Activation of Lyn Tyrosine Kinase through Decreased Membrane Cholesterol Levels during a Change in Its Membrane Distribution upon Cell Detachment

Cellular membranes, which can serve as scaffolds for signal transduction, dynamically change their characteristics upon cell detachment. Src family kinases undergo post-translational lipid modification and are involved in a wide range of signaling events at the plasma membrane, such as cell proliferation, cell adhesion, and survival. Previously, we showed the differential membrane distributions among the members of Src family kinases by sucrose density gradient fractionation. However, little is known about the regulation of the membrane distribution of Src family kinases upon cell detachment. Here, we show that cell detachment shifts the main peak of the membrane distribution of Lyn, a member of Src family kinase, from the low density to the high density membrane fractions and enhances the kinase activity of Lyn. The change in Lyn distribution upon cell detachment involves both dynamin activity and a decrease in membrane cholesterol. Cell detachment activates Lyn through decreased membrane cholesterol levels during a change in its membrane distribution. Furthermore, cholesterol incorporation decreases Lyn activity and reduces the viability of suspension cells. These results suggest that cell detachment-induced Lyn activation through the change in the membrane distribution of Lyn plays an important role in survival of suspension cells.     

The promoting role of lysosome-localized c-Src in autophagosome-lysosome fusion

Src-family kinases (SFKs), such as c-Src, Lyn and Fyn, belong to non-receptor-type tyrosine kinases and play key roles in cell proliferation, adhesion, and migration. SFKs are anchored to the plasma membrane, Golgi membranes and lysosomal membranes through lipid modifications. Although the functions of SFKs being localized to the plasma membrane are intensively studied, those of SFKs being localized to organelle membranes are poorly understood. Here, we show that, among SFKs, c-Src in particular is involved in a decrease in the amount of LC3-II. c-Src and non-palmitoylated Lyn [Lyn(C3S) (cysteine-3 → serine-3)], which are localized onto lysosomes, decrease the amount of LC3-II and treatment with SFK inhibitors increases the amount of LC3-II, suggesting the importance of SFKs' lysosomal localization for a change of autophagic flux in a kinase activity-dependent manner. Colocalization of LC3-II with the lysosome-associated membrane protein LAMP1 shows that lysosome-localized SFKs promote the fusion of autophagosomes with lysosomes. Lysosome-localized SFKs play a positive role in the maintenance of cell viability under starvation conditions, which is further supported by knockdown of c-Src. Therefore, our results suggest that autophagosome-lysosome fusion is promoted by lysosome-localized c-Src, leading to cell survival under starvation conditions.     

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.     

Rapid trafficking of c-Src, a non-palmitoylated Src-family kinase, between the plasma membrane and late endosomes/lysosomes

Src-family kinases (SFKs) are co-expressed with multiple combinations of each member in a single cell and involved in various signalings. Recently, we showed by sucrose-density gradient fractionation that the subcellular distribution of c-Src is distinct from that of Lyn. However, little is known about the trafficking of c-Src in living cells. Here, we show by time-lapse monitoring combined with photobleaching techniques that c-Src, a non-palmitoylated SFK, is rapidly exchanged between the plasma membrane and intracellular organelles representing late endosomes/lysosomes possibly through its cytosolic release. Although Lyn, a palmitoylated SFK, is exocytosed to the plasma membrane via the Golgi apparatus along the secretory pathway, lack of palmitoylation directs Lyn away from the exocytotic transport to the c-Src-type trafficking between the plasma membrane and late endosomes/lysosomes. Intriguingly, c-Src and a non-palmitoylated Lyn mutant are efficiently delivered and immobilized to focal adhesions when their SH2 domains are able to mediate protein-protein interactions in place of intramolecular bindings. However, palmitoylation of Lyn inhibits its recruitment to focal adhesions. These results suggest that palmitoylation of SFKs is critical for SFK localization and trafficking and implicate that two distinct trafficking pathways for SFKs may be involved in SFKs' specific functions.     

Involvement of Golgi-associated Lyn tyrosine kinase in the translocation of annexin II to the endoplasmic reticulum under oxidative stress

Src-family tyrosine kinases, known to participate in signaling pathways of a variety of receptors at the plasma membrane, are found in cellular endomembranes such as the Golgi apparatus and endosomes. Recently, we showed that Lyn, a member of the Src kinases, accumulates on the Golgi apparatus and then traffics to the plasma membrane. We show here that a majority of endogenous Lyn but not c-Src is accumulated in Golgi-enriched heavy-membrane fractions on a sucrose-density gradient, whereas a small amount of endogenous Lyn is present in light-membrane fractions containing the plasma membrane. Inducible expression of kinase-active Lyn, which biosynthetically reaches the Golgi apparatus, triggers tyrosine phosphorylation of proteins including annexin II. Coimmunoprecipitation analyses reveal that Lyn physically associates with annexin II, and an in vitro kinase assay shows that Lyn phosphorylates annexin II directly. Furthermore, stimulation of cells with H2O2 induces tyrosine phosphorylation of annexin II on the Golgi apparatus in a manner that is dependent on the kinase activity of Src kinases, leading to the translocation of annexin II from the Golgi apparatus to the endoplasmic reticulum. Thus, these results suggest that endomembranes containing the Golgi apparatus where Lyn is anchored can serve as a signaling platform under oxidative stress.     

Hsp90 is essential for the synthesis and subsequent membrane association, but not the maintenance, of the Src-kinase p56(lck)

Tyrosine kinases of the Src family are synthesized as cytosolic proteins that subsequently translocate to membranes. Little is known of the mechanisms responsible for targeting these proteins to membranes, although a role for the cytosolic chaperone Hsp90 has been proposed. Here, we have studied the involvement of Hsp90 in the synthesis, membrane binding, and maintenance of the Src-kinase Lck. Using specific inhibitors of Hsp90, geldanamycin and radicicol, we found that functional Hsp90 is essential for the stability of newly synthesized, but not mature, Lck. Similar results were obtained for two other Src-kinases, c-Src and Lyn. In contrast, LckY505F and LckDeltaSH2, constitutively active Lck mutants lacking the C-terminal regulatory tyrosine or the entire Src homology 2 domain, respectively, required Hsp90 activity to stabilize the mature proteins. Lck synthesized in the absence of Hsp90 activity was degraded within 30-45 min. This unstable Lck was myristoylated normally but did not associate with membranes or CD4, interactions that normally start within minutes of the completion of Lck synthesis. A construct composed of the N-terminal unique domain of Lck fused to green fluorescent protein did not require Hsp90 activity during synthesis. In addition, this protein associated with membranes efficiently in the absence of Hsp90 activity. Together these data suggest that interaction with Hsp90 is necessary for the correct synthesis and subsequent membrane binding of Lck. However, Hsp90 does not appear to play a direct role in Lck membrane, or CD4, association.     

Affinity of Src family kinase SH3 domains for HIV Nef in vitro does not predict kinase activation by Nef in vivo

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.     

HIV-1 Nef selectively activates Src family kinases Hck, Lyn, and c-Src through direct SH3 domain interaction

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.     

Src-Tyrosine kinases are major agents in mitochondrial tyrosine phosphorylation

Mitochondrial tyrosine phosphorylation is emerging as an important mechanism in regulating mitochondrial function. This article, aimed at identifying which kinases are the major agents in mitochondrial tyrosine phosphorylation, shows that this role should be attributed to Src family members. Indeed, various members of this family, for example, Fgr, Fyn, Lyn, c-Src, are constitutively present in the internal structure of mitochondria as well as Csk, a key enzyme in the regulation of the activity of this family. By means of different approaches, biochemical fractioning, Western blotting and immunogold analysis "in situ" of phosphotyrosine signaling, evidence is reported on the existence of a signal transduction pathway from plasma membrane to mitochondria, resulting in increasing Src-dependent mitochondrial tyrosine phosphorylation. The activation of Src kinases at mitochondrial level is associated with the proliferative status where several mitochondrial proteins are specifically tyrosine-phosphorylated.     

c-Src mediates mitogenic signals and associates with cytoskeletal proteins upon vascular endothelial growth factor stimulation in Kaposi's sarcoma cells

Vascular endothelial growth factor (VEGF) appears to be a critical cytokine modulating the growth and spread of Kaposi's sarcoma (KS). Furthermore, infection with the KS herpes virus results in up-regulation of VEGF and triggering of VEGF receptor activation. The molecular mechanisms regulating such cytokine-driven proliferation of KS cells are not well characterized. We investigated the role of Src-related tyrosine kinases in VEGF-mediated signaling in model KS 38 tumor cells. VEGF stimulation specifically activated c-Src kinase activity but not that of other related Src kinases such as Lyn, Fyn, or Hck in KS cells. Pyrazolopyrimidine, a selective inhibitor of Src family tyrosine kinases, significantly blocked the VEGF-induced growth of KS cells. Further studies using mutants of c-Src kinase revealed that Src mediates mitogen-activated protein kinase activation induced by VEGF. We also observed that VEGF stimulation resulted in increased tyrosine phosphorylation of the focal adhesion components paxillin and p130cas. Furthermore, VEGF induction enhanced the complex formation between Src kinase and paxillin. Src kinase appears to play an important functional role in VEGF-induced signaling in KS cells and may act to link pathways from the VEGF receptor to mitogen-activated protein kinase and cytoskeletal components, thereby effecting tumor proliferation and migration.     

Differential mitotic activation of endogenous c-Src, c-Yes, and Lyn in HeLa cells

Src-family tyrosine kinases (SFKs) play an important role in mitosis. Despite overlapping expression of multiple SFK members, little is known about how individual SFK members are activated in M phase. Here, we examined mitotic activation of endogenous c-Src, c-Yes, and Lyn, which are co-expressed in HeLa cells. c-Src, c-Yes, and Lyn were activated at different levels in M phase, and the activation was inhibited by Cdc2 inactivation. Mitotic c-Src and c-Yes exhibited normal- and retarded-electrophoretic-mobility forms on SDS-polyacrylamide gels, whereas Lyn did not show mobility retardation. Like c-Src, the retardation of electrophoretic mobility of c-Yes was caused by Cdc2-mediated phosphorylation. The normal- and retarded-mobility forms of c-Src were comparably activated, but activation of the retarded-mobility form of c-Yes was higher than that of the normal-mobility form of c-Yes. Thus, these results suggest that endogenous c-Src, c-Yes, and Lyn are differentially activated through Cdc2 activation during M phase.     

Translocation of the Csk homologous kinase (Chk/Hyl) controls activity of CD36-anchored Lyn tyrosine kinase in thrombin-stimulated platelets.

Chk/Hyl is a recently isolated non-receptor tyrosine kinase with greatest homology to a ubiquitous negative regulator of Src family kinases, Csk. To understand the significance of co-expression of Chk and Csk in platelets, we examined the subcellular localization of each protein. Chk, but not Csk, was completely translocated from the Triton X-100-soluble to the Triton X-100-insoluble cytoskeletal fraction within 10 s of thrombin stimulation. Chk and Lyn, but not Csk and c-Src, co-fractionated in the higher density lysate fractions of resting platelets, with Chk being found to localize close to CD36 (membrane glycoprotein IV)-anchored Lyn. The kinase activity of co-fractionated Lyn was suppressed 3-fold. In vitro phosphorylation assays showed that Chk suppressed Lyn activity by phosphorylating its C-terminal negative regulatory tyrosine. Upon stimulation of platelets with thrombin, the rapid and complete translocation of Chk away from Lyn caused concomitant activation of Lyn. This activation was accompanied by dephosphorylation of Lyn at its C-terminal negative regulatory tyrosine in cooperation with a protein tyrosine phosphatase. These results suggest that Chk, but not Csk, may function as a translocation-controlled negative regulator of CD36-anchored Lyn in thrombin-induced platelet activation.     

Trafficking of Lyn through the Golgi caveolin involves the charged residues on alphaE and alphaI helices in the kinase domain

Src-family kinases, known to participate in signaling pathways of a variety of surface receptors, are localized to the cytoplasmic side of the plasma membrane through lipid modification. We show here that Lyn, a member of the Src-family kