Src激酶与脑血管病

Src家族激酶(Src family kinase,SFK)是最大的非受体酪氨酸激酶家族,在调控细胞形态、增殖、生长、黏附和运动方面发挥着关键作用.Src激酶的激活与细胞表面许多信号输入相耦联,包括生长因子、细胞因子、免疫细胞受体、G蛋白耦联受体、整合素以及其他细胞黏附分子.此外,作为连接细胞内和细胞外许多重要信号通路的膜结合开关分子,Src激酶在脑血管病的发生中也起着重要作用.     

Robust protein protein interactions in crowded cellular environments

The capacity of proteins to interact specifically with one another underlies our conceptual understanding of how living systems function. Systems-level study of specificity in protein-protein interactions is complicated by the fact that the cellular environment is crowded and heterogeneous; interaction pairs may exist at low relative concentrations and thus be presented with many more opportunities for promiscuous interactions compared with specific interaction possibilities. Here we address these questions by using a simple computational model that includes specifically designed interacting model proteins immersed in a mixture containing hundreds of different unrelated ones; all of them undergo simulated diffusion and interaction. We find that specific complexes are quite robust to interference from promiscuous interaction partners only in the range of temperatures T(design) > T > T(rand). At T > T(design), specific complexes become unstable, whereas at T < T(rand), formation of specific complexes is suppressed by promiscuous interactions. Specific interactions can form only if T(design) > T(rand). This condition requires an energy gap between binding energy in a specific complex and set of binding energies between randomly associating proteins, providing a general physical constraint on evolutionary selection or design of specific interacting protein interfaces. This work has implications for our understanding of how the protein repertoire functions and evolves within the context of cellular systems.     

Utilization of host SR protein kinases and RNA-splicing machinery during viral replication

Although the viral genome is often quite small, it encodes a broad series of proteins. The virus takes advantage of the host-RNA-processing machinery to provide the alternative splicing capability necessary for the expression of this proteomic diversity. Serine-arginine-rich (SR) proteins and the kinases that activate them are central to this alternative splicing machinery. In studies reported here, we use the HIV genome as a model. We show that HIV expression decreases overall SR protein/activity. However, we also show that HIV expression is significantly increased (20-fold) when one of the SR proteins, SRp75 is phosphorylated by SR protein kinase (SRPK)2. Thus, inhibitors of SRPK2 and perhaps of functionally related kinases, such as SRPK1, could be useful antiviral agents. Here, we develop this hypothesis and show that HIV expression down-regulates SR proteins in Flp-In293 cells, resulting in only low-level HIV expression in these cells. However, increasing SRPK2 function up-regulates HIV expression. In addition, we introduce SR protein phosphorylation inhibitor 340 (SRPIN340), which preferentially inhibits SRPK1 and SRPK2 and down-regulates SRp75. Although an isonicotinamide compound, SPRIN340 (or its derivatives) remain to be optimized for better specificity and lower cytotoxicity, we show here that SRPIN340 suppresses propagation of Sindbis virus in plaque assay and variably suppresses HIV production. Thus, we show that SRPK, a well known kinase in the cellular RNA-processing machinery, is used by at least some viruses for propagation and hence suggest that SRPIN340 or its derivatives may be useful for curbing viral diseases.     

Synthetic peptide fragment of src gene product inhibits the src protein kinase and crossreacts immunologically with avian onc kinases and cellular phosphoproteins.

All the known avian sarcoma viruses have associated protein kinase activities that phosphorylate tyrosine residues of their target proteins. A decapeptide fragment of pp60src of Rous sarcoma virus (RSV), residues 415-424, and an analog of that sequence have been chemically synthesized by solid-phase methods. The two decapeptides were not phosphorylated by pp60src of RSV, P90 of Y73 avian sarcoma virus, or P140 of Fujinami sarcoma virus. However, both peptides were able to inhibit competitively the kinase activities associated with the transforming proteins. Antiserum was raised against one of the peptides and IgG was purified from the serum by affinity chromatography. The antibody was able to precipitate pp60src of RSV as well as P90 of Y73 virus from cells infected with these viruses. The antibody also precipitated a number of high molecular weight phosphoproteins from normal chicken and rat fibroblasts and from several lines of virus-transformed cells.     

Identification and activation of Src family kinases in primary megakaryocytes

OBJECTIVES: We have recently shown that the Src family of tyrosine kinases (SFKs) are activated by TPO stimulation in both primary megakaryocytic progenitors and a hematopoietic cells line (BaF3) expressing the TPO receptor (Mpl). In this study, we examine which of the eight Src family members are expressed in primary megakaryocytes (MKs) and determine which of these become activated in response to TPO. MATERIALS AND METHODS: High-density oligonucleotide microarrays were used to compare the gene expression profiles of Src kinases from undifferentiated hematopoietic progenitors (CD34+/CD38(lo)) and after in vitro megakaryocytic differentiation. Western blot analysis of lysates from purified, mature murine MKs identified which of SFKs are present. Finally, in vitro kinase assays determined which of the SFKs in primary MKs are activated by TPO stimulation. RESULTS: Array profiles demonstrate that Fyn, Lyn, Fgr, Hck, Src, and Yes are all expressed in cultured human MKs (Fyn, Lyn>Src, Yes, Fgr, Hck). Similarly, Western blots of murine MKs identified the same six SFKs (Fyn, Fgr, Hck, Lyn, Src, and Yes). Of these, only Fyn and Lyn demonstrate increased kinase activity after TPO stimulation. Interestingly, gene expression analysis indicates that, among the SFKs, Fyn expression is uniquely upregulated during MK development. CONCLUSION: These results provide the first direct evidence that two Src kinases are activated in primary MKs, Fyn and Lyn. The fact that only Fyn expression is significantly upregulated during MK differentiation suggests variable gene regulation. Specificity of the TPO signaling cascade is demonstrated by the selective activation of Fyn and Lyn.     

Position of Src tyrosine kinases in the interaction between angiotensin II and endothelin in in vivo vascular protein synthesis

OBJECTIVES: Endothelin is a necessary intermediate in the trophic action of angiotensin II during hypertension-induced resistance artery remodeling in vivo. Since Src tyrosine kinases can be activated by both agonists, we studied their role in the trophic action of angiotensin II, endothelin and their interaction in rat small mesenteric arteries. METHODS AND RESULTS: Twenty-six hour infusion of high-dose angiotensin II (400 ng/kg per min) or endothelin (5 pmol/kg per min) via osmotic pumps significantly enhanced vascular protein synthesis in vivo. When angiotensin II was used as the trophic stimulus, treatment with a Src tyrosine kinase inhibitor (PP2, 0.5 mg/kg, starting at 21 h of the 26-h stimulation) produced a significant attenuation of extracellular regulated kinase 1 (ERK 1) phosphorylation and of protein synthesis. However, PP2 administered at 21 h or throughout the 26-h infusion did not abrogate the elevation of protein synthesis induced by endothelin. Moreover, endothelin did not enhance the phosphorylation of ERK 1/2 in small mesenteric arteries. We confirmed that angiotensin II stimulated the expression of prepro-endothelin mRNA in small mesenteric arteries in a Src-dependent manner, as the response was inhibited by PP2. To support the specific inhibitory activity of PP2 on Src tyrosine kinases in vivo, angiotensin II-induced phosphorylation of cortactin, a Src-specific substrate, was inhibited by PP2. CONCLUSION: Src tyrosine kinases represent an important signaling element in angiotensin II-induced endothelin production in small arteries in vivo. However, Src tyrosine kinases did not appear to contribute to the trophic signaling of endothelin, suggesting that they lie upstream of endothelin in the angiotensin II-endothelin-protein synthesis cascade.     

Signaling via Src family kinases is required for normal internalization of the receptor c-Kit.

Stem cell factor (SCF) exerts its biological effects by binding to a specific receptor, the tyrosine kinase c-Kit, which is expressed on the cell surface. Although normal cellular trafficking of growth factor receptors may play a critical role in the modulation of receptor function, the mechanisms that regulate the distribution of c-Kit on the cell surface and the internalization of c-Kit have not been fully defined. We investigated whether signal transduction via Src family kinases is required for normal c-Kit trafficking. Treatment of the SCF-responsive human hematopoietic cell line MO7e with the inhibitor of Src family kinases PP1 blocked SCF-induced capping of c-Kit and internalization of c-Kit. c-Kit was able to associate with clathrin in the presence of PP1, suggesting that entry of c-Kit into clathrin-coated pits occurs independently of Src family kinases. SCF-induced internalization of c-Kit was also diminished in the D33-3 lymphoid cell line in which expression of Lyn kinase was disrupted by homologous recombination. These results indicate that Src family kinases play a role in ligand-induced trafficking of c-Kit.     

tRNA-tRNA interactions within cellular ribosomes.

We describe an assay that converts the effects of tRNA-tRNA contacts at two particular codons into a quantitative effect on beta-galactosidase level. The assay measures the separate and combined efficiency of suppression at adjacent nonsense codons in vivo using a set of specially created homologous messages. In a survey of distal anticodon arm substitutions, we find that particular mutant tRNAs occupying the P-site reduce the apparent efficiency of the suppressor tRNA reading the A-site codon by factors of 2-170. By using measured tRNA-tRNA distances and the crystallographic tRNA structure, we propose a model of the tRNA-tRNA-mRNA complex. In the model, the anticodon loops of the P-site and A-site tRNAs contact one another in a way that is consistent with our combined tRNA efficiency data. These results suggest that tRNA-tRNA interactions that modulate tRNA action are an inevitable feature of translation.     

Inhibition of the Src and Jak kinases protects against lipopolysaccharide-induced acute lung injury

The cascade of cellular and molecular pathways mediating acute lung injury is complex and incompletely defined. Although the Src and Jak family of kinases is upregulated in LPS-induced murine lung injury, their role in the development of lung injury is unknown. Here we report that systemic inhibition of these kinases using specific small molecule inhibitors (PP2, SU6656, tyrphostin A1) significantly attenuated LPS-induced lung injury, as determined by histologic and capillary permeability assays. These inhibitors blocked LPS-dependent cytokine and chemokine production in the lung and in the serum. In contrast, lung-targeted inhibition of these kinases in the airway epithelium via adenoviral-mediated gene transfer of dominant negative Src or of suppressor of cytokine signaling (SOCS-1) disrupted lung cytokine production but had no effect on systemic cytokine production or lung vascular permeability. Mice were significantly protected from lethal LPS challenge by the small molecule inhibitors of Jak and Src kinase. Importantly, this protection was still evident even when the inhibitors were administered 6 hours after LPS challenge. Taken together, these observations suggest that Jak and Src kinases participate in acute lung injury and verify the potential of this class of selective tyrosine kinase inhibitors to serve as novel therapeutic agents for this disease.     

Clathrin-coated vesicles bearing GAIP possess GTPase-activating protein activity in vitro.

Galpha-interacting protein (GAIP) is a member of the RGS (regulators of G protein signaling) family, which serve as GAPs (GTPase-activating proteins) for Galpha subunits. Previously, we demonstrated that GAIP is localized on clathrin-coated vesicles (CCVs). Here, we tested whether GAIP-enriched vesicles could accelerate the GTPase activity of Galphai proteins. A rat liver fraction containing vesicular carriers (CV2) was enriched (4.5x) for GAIP by quantitative immunoblotting, and GAIP was detected on some of the vesicles in the CV2 fraction by immunoelectron microscopy. When liver fractions were added to recombinant Galphai3 and tested for GAP activity, only the CV2 fraction contained GAP activity. Increasing amounts of CV2 increased the activity, whereas immunodepletion of the CV2 fraction with an antibody against the C terminus of GAIP decreased GAP activity. CCV fractions were prepared from rat liver by using a protocol that maintains the clathrin coats. GAIP was enriched in these fractions and was detected on CCVs by immunogold labeling. Addition of increasing amounts of CCV to recombinant Galphai3 protein increased the GTPase activity. We conclude that CCVs possess GAP activity for Galphai3 and that membrane-associated GAIP is capable of interacting with Galphai3. The reconstitution of the interaction between a heterotrimeric G protein and GAIP on CCVs provides biochemical evidence for a model whereby the G protein and its GAP are compartmentalized on different membranes and come into contact at the time of vesicle fusion. Alternatively, they may be located on the same membrane and segregate at the time of vesicle budding.     

Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein.

Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes. Vesicle budding that results from the interaction of cytoplasmic coat proteins (coatomer and clathrin) with intracellular organelles requires a type of GTP-binding protein termed ADP-ribosylation factor (ARF). The GTPase cycle of ARF proteins that allows the uncoating and fusion of a transport vesicle with a target membrane is mediated by ARF-dependent GTPase-activating proteins (GAPs). A previously identified yeast protein, Gcs1, exhibits structural similarity to a mammalian protein with ARF-GAP activity in vitro. We show herein that the Gcs1 protein also has ARF-GAP activity in vitro using two yeast Arf proteins as substrates. Furthermore, Gcs1 function is needed for the efficient secretion of invertase, as expected for a component of vesicle transport. The in vivo role of Gcs1 as an ARF GAP is substantiated by genetic interactions between mutations in the ARF1/ARF2 redundant pair of yeast ARF genes and a gcs1-null mutation; cells lacking both Gcs1 and Arf1 proteins are markedly impaired for growth compared with cells missing either protein. Moreover, cells with decreased levels of Arf1 or Arf2 protein, and thus with decreased levels of GTP-Arf, are markedly inhibited for growth by increased GCS1 gene dosage, presumably because increased levels of Gcs1 GAP activity further decrease GTP-Arf levels. Thus by both in vitro and in vivo criteria, Gcs1 is a yeast ARF GAP.     

The Arf GTPase-activating protein ASAP1 regulates the actin cytoskeleton

Arf family GTP-binding proteins are best characterized as regulators of membrane traffic, but recent studies indicate an additional role in cytoskeletal organization. An Arf GTPase-activating protein of the centaurin beta family, ASAP1 (also known as centaurin beta4), binds Arf and two other known regulators of the actin cytoskeleton, the tyrosine kinase Src and phosphatidylinositol 4,5-bisphosphate. In this paper, we show that ASAP1 localizes to focal adhesions and cycles with focal adhesion proteins when cells are stimulated to move. Overexpression of ASAP1 altered the morphology of focal adhesions and blocked both cell spreading and formation of dorsal ruffles induced by platelet-derived growth factor (PDGF). On the other hand, ASAP1, with a mutation that disrupted GTPase-activating protein activity, had a reduced effect on cell spreading and increased the number of cells forming dorsal ruffles in response to PDGF. These data support a role for an Arf GTPase-activating protein, ASAP1, as a regulator of cytoskeletal remodeling and raise the possibility that the Arf pathway is a target for PDGF signaling.     

Ras GTPase-activating protein: a substrate and a potential binding protein of the protein-tyrosine kinase p56lck.

Ras GTPase-activating protein (GAP) is a cytoplasmic factor that regulates the GTPase activity of p21ras. Phosphorylation of GAP on tyrosine has recently been reported by several groups and may be an important step in linking signaling pathways involving p21ras and protein-tyrosine kinases. p56lck, a src-like protein-tyrosine kinase, seems to play a crucial role in T-cell development and T-cell activation. However, the molecular mechanisms of T-cell signaling involving p56lck and the substrates of p56lck have not yet been identified. To test whether GAP is a substrate of p56lck, in vitro kinase reactions were performed with purified, recombinant GAP and p56lck. We found that GAP became specifically phosphorylated on tyrosine within one tryptic peptide. Furthermore, coimmunoprecipitation studies provided evidence that the tyrosine-phosphorylated form of GAP is bound to p56lck. These results suggest that in T cells the function of GAP might be regulated through its phosphorylation on tyrosine and binding to the protein-tyrosine kinase p56lck.     

Rabbit ovarian protein kinases. III. Gonadotrophin-induced activation of soluble adenosine 3',5'-monophosphate-dependent protein kinases.

Cytosol of mature estrous rabbit follicles contains a single species of protein kinase, protein kinase 3, which can be classified as a type II cAMP-dependent protein kinase. Cytosol of functional rabbit corpora lutea (CL) contains, in addition to protein kinase 3, a second species of kinase activity, protein kinase 2, which can be classified as a type I cAMP-dependent protein kinase. These conclusions are based upon the relative dissociation and reassociation characteristics of the two holoenzymes in the presence and absence of 0.5 M NaCl after in vitro dissociation by cAMP, upon the effect of MgATP on salt- and basic protein-induced dissociation, and upon their relative elution from DEAE-cellulose. Protein kinase 3 in mature estrous rabbit follicles was rapidly activated after an iv injection of hCG. The activation was demonstrated by an increase of the protein kinase activity ratio as well as by the appearance of the free catalytic subunit of protein kinase upon Sephadex gel filtration. Maximal activation occurred within 10 min of in vivo hormone administration and required ovulatory doses of hormones with LH-like activity. Neither PRL, ACTH, epinephrine, nor a highly purified preparation of FSH promoted activation of the follicular protein kinase 3. Demonstration of protein kinase activation in follicles was achieved in the presence of 0.5 M NaCl in the homogenization media. After an iv injection of hCG, a partial activation of luteal protein kinases 2 and 3 was demonstrated, as reflected by the increase of the protein kinase activity ratio. These results implicate an important role for cAMP-dependent protein kinase 3 in LH action in rabbit ovarian follicles and for cAMP-dependent protein kinases 2 and 3 in LH action in rabbit CL.     

Src family kinase and adenosine differentially regulate multiple MAP kinases in ischemic myocardium: modulation of MAP kinases activation by ischemic preconditioning.

Recent studies suggest that ischemia activates Src and members of the mitogen-activated protein (MAP) kinase superfamily and their downstream effectors, including big MAP kinase 1 (BMK1) and p90 ribosomal S6 kinase (p90RSK). It has also been reported that adenosine is released during ischemia and involved in triggering the protective mechanism of ischemic preconditioning. To assess the roles of Src and adenosine in ischemia-induced MAP kinases activation, we utilized the Src inhibitor PP2 (4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and the adenosine receptor antagonist 8-(p-sulfophenyl) theophylline (SPT) in perfused guinea pig hearts. PP2 (1 microm) inhibited ischemia-induced Src, BMK1 and JNK activation but not JAK2 and p38 activation. SPT inhibited ischemia-mediated p38 and JNK activation. These results demonstrate that Src family kinase and adenosine regulate MAP kinases by parallel pathways. Preconditioning significantly improved both recovery of developed pressure and dp/dt in isolated guinea pig hearts. Since the protective effect of preconditioning was blocked by PP2 (1 microm) and SPT (50 microm), we next investigated the regulation of Src, MAP kinases and p90RSK during preconditioning. The activity and time course of ERK1/2 was not changed, but p90RSK activation by reperfusion was completely inhibited by preconditioning. In contrast, the activation by ischemia of Src, BMK1, p38 and JNK was significantly faster in preconditioned hearts. Maximal BMK1 activation by ischemia was also significantly enhanced by preconditioning. These data suggest important roles for Src family kinases and adenosine in mediating preconditioning, and suggest specific roles for individual MAP kinases in preconditioning.