine kinase catalytic domain of v-Src, various combinations of domains have been expressed in bacteria as fusion proteins. Constructs containing the isolated catalytic domain, SH2 + catalytic domain, and SH3 + SH2 + catalytic domains were active in autophosphorylation assays. For the catalytic domain of v-Src, but not for v-Abl, addition of exogenous Src SH3-SH2 domains stimulated the autophosphorylation activity. In contrast to results for autophosphorylation, constructs containing Src homology domains were more active towards a synthetic peptide substrate than the isolated catalytic domain. The ability of the SH2 and SH3 domains of v-Src to stabilize an active enzyme conformation was also confirmed by refolding after denaturation in guanidinium hydrochloride. Collectively the data suggest that, in addition to their roles in intermolecular protein-protein interactions, the Src homology regions of v-Src exert a positive influence on tyrosine kinase function, potentially by maintaining an active conformation of the catalytic domain. 相似文献
16.
José Manuel García-Martínez Annarica Calcabrini Lorena González Esther Martín-Forero María Teresa Agulló-Ortuño Valérie Simon Harriet Watkin Steve M. Anderson Serge Roche Jorge Martín-Pérez 《Cellular signalling》2010,22(3):415-426
The cytokine prolactin (PRL) plays important roles in the proliferation and differentiation of the mammary gland and it has been implicated in tumorigenesis. The prolactin receptor (PRLR) is devoid of catalytic activity and its mitogenic response is controlled by cytoplasmic tyrosine kinases of the Src (SFK) and Jak families. How PRLR uses these kinases for signaling is not well understood. Previous studies indicated that PRLR-induced Jak2 activation does not require SFK catalytic activity in favor of separate signaling operating on this cellular response. Here we show that, nevertheless, PRLR requires Src-SH2 and -SH3 domains for Jak2 signaling. In W53 lymphoid cells, conditional expression of two c-Src non-catalytic mutants, either SrcK295M/Y527F or Src?K, whose SH3 and SH2 domains are exposed, controls Jak2/Stat5 activation by recruiting Jak2, avoiding its activation by endogenous active SFK. In contrast, the kinase inactive SrcK295M mutant, with inaccessible SH3 and SH2 domains, does not. Furthermore, all three mutants attenuate PRLR-induced Akt and p70S6K activation. Accordingly, PRLR-induced Jak2/Stat5 signaling is inhibited in MCF7 breast cancer cells by Src depletion, expression of SrcK295M/Y527F or active Src harboring an inactive SH2 (SrcR175L) or SH3 domain (SrcW118A). Finally, Jak2/Stat5 pathway is also reduced in Src?/? mice mammary glands. We thus conclude that, in addition to Akt and p70S6K, SFK regulate PRLR-induced Jak2 signaling through a kinase-independent mechanism. 相似文献
17.
The crystal structures of three Src-family tyrosine kinases have been determined recently. The structure of the catalytic domain of Lck has been determined in the active autophosphorylated state. The structures of larger constructs of c-Src and Hck, containing the SH3, SH2 and catalytic domains, as well as a C-terminal regulatory tail, have been determined in the down-regulated state, phosphorylated in the C-terminal tail. A comparison of these structures leads to an unanticipated mechanism for the regulation of catalytic activity by cooperative interactions between the SH2, SH3 and catalytic domains. 相似文献
18.
ACK1 (activated Cdc42-associated kinase 1) is a nonreceptor tyrosine kinase and the only tyrosine kinase known to interact with Cdc42. To characterize the enzymatic properties of ACK, we have expressed and purified active ACK using the baculovirus/Sf9 cell system. This ACK1 construct contains (from N to C terminus) the kinase catalytic domain, SH3 domain, and Cdc42-binding Cdc42/Rac interactive binding (CRIB) domain. We characterized the substrate specificity of ACK1 using synthetic peptides, and we show that the specificity of the ACK1 catalytic domain most closely resembles that of Abl. Purified ACK1 undergoes autophosphorylation, and autophosphorylation enhances kinase activity. We identified Tyr284 in the activation loop of ACK1 as the primary autophosphorylation site using mass spectrometry. When expressed in COS-7 cells, the Y284F mutant ACK1 showed dramatically reduced levels of tyrosine phosphorylation. Although the SH3 and CRIB domains of purified ACK1 are able to bind ligands (a polyproline peptide and Cdc42, respectively), the addition of ligands did not stimulate tyrosine kinase activity. To characterize potential interacting partners for ACK1, we screened several SH2 and SH3 domains for their ability to bind to full-length ACK1 or to the catalytic-SH3-CRIB construct. ACK1 interacts most strongly with the SH3 domains of Src family kinases (Src or Hck) via its C-terminal proline-rich domain. Co-expression of Hck with kinase-inactive ACK1(K158R) in mammalian cells resulted in tyrosine phosphorylation of ACK1, suggesting that ACK1 is a substrate for Hck. Our data suggest that Hck is a novel binding partner for ACK1 that can regulate ACK1 activity by phosphorylation. 相似文献
19.
Mandal A Shahidullah M Beimgraben C Delamere NA 《Journal of cellular physiology》2011,226(10):2555-2561
Previous studies show Src family kinase (SFK) activation is involved in a response that stimulates Na,K-ATPase. Here, we tested whether SFK activation is involved in the Na,K-ATPase response to endothelin-1 (ET-1). Intact porcine lenses were exposed to 100 nM ET-1 for 5-30 min. Then, the epithelium was removed and used for Na,K-ATPase activity measurement and Western blot analysis of SFK activation. Na,K-ATPase activity was reduced by ~30% in lenses exposed to ET-1 for 15 min. The response was abolished by the SFK inhibitor PP2 or the ET receptor antagonist, PD145065. Activation of a ~61 kDa SFK was evident from an increase in Y416 phosphorylation, which reached a maximum at 15 min ET-1 treatment, and a decrease in Y527 phosphorylation. PP2 prevented SFK activation. Since Fyn, Src, Hck, and Yes may contribute to the observed 61 kDa band, these SFKs were isolated by immunoprecipitation and analyzed. Based on Y416 phosphorylation, ET-1 appeared to activate Fyn, while Src and Hck were inhibited and Yes was unaltered. ET-1 requires SFK activation to cause Na,K-ATPase inhibition. ET-1 elicits a different pattern of SFK activation from that reported earlier for purinergic agonists that stimulate Na,K-ATPase activity and activate Src. In the ET-1 response Src is inhibited and Fyn is activated. The findings suggest SFK phosphorylation is involved in a regulatory mechanism for Na,K-ATPase. Knowing this may help us understand drug actions on Na,K-ATPase. Faulty regulation of Na,K-ATPase in the lens could contribute to cataract formation since an abnormal sodium content is associated with lens opacification. 相似文献
20.
Nef is an HIV accessory protein required for high-titer viral replication and AIDS progression. Previous studies have shown that the SH3 domains of Hck and Lyn bind to Nef via proline-rich sequences in vitro, identifying these Src-related kinases as potential targets for Nef in vivo. Association of Nef with Hck causes displacement of the intramolecular interaction between the SH3 domain and the SH2-kinase linker, leading to kinase activation both in vitro and in vivo. In this study, we investigated whether interaction with Nef induces activation of other Src family kinases (Lyn, Fyn, Src, and Lck) following coexpression with Nef in Rat-2 fibroblasts. Coexpression with Nef induced Hck kinase activation and fibroblast transformation, consistent with previous results. In contrast, coexpression of Nef with Lyn was without effect, despite equivalent binding of Nef to full-length Lyn and Hck. Furthermore, Nef was found to suppress the kinase and transforming activities of Fyn, the SH3 domain of which exhibits low affinity for Nef. Coexpression with Nef did not alter c-Src or Lck tyrosine kinase or transforming activity in this system. Differential modulation of Src family members by Nef may produce unique downstream signals depending on the profile of Src kinases expressed in a given cell type. 相似文献