Src Family Kinases Hck Research Articles

Ceramide-mediated stimulation of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) accumulation in murine macrophages requires tyrosine kinase activity.

In macrophages, bacterial lipopolysaccharide (LPS) has been noted to mimic certain effects of the sphingolipid ceramide, suggesting that ceramide may be involved in macrophage activation by LPS and/or that LPS utilizes ceramide-related signaling pathways. Putative downstream targets of ceramide include a ceramide-activated (serine/threonine) protein kinase (CAPK) and phosphatase (CAPP). However, the potential role of tyrosine phosphorylation pathways in macrophage response to ceramide has not been examined. Herein we report that cell-permeable analogs of ceramide up-regulate both inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) production in RAW 264.7 murine macrophages. Herbimycin A and genistein, potent natural inhibitors of protein tyrosine (but not serine/threonine) phosphorylation, block ceramide-induced iNOS and TNF production. Furthermore, the highly src-family selective pyrazolopyrimidine inhibitor PP1 also blocks ceramide-induced iNOS and TNF production in RAW 264.7 cells. We found that PP1 also inhibits ceramide-mediated tyrosine phosphorylation of the src-family kinase hck. These data indicate that src-related tyrosine kinases play a critical role in macrophage activation by ceramide.

Kinase pathways in chemoattractant-induced degranulation of neutrophils: the role of p38 mitogen-activated protein kinase activated by Src family kinases.

The aim of the present study was to investigate the role of tyrosine phosphorylation pathways in fMLP-induced exocytosis of the different secretory compartments (primary and secondary granules, as well as secretory vesicles) of neutrophils. Genistein, a broad specificity tyrosine kinase inhibitor, blocked the exocytosis of primary and secondary granules, but had only a marginal effect on the release of secretory vesicles. Genistein also inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPK), raising the possibility that inhibition of ERK and/or p38 MAPK might be responsible for the effect of the drug on the degranulation response. Indeed, SB203580, an inhibitor of p38 MAPK, decreased the release of primary and secondary granules, but not that of secretory vesicles. However, blocking the ERK pathway with PD98059 had no effect on any of the exocytic responses tested. PP1, an inhibitor of Src family kinases, also attenuated the release of primary and secondary granules, and neutrophils from mice deficient in the Src family kinases Hck, Fgr, and Lyn were also defective in secondary granule release. Furthermore, activation of p38 MAPK was blocked by both PP1 and the hck-/-fgr-/-lyn-/- mutation. Taken together, our data indicate that fMLP-induced degranulation of primary and secondary granules of neutrophils is mediated by p38 MAPK activated via Src family tyrosine kinases. Although piceatannol, a reportedly selective inhibitor of Syk, also prevented degranulation and activation of p38 MAPK, no fMLP-induced phosphorylation of Syk could be observed, raising doubts about the specificity of the inhibitor.

Impaired integrin-mediated signal transduction, altered cytoskeletal structure and reduced motility in Hck/Fgr deficient macrophages.

Integrin-mediated adhesion of monocytes and macrophages initiates a signal transduction pathway that leads to actin cytoskeletal reorganization, cell migration and immunologic activation. This signaling pathway is critically dependent on tyrosine kinases. To investigate the role of the Src-family of tyrosine kinases in integrin signal transduction, we have examined the adhesive properties of macrophages isolated from hck-/-fgr-/- double knockout mice which lack two of the three predominant Src-family kinases expressed in myeloid cells. Previous examination of polymorphonuclear leukocytes from these animals indicated that these kinases were critical in initiating the actin cytoskeletal rearrangements that lead to respiratory burst and granule secretion following integrin ligation. Double mutant peritoneal exudate macrophages demonstrated markedly reduced tyrosine phosphorylation responses compared to wild-type cells following plating on fibronectin, collagen or vitronectin-coated surfaces. Tyrosine phosphorylation of several actin-associated proteins (cortactin, paxillin, and tensin), as well as the Syk and Pyk2 tyrosine kinases, were all significantly reduced in double mutant cells. The subcellular localization of focal-adhesion associated proteins was also dramatically altered in mutant macrophages cultured on fibronectin-coated surfaces. In wild-type cells, filamentous actin, paxillin, and talin were concentrated along leading edges of the plasma membrane, suggesting that these proteins contribute to cellular polarization during migration in culture. Double mutant cells failed to show the polarized subcellular localization of these proteins. Likewise, double mutant macrophages failed to form normal filopodia under standard culture conditions. Together, these signaling and cytoskeletal defects may contribute to the reduced motility observed in in vitro assays. These data provide biochemical and morphological evidence that the Src-family kinases Hck and Fgr are required for normal integrin-mediated signal transduction in murine macrophages.

Adhesion-Dependent Degranulation of Neutrophils Requires the Src Family Kinases Fgr and Hck

Polymorphonuclear neutrophils (PMN) adherent to integrin ligands respond to inflammatory mediators by reorganizing their cytoskeleton and releasing reactive oxygen intermediates. As Src family tyrosine kinases are implicated in these responses, we investigated their possible role in regulating degranulation. Human PMN incubated on fibrinogen released lactoferrin in response to TNF-alpha and this response was inhibited by PP1, a Src family tyrosine kinase inhibitor. This drug had no effect on lactoferrin secretion induced by PMA, an adhesion-independent agonist of PMN degranulation. However, PP1 blocked secretion in PMN plated on plain tissue culture plastic, a surface inducing PMN spreading in the absence of any stimulus. Double knockout hck-/- fgr-/- PMN adherent to collagen or fibrinogen failed to release lactoferrin in response to TNF-alpha but responded to PMA as wild-type PMN. Degranulation induced by spreading over tissue culture plastic was also defective in hck-/- fgr-/- PMN. Defective adhesion-dependent degranulation required the absence of both kinases, because single knockout fgr-/- or hck-/- PMN responded as wild-type cells. Analysis of lactoferrin secretion in hck-/- fgr-/- or PP1-treated, suspended PMN showed that Src kinases are not implicated in degranulation dependent on activation of protein kinase C or increase in intracellular free Ca2+ but may play a role in the response to FMLP of cytochalasin B-treated PMN. These findings identify a role for Src family kinases in a signaling pathway leading to granule-plasma membrane fusion and suggest that Fgr and Hck would be targets for pharmacological control of adhesion-dependent degranulation in the inflammatory site.

Hypertonicity induces shedding of L-selectin: a role for p38 activation

Hemorrhagic shock predisposes to adult respiratory distress syndrome, which frequently results in prolonged ICU stay and carries a 50% mortality. We have previously shown that resuscitation with hypertonic saline (NaCl 7.5%) attenuates the post-hemorrhage lung injury by preventing neutrophil (PMN) sequestration. This beneficial effect was due to multiple effects on PMN function and included shedding of the PMN adhesion molecule L-selectin. The aim of the present study was to investigate the signalling pathway underlying this immunological effect. Isolated human PMN were treated with either iso (290 mOsm) or hypertonic (500 mOsm) medium, for up to 2 h. Hypertonicity induced extensive tyrosine phosphorylation in multiple bands. The broad-spectrum inhibitor genistein, abrogated this effect and concomitantly prevented the hypertonic (HT) shedding of L-selectin (graph). In order to characterize the tyrosine kinases involved in this process, we investigated which kinases were phosphorylated, upon shrinkage, and then whether pharmacological inhibition prevented shedding. We found that the non-receptor tyrosine kinases Syk, Pyk-2 and the Src-family kinase Hck were strongly phosphorylated upon shrinkage. However, PP1, a Src-family inhibitor, prevented their phosphorylation but not the HT shedding of L-selectin, suggesting that this effect is independent of Src activation. Next, we found that the p38 was activated upon hypertonic shrinkage in a genistein sensitive but PP1 insensitive way. Moreover, the inhibition of p38 activation by SB203580 significantly reduced the HT shedding suggesting that p38 is involved in this process (graph). The LPS- and FMLP-induced shedding of L-selectin was also abrogated by SB203580. THUS: Hypertonicity induces a unique pattern of tyrosine phosphorylation in human neutrophils, involving a variety of kinases, most Src-dependent. However, the hypertonic shedding of L-selectin seems to be selectively coupled to p38 activation. In fact, p38 appears to be a central mediator of L-selectin shedding induced by various stimuli. Hypertonicity-induced, p38-mediated L-selectin shedding appears to have an imporant role in the beneficial immune modulatory effect of hypertonicity, preventing neutrophil lung sequestration and cell-mediated tissue damage. Figure

CD43 is associated with tyrosine kinase activity in human neutrophils.

CD43 appears to be involved in signal transduction, however, the mechanism of this function is unknown. Protein kinase activity was detected in neutrophils associated with CD43. Most of the protein kinase activity associated with these antigens was tyrosine kinase activity. The src family kinases lyn and hck were found to account for much of the associated tyrosine kinase activity. The data suggest that associated tyrosine kinase activity may play a role in signal transduction via CD43 to regulate other cell functions.

Tyrosine kinase activity is associated with CD44 in human neutrophils

CD44 appears to be involved in signal transduction, however, the mechanism of this function is unclear. Protein kinase activity was detected in neutrophils associated with CD44. Most of the protein kinase activity associated with these antigens was tyrosine kinase activity. Src family kinases lyn and hck were found to account for much of the associated tyrosine kinase activity. The data suggest that associated tyrosine kinase activity may play a role in signal transduction from CD44 in neutrophils to regulate other cell functions.

Intramolecular Regulatory Interactions in the Src Family Kinase Hck Probed by Mutagenesis of a Conserved Tryptophan Residue

Intramolecular interactions between the Src homology domains (SH2 and SH3) and the catalytic domains of Src family kinases result in repression of catalytic activity. The crystal structure of the Src family kinase Hck, with its regulatory domains intact, has been solved. It predicts that a conserved residue, Trp260, at the end of the linker between the SH2 and the catalytic domains plays an important role in regulation by the SH3 and SH2 domains. We have mutated this residue and compared the activities of C-terminally phosphorylated wild type Hck and W260A Hck. The W260A mutant has a higher specific activity than wild type Hck. The W260A mutant requires autophosphorylation at Tyr416 for full activity, but it is not activated by ligand binding to the SH3 or SH2 domains. This mutation also changes the accessibility of the SH2 and SH3 domains to their cognate peptide ligands. Our results indicate that Trp260 plays a critical role in the coupling of the regulatory domains to the catalytic domain, as well as in positioning the ligand binding surfaces.

A beta 1 integrin signaling pathway involving Src-family kinases, Cbl and PI-3 kinase is required for macrophage spreading and migration.

We have used mutant macrophages which are deficient in expression of Src-family kinases to define an integrin signaling pathway that is required for macrophage adhesion and migration. Following ligation of surface integrins by fibronectin, the p120(c-cbl) (Cbl) protein rapidly becomes tyrosine phosphorylated and associated with the Src-family kinases Fgr and Lyn. In hck-/-fgr-/-lyn-/- triple mutant cells, which are defective in spreading on fibronectin-coated surfaces in vitro and show impaired migration in vivo, Cbl tyrosine phosphorylation is blocked, Cbl protein levels are low, adhesion-dependent translocation of Cbl to the membrane is impaired and Cbl-associated, membrane-localized phosphatidylinositol 3 (PI-3)-kinase activity is dramatically reduced. In contrast, adhesion-dependent activation of total cellular PI-3 kinase activity is normal in mutant cells, demonstrating that it is the membrane-associated fraction of PI-3 kinase which is most critical in regulating actin cytoskeletal rearrangements that lead to cell spreading. Treatment of wild-type cells with the Src-family-specific inhibitor PP1, Cbl antisense oligonucleotides or pharmacological inhibitors of PI-3 kinase blocks cell spreading on fibronectin surfaces. These data provide a molecular description for the role of Src-family kinases Hck, Fgr and Lyn in beta 1-integrin signal transduction in macrophages.

Resistance to endotoxic shock and reduced neutrophil migration in mice deficient for the Src-family kinases Hck and Fgr.

Signal transduction through the leukocyte integrins is required for the processes of firm adhesion, activation, and chemotaxis of neutrophils during inflammatory reactions. Neutrophils isolated from knockout mice that are deficient in the expression of p59/61(hck) (Hck) and p58(c-fgr) (Fgr), members of the Src-family of protein tyrosine kinases, have been shown to be defective in adhesion mediated activation. Cells from these animals have impaired induction of respiratory burst and granule secretion following plating on surfaces that crosslink beta2 and beta3 integrins. To determine if the defective function of hck-/-fgr-/- neutrophils observed in vitro also results in impaired inflammatory responses in vivo, we examined responses induced by lipopolysaccharide (LPS) injection in these animals. The hck-/-fgr-/- mice showed marked resistance to the lethal effects of high-dose LPS injection despite the fact that high levels of serum tumor necrosis factor alpha and interleukin 1alpha were detected. Serum chemistry analysis revealed a marked reduction in liver and renal damage in mutant mice treated with LPS, whereas blood counts showed a marked neutrophilia that was not seen in wild-type animals. Direct examination of liver sections from mutant mice revealed reduced neutrophil migration into the tissue. These data demonstrate that defective integrin signaling in neutrophils, caused by loss of Hck and Fgr tyrosine kinase activity, results in impaired inflammation-dependent tissue injury in vivo.

The Src Family Kinase Hck Interacts with Bcr-Abl by a Kinase-independent Mechanism and Phosphorylates the Grb2-binding Site of Bcr

bcr-abl, the oncogene causing chronic myeloid leukemia, encodes a fusion protein with constitutively active tyrosine kinase and transforming capacity in hematopoietic cells. Various intracellular signaling intermediates become activated and/or associate by/with Bcr-Abl, including the Src family kinase Hck. To elucidate some of the structural requirements and functional consequences of the association of Bcr-Abl with Hck, their interaction was investigated in transiently transfected COS7 cells. Neither the complex formation of Hck kinase with Bcr-Abl nor the activation of Hck by Bcr-Abl was dependent on the Abl kinase activity. Both inactivating point mutations of Hck and dephosphorylation of Hck enhanced its complex formation with Bcr-Abl, indicating that their physical interaction was negatively regulated by Hck (auto)phosphorylation. Finally, experiments with a series of kinase negative Bcr-Abl mutants showed that Hck phosphorylated Bcr-Abl and induced the binding of Grb2 to Tyr177 of Bcr-Abl. Taken together, our results suggest that Bcr-Abl preferentially binds inactive forms of Hck by an Abl kinase-independent mechanism. This physical interaction stimulates the Hck tyrosine kinase, which may then phosphorylate the Grb2-binding site in Bcr-Abl.

Lipopolysaccharide (LPS)-induced macrophage activation and signal transduction in the absence of Src-family kinases Hck, Fgr, and Lyn.

Lipopolysaccharide (LPS) stimulates immune responses by interacting with the membrane receptor CD14 to induce the generation of cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and IL-6. The mechanism by which the LPS signal is transduced from the extracellular environment to the nuclear compartment is not well defined. Recently, an increasing amount of evidence suggests that protein tyrosine kinases especially the Src-family kinases Hck, Fgr, and Lyn, play important roles in LPS signaling. To directly address the physiological function of Hck, Fgr and Lyn in LPS signaling, a genetic approach has been used to generate null mutations of all three kinases in a single mouse strain. hck-/-fgr-/-lyn-/- mice are moderately healthy and fertile; macrophages cultured from these mice express normal levels of CD14 and no other Src-family kinases were detected. Although the total protein phosphotyrosine level is greatly reduced in macrophages derived from hck-/-fgr-/-lyn-/- mice, functional analyses indicate that both elicited peritoneal (PEMs) and bone marrow-derived macrophages (BMDMs) from triple mutant mice have no major defects in LPS-induced activation. Nitrite production and cytokine secretion (IL-1, IL-6, and TNF-alpha) are normal or even enhanced in hck-/-fgr-/-lyn-/- macrophages after LPS stimulation. The development of tumor cell cytotoxicity is normal in triple mutant BMDMs and only partially impaired in PEMs after LPS stimulation. Furthermore, the activation of the ERK1/2 and JNK kinases, as well as the transcription factor NF-kappaB, are the same in normal and mutant macrophages after LPS stimulation. The current study provides direct evidence that three Src-family kinases Hck, Fgr, and Lyn are not obligatory for LPS-initiated signal transduction.

Deficiency of Src family kinases Fgr and Hck results in activation of erythrocyte K/Cl cotransport.

Src-family kinases play a central role in regulation of hematopoietic cell functions. We found that mouse erythrocytes express the Src-family kinases Fgr and Hck, as well as Lyn. To directly test whether Fgr and Hck play any role in erythrocyte function, we analyzed red cells isolated from fgr-/-, hck-/-, and fgr-/- hck-/- knock-out mice. Mean corpuscular hemoglobin concentration and median density are increased, while K content is decreased, in fgr-/- hck-/- double-mutant erythrocytes compared with wild-type, fgr-/-, or hck-/- erythrocytes. Na/K pump and Na/K/Cl cotransport were not altered, but K/Cl cotransport activity was significantly and substantially higher (approximately three-fold) in fgr-/- hck-/- double-mutant erythrocytes. This enhanced K/Cl cotransport activity did not depend on cell age. In fact, in response to bleeding, K/Cl cotransport activity increased in parallel with reticulocytosis in wild-type erythrocytes, while abnormal K/Cl cotransport did not change as a consequence of reticulocytosis in fgr-/- hck-/- double-mutant erythrocytes. Okadaic acid, an inhibitor of a phosphatase that has been implicated in activation of the K/Cl cotransporter, inhibited K/Cl cotransport in wild-type and fgr-/- hck-/- double-mutant erythrocytes to a comparable extent. In contrast, staurosporine, an inhibitor of a kinase that has been suggested to negatively regulate this same phosphatase enhanced K/Cl cotransport in wild-type but not in fgr-/- hck-/- double-mutant erythrocytes. On the basis of these findings, we propose that Fgr and Hck are the kinases involved in the negative regulation of the K/Cl cotransporter-activating phosphatase. Abnormality of erythrocyte K/Cl cotransport in fgr-/- hck-/- double-mutant animals represents the first demonstration that Src-family kinases may be involved in regulation of membrane transport.

CD63 associates with tyrosine kinase activity and CD11/CD18, and transmits an activation signal in neutrophils.

As a member of the tetraspan family, it has been hypothesized that CD63 may be associated with signal transduction; however, its role in leukocyte function is unknown. To examine the potential ability of CD63 to activate neutrophils, the effects of five CD63 mAbs, AHN-16, -16.1, -16.2, -16.3, and -16.5, were examined for their ability to alter neutrophil adhesion to HUVEC monolayers. These CD63 Abs increased neutrophil adhesion to resting and TNF-stimulated HUVEC monolayers. This increase in neutrophil adhesion caused by CD63 Abs was blocked by a CD18 Ab and was associated with up-regulation of CD11/CD18 and down-regulation of CD62L on the neutrophil surface. CD11/CD18 was also found to be associated with CD63. This increase in neutrophil adhesion required physiologic extracellular calcium concentrations at or near the time of CD63 Ab binding. The incubation of CD63 Abs with cells in the absence of calcium for 10 min before repletion of calcium resulted in no increase in neutrophil adhesion. Protein kinase activity was detected in neutrophils associated with CD63. Most of the protein kinase activity associated with these Ags was tyrosine kinase activity, with a lesser amount of threonine and serine kinase activities. Src family kinases Lyn and Hck accounted for much of the associated tyrosine kinase activity. The data suggest that CD63 Ab binding to the neutrophil surface triggers a transient activation signal that requires extracellular calcium and regulates the adhesive activity of CD11/CD18. Associated protein kinase activity may play a role in signal transduction by CD63 to regulate other cell functions.