Identification of tyrosine residues on ELMO1 that are phosphorylated by the Src-family kinase Hck

The SH3 and SH2 domains of hematopoietic cell kinase (Hck) play important roles in substrate targeting. To identify new components of Hck signaling pathways, we identified proteins that bind to the SH3 domain of Hck (Scott et al. (2002) J. Biol. Chem. 277, 28238). One such protein was ELMO1, the mammalian orthologue of the Caenorhabditis elegans gene, ced-12. ELMO1 is an approximately 80-kD protein containing a PH domain and a C-terminal Pro-rich sequence. In C. elegans, ced-12 is required for the engulfment of dying cells and for cell migration. In mammalian fibroblasts, ELMO1 binds to Dock180, and functions upstream of Rac during phagocytosis and cell migration. We previously showed that ELMO1 binds directly to the Hck SH3 domain and is phosphorylated by Hck. In this study, we used mass spectrometry to identify the following sites of ELMO1 phosphorylation: Tyr 18, Tyr 216, Tyr 511, Tyr 395, and Tyr 720. Mutant forms of ELMO1 lacking these sites were defective in their ability to promote phagocytosis and migration in fibroblasts. Single tyrosine mutations showed that Tyr 511 is particularly important in mediating these biological effects. These mutants displayed comparable binding to Dock180 and Crk as wild-type ELMO1, but gave a lowered activation of Rac. The data suggest that Src family kinase mediated tyrosine phosphorylation of ELMO1 might represent an important regulatory mechanism that controls signaling through the ELMO1/Crk/Dock180 pathway.     

Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts.

BACKGROUND: Fyn and c-Src are two of the most widely expressed Src-family kinases. Both are strongly implicated in the control of cytoskeletal organization and in the generation of integrin-dependent signalling responses in fibroblasts. These proteins are representative of a large family of tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well established, no corresponding phosphatases have been identified that, under physiological conditions, function as positive regulators of c-Src and Fyn in fibroblasts. RESULTS: Receptor protein tyrosine phosphatase alpha (RPTPalpha) was inactivated by homologous recombination. Fibroblasts derived from these RPTPalpha-/- mice had impaired tyrosine kinase activity of both c-Src and Fyn, and this was accompanied by a concomitant increase in c-Src Tyr527 phosphorylation. RPTPalpha-/- fibroblasts also showed a reduction in the rate of spreading on fibronectin substrates, a trait that is a phenocopy of the effect of inactivation of the c-src gene. In response to adhesion on a fibronectin substrate, RPTPalpha-/- fibroblasts also exhibited characteristic deficiencies in integrin-mediated signalling responses, such as decreased tyrosine phosphorylation of the c-Src substrates Fak and p 130(cas), and reduced activation of extracellular signal regulated (Erk) MAP kinases. CONCLUSIONS: These observations demonstrate that RPTPalpha functions as a physiological upstream activator of Src-family kinases in fibroblasts and establish this tyrosine phosphatase as a newly identified regulator of integrin signalling.     

Pertussis toxin activates tyrosine kinase signaling cascade in myelomonocytic cells: a mechanism for cell adhesion

Pertussis toxin (PTX) has recently been shown to specifically bind to CD14 to promote myelomonocytic cell adhesion to serum. The present study investigated the signaling mechanisms responsible for PTX-induced differentiated U937 cell adhesion. PTX-induced myelomonocytic cell adhesion was blocked by genistein or tyrphostin-47 (two protein tyrosine kinase inhibitors), LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor), or PD098059 (a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor). PTX induced a rapid tyrosine phosphorylation of several discrete cytoplasmic proteins, which could be inhibited by genistein or tyrphostin 47. In addition, PTX induced phosphorylation of Akt and of ERK2, which could be completely blocked by LY294002 and PD098059, respectively, and by genistein or tyrphostin 47 as well. All of these PTX-induced signaling events could be reproduced using purified PTX B-oligomer (PTX-B) alone. Our data show that PTX can activate tyrosine kinase signaling cascade, including the downstream PI3K and ERK/MAPK pathways, in myelomonocytic cells to induce cell adhesion to serum.     

Specific oncogenic activity of the Src-family tyrosine kinase c-Yes in colon carcinoma cells

c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of β-catenin at cell membranes and a reduction of expression of β-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in β-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src.     

CD19 amplifies B lymphocyte signal transduction by regulating Src-family protein tyrosine kinase activation.

Ligation of the B cell Ag receptor (BCR) induces cellular activation by stimulating Src-family protein tyrosine kinases (PTKs) to phosphorylate members of the BCR complex. Subsequently, Src-family PTKs, particularly Lyn, are proposed to phosphorylate and bind CD19, a cell-surface costimulatory molecule that regulates mature B cell activation. Herein, we show that B cells from CD19-deficient mice have diminished Lyn kinase activity and BCR phosphorylation following BCR ligation. Tyrosine phosphorylation of other Src-family PTKs was also decreased in CD19-deficient B cells. In wild-type B cells, CD19 was constitutively complexed with Vav, Lyn, and other Src-family PTKs, with CD19 phosphorylation and its associations with Lyn and Vav increased after BCR ligation. Constitutive CD19/Lyn/Vav complex signaling may therefore be responsible for the establishment of baseline signaling thresholds in B cells before Ag receptor ligation, in addition to accelerating signaling following BCR engagement or other transmembrane signals. In vitro kinase assays using purified CD19 and purified Lyn revealed that the kinase activity of Lyn was significantly increased when coincubated with CD19. Thus, constitutive and induced CD19/Lyn complexes are likely to regulate basal signaling thresholds and BCR signaling by amplifying the kinase activity of Lyn and other Src-family PTKs. These in vivo and in vitro findings demonstrate a novel mechanism by which CD19 regulates signal transduction in B lymphocytes. The absence of this CD19/Src-family kinase amplification loop may account for the hyporesponsive phenotype of CD19-deficient B cells.     

Pertussis toxin activates protein kinase C and a tyrosine protein kinase in the human T cell line Jurkat

Pertussis toxin activates T lymphocytes by a mechanism that is independent of its ADP-ribosylation activity. The toxin stimulates increases in diacylglycerol and intracellular calcium apparently by interacting with a cell surface receptor. Consistent with the production of these second messengers we have found that pertussis toxin activates protein kinase C in the Jurkat cell line. The toxin was also found to activate a tyrosine protein kinase in these cells in a manner similar to that observed with phytohemagglutinin. These results provide evidence that the mechanism of activation of T cells by pertussis toxin involves stimulating the activity of protein kinase C and a tyrosine protein kinase.     

Endogenous and synthetic inhibitors of the Src-family protein tyrosine kinases

Src-family kinases (SFKs) are protooncogenic enzymes controlling mammalian cell growth and proliferation. The activity of SFKs is primarily regulated by two tyrosine phosphorylation sites: autophosphorylation of a conserved tyrosine (Y(A)) in the kinase domain results in activation while phosphorylation of the regulatory tyrosine (Y(T)) near the C-terminus leads to inactivation. The phosphorylated Y(T) (pY(T)) engages in intramolecular interactions that stabilise the inactive conformation of SFKs. These inhibitory intramolecular interactions include the binding of pY(T) to the SH2 domain and the binding of the SH2-kinase linker to the SH3 domain. Thus, SFKs are active upon (i) disruption of the inhibitory intramolecular interactions, (ii) autophosphorylation of Y(A) and/or (iii) dephosphorylation of pY(T). Since aberrant activation of SFKs contributes to cancer, SFKs in normal cells are kept inactive by multiple endogenous inhibitors classified as catalytic and non-catalytic inhibitors. The catalytic inhibitors include C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) that phosphorylate Y(T) of SFKs, as well as the protein tyrosine phosphatases that dephosphorylate pY(A) of the activated SFKs. The non-catalytic inhibitors inactivate SFKs by direct binding. CHK is unique among these inhibitors because it employs both catalytic and non-catalytic mechanisms to inhibit SFKs. Other known non-catalytic inhibitors include WASP, caveolin and RACK1, which function to down-regulate SFKs in specific subcellular locations. This review discusses how the various endogenous SFK inhibitors cooperate to regulate SFKs in normal cells. As chemical compounds that can selectively inhibit SFKs in vivo are potential anti-cancer therapeutics, this review also discusses how investigation into the inhibitory mechanisms of the endogenous inhibitors will benefit the design and screening of these compounds.     

The tyrosine kinase Hck is an inhibitor of HIV-1 replication counteracted by the viral vif protein

The virus infectivity factor (Vif) protein facilitates the replication of human immunodeficiency virus type 1 (HIV-1) in primary lymphocytes and macrophages. Its action is strongly dependent on the cellular environment, and it has been proposed that the Vif protein counteracts cellular activities that would otherwise limit HIV-1 replication. Using a glutathione S-transferase pull-down assay, we identified that Vif binds specifically to the Src homology 3 domain of Hck, a tyrosine kinase from the Src family. The interaction between Vif and the full-length Hck was further assessed by co-precipitation assays in vitro and in human cells. The Vif protein repressed the kinase activity of Hck and was not itself a substrate for Hck phosphorylation. Within one single replication cycle of HIV-1, Hck was able to inhibit the production and the infectivity of vif-deleted virus but not that of wild-type virus. Accordingly, HIV-1 vif- replication was delayed in Jurkat T cell clones stably expressing Hck. Our data demonstrate that Hck controls negatively HIV-1 replication and that this inhibition is suppressed by the expression of Vif. Hck, which is present in monocyte-macrophage cells, represents the first identified cellular inhibitor of HIV-1 replication overcome by Vif.     

Crystal structure of Hck in complex with a Src family-selective tyrosine kinase inhibitor.

The crystal structure of the autoinhibited form of Hck has been determined at 2.0 A resolution, in complex with a specific pyrazolo pyrimidine-type inhibitor, PP1. The activation segment, a key regulatory component of the catalytic domain, is unphosphorylated and is visualized in its entirety. Tyr-416, the site of activating autophosphorylation in the Src family kinases, is positioned such that access to the catalytic machinery is blocked. PP1 is bound at the ATP-binding site of the kinase, and a methylphenyl group on PP1 is inserted into an adjacent hydrophobic pocket. The enlargement of this pocket in autoinhibited Src kinases suggests a route toward the development of inhibitors that are specific for the inactive forms of these proteins.     

Shear stress activates Tie2 receptor tyrosine kinase in human endothelial cells

The receptor tyrosine kinase (RTK) Tie2 is expressed predominantly on endothelial cells. Tie2 is critical for vasculogenesis during development and could be important for maintaining endothelial cell survival and integrity in adult blood vessels. Although most RTKs are activated by shear stress in the absence of ligand activation, the effect of shear stress on Tie2 is unknown. Therefore, we examined the effect of shear stress on Tie2 phosphorylation in primary cultured endothelial cells. Interestingly, shear stress (20 dyne/cm(2)) produced a rapid, marked, and sustained Tie2 phosphorylation, while it produced a rapid but slight and transient phosphorylation of insulin receptor and VEGF receptor 2 (Flk1). In addition, Tie2 phosphorylation in response to shear stress was velocity-dependent, while phosphorylation of insulin receptor and Flk1 was not. Shear stress also produced Akt phosphorylation in a time-, velocity-, and PI 3-kinase-dependent manner. Accordingly, shear stress suppressed serum deprivation-induced endothelial cell apoptosis. Taken together, our results indicated that activation of Tie2/PI 3-kinase/Akt in response to shear stress could be an important signaling cascade for maintaining endothelial survival and integrity in blood vessels.     

Growth signal erythropoietin activates the same tyrosine kinases as interleukin 3, but activates only one tyrosine kinase as differentiation signal.

By Western blotting with anti-phosphotyrosine-specific antibody, we demonstrated that both erythropoietin (Epo) and interleukin 3 (IL3) induce rapid and transient tyrosine phosphorylation of a common set of proteins of 45, 55, 69, 87, 90, 95 and 160 KDa as a growth signal in Epo- and IL3-dependent FD-M6 cells. In contrast, only two proteins of 87 and 90 KDa were transiently phosphorylated in Epo-induced erythroid differentiation of SKT6 cells. Furthermore, no tyrosine phosphorylation was observed in dimethyl sulfoxide-induced differentiation of SKT6 cells. Taken together with other observations, these results indicate that Epo, IL3 and GM-CSF activate the same tyrosine protein kinases as growth signal and that Epo-induced differentiation signal uses only a part of the tyrosine kinase pathway.     

Gambogic acid activates AMP-activated protein kinase in mammalian cells

AMP-activated protein kinase (AMPK) plays a key role in maintaining intracellular and whole-body energy homeostasis. Activation of AMPK has been shown to ameliorate the symptoms of metabolic diseases, such as type 2 diabetes and obesity. Here we show that gambogic acid (GB), a known antitumor agent, activates AMPK by increasing the phosphorylation of AMPKα and its downstream substrate ACC in various cell lines. Further study revealed that GB stimulated AMPK activity independent of upstream kinases. Moreover, the AMPK inhibitor, compound C, has no effects on the GB-induced AMPK activation. We also found that GB promptly increased intracellular ROS level, and antioxidants attenuated the ROS production. Interestingly, only the thiol antioxidants significantly abolished GB-enhanced AMPK activation. In addition, analysis of binding and dissociation kinetics indicated that GB bound to the AMPKα subunit. Collectively, these results suggest that GB may be a novel direct activator of AMPK.     

Association of Src-family protein tyrosine kinases with sphingolipids in rat cerebellar granule cells differentiated in culture

Src family kinases play a relevant role in the development and differentiation of neuronal cells. They are abundant in sphingolipid-enriched membrane domains of many cell types, and these domains are hypothesized to function in bringing together molecules important to signal transduction. We studied the association of Src family tyrosine kinases and their negative regulatory kinase, Csk, with sphingolipids in sphingolipid-enriched domains of rat cerebellar granule cells differentiated in culture. We find that c-Src, Lyn and Csk are enriched in the sphingolipid-enriched fraction prepared from these cells. Coimmunoprecipitation experiments show that these and sphingolipids are part of the same domain. Cross-linking experiments with a photoactivable, radioactive GD1b derivative show that c-Src and Lyn, which are anchored to the membrane via a myristoyl chain, associate directly with GD1b. Csk, which is not inserted in the hydrophobic core of the membrane, is not photolabeled by this ganglioside. These results suggest that lipid-lipid, lipid-protein, and protein-protein interactions cooperate to maintain domain structure. We hypothesize that such interactions might play a role in the process of neuronal differentiation.     

Association of Src-family protein tyrosine kinases with sphingolipids in rat cerebellar granule cells differentiated in culture

Src family kinases play a relevant role in the development and differentiation of neuronal cells. They are abundant in sphingolipid-enriched membrane domains of many cell types, and these domains are hypothesized to function in bringing together molecules important to signal transduction. We studied the association of Src family tyrosine kinases and their negative regulatory kinase, Csk, with sphingolipids in sphingolipid-enriched domains of rat cerebellar granule cells differentiated in culture. We find that c-Src, Lyn and Csk are enriched in the sphingolipid-enriched fraction prepared from these cells. Coimmunoprecipitation experiments show that these and sphingolipids are part of the same domain. Cross-linking experiments with a photoactivable, radioactive GD1b derivative show that c-Src and Lyn, which are anchored to the membrane via a myristoyl chain, associate directly with GD1b. Csk, which is not inserted in the hydrophobic core of the membrane, is not photolabeled by this ganglioside. These results suggest that lipid–lipid, lipid–protein, and protein–protein interactions cooperate to maintain domain structure. We hypothesize that such interactions might play a role in the process of neuronal differentiation.     

Pervanadate-induced reverse translocation and tyrosine phosphorylation of phorbol ester-stimulated protein kinase C betaII are mediated by Src-family tyrosine kinases in porcine neutrophils

Protein kinase C (PKC), upon activation, translocates from the cytosol to the plasma membrane. Phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, is known to induce irreversible translocation of PKC to the plasma membrane, in contrast to the reversible translocation resulting from physiological stimuli and subsequent rapid return to the cytosol (reverse translocation). However, we have previously shown that tyrosine phosphatase (PTPase) inhibitors induce reverse translocation of PMA-stimulated PKCbetaII in porcine polymorphonuclear leukocytes (PMNs). In the present study, we showed that pervanadate, a potent PTPase inhibitor, also induces tyrosine phosphorylation of PMA-stimulated PKCbetaII in porcine PMNs. Furthermore, PP2, a specific inhibitor of Src-family tyrosine kinases (PTKs), was found to inhibit both pervanadate-induced reverse translocation and tyrosine phosphorylation of PMA-stimulated PKCbetaII, suggesting that these two pervanadate-induced responses are mediated by Src-family PTKs. Our findings provide novel insight into the relationship between the subcellular localization and tyrosine phosphorylation of PKC.     

Prostaglandin E1 activates a chloride current in Jurkat T lymphocytes via cAMP-dependent protein kinase.

Patch-clamp studies have identified a cAMP-dependent Cl- conductance in lymphocytes that is defectively regulated in cystic fibrosis. In this study we used 125I efflux and whole-cell patch-clamp studies to investigate whether prostaglandin E1 (PGE1), an agonist that generates intracellular cAMP in Jurkat T lymphocytes, activates a Cl- conductance. Stimulation of T cells by externally applied PGE1 stimulated 125I efflux and activated a slowly developing membrane current. When external and internal Cl- were about equal, the current reversed at about zero mV, but when external Cl- was lowered from 157 to 7 mM the reversal potential shifted 75 mV in the positive direction, demonstrating that the current carrier was Cl-. In addition, the current was blocked by 10 microM 5-nitro-2(3-phenylpropylamino) benzoic acid (NPPB), a potent Cl- channel blocker. A membrane-permeable cAMP analog mimicked the effect of PGE1, whereas intracellular application of a cAMP antagonist Rp-cAMP blocked the effect of PGE1. Addition of purified catalytic subunit of cAMP-dependent protein kinase (PKA) plus ATP to the recording pipette also activated a similar current, whereas internally applied Walsh inhibitor, the synthetic peptide inhibitor of PKA, blocked the PGE1 effect. These results suggest that PGE1, acting through PKA, activates a Cl- current in Jurkat T cells.     

Complement activates the c-Jun N-terminal kinase/stress-activated protein kinase in glomerular epithelial cells

In the rat passive Heymann nephritis model of membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 activates cytosolic phospholipase A(2) (cPLA(2)), and products of cPLA(2)-mediated phospholipid hydrolysis modulate GEC injury and proteinuria. In the present study, we demonstrate that C5b-9 activates c-Jun N-terminal kinase (JNK) in cultured rat GECs and that JNK activity is increased in glomeruli isolated from proteinuric rats with passive Heymann nephritis, as compared with control rats. Stable overexpression of cPLA(2) in GECs amplified complement-induced release of arachidonic acid (AA) and JNK activity, as compared with neo (control) GECs. Activation of JNK was not affected by indomethacin. Incubation of GECs with complement stimulated production of superoxide, and pretreatment with the antioxidants, N-acetylcysteine, glutathione, and alpha-tocopherol as well as with diphenylene iodonium, an inhibitor of the NADPH oxidase, inhibited complement-induced JNK activation. Conversely, H(2)O(2) activated JNK, whereas exogenously added AA stimulated both superoxide production and JNK activity. Overexpression of a dominant-inhibitory JNK mutant or treatment with diphenylene iodonium exacerbated complement-dependent GEC injury. Thus, activation of cPLA(2) and release of AA facilitate complement-induced JNK activation. AA may activate the NADPH oxidase, leading to production of reactive oxygen species, which in turn mediate the activation of JNK. The functional role of JNK activation is to limit or protect GECs from complement attack.     

Mercuric chloride induced membrane damage in tomato cultured cells

Suspension cultures of tomato cells were used to study the membrane injury by the toxic concentration of mercuric chloride. Assessment of electrolyte leakage, UV-absorbance of the tissue leachates, relative leakage ratio, injury index, membrane lipid peroxidation, lipoxygenase activity, α-amino nitrogen and total soluble carbohydrate contents showed the extent of membrane damage as a function of the increasing concentration of mercuric chloride. It is suggested that the selected parameters can be used as qualitative tests for determination of stress-induced membrane damage     

Okadaic acid activates microtubule-associated protein kinase in quiescent fibroblastic cells

Okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A, and is a strong tumor promoter that is not an activator of protein kinase C. Treatment of quiescent cultures of rat fibroblastic 3Y1 cells with okadaic acid induced marked activation of a kinase activity that phosphorylated microtubule-associated protein (MAP) 2 and myelin basic protein, but not histone or casein, in vitro. This activated kinase eluted at approximately 0.15 M NaCl on a DEAE-cellulose column and its apparent molecular mass was determined to be approximately 40 kDa by gel filtration. Detection of the kinase activity in polyacrylamide gels containing substrate proteins after sodium dodecyl sulfate gel electrophoresis revealed that the okadaic-acid-activated kinase activity resided mainly in two closely related polypeptides with apparent molecular mass approximately 40 kDa. The characteristics of this kinase were indistinguishable from those of the mitogen-activated MAP kinase in the same cells. The okadaic-acid-activated MAP kinase was deactivated by protein phosphatase 2A treatment in vitro. These results suggest that MAP kinase is negatively regulated by protein phosphatases 1 and/or 2A in quiescent cells and therefore can be activated by inhibiting these protein phosphatases. Interestingly, the okadaic-acid-induced activation of MAP kinase was transient and epidermal-growth-factor-induced activation was also transient, even in the presence of okadaic acid. These data may imply that protein phosphatases 1 and 2A are not involved in the deactivation of MAP kinase in cells.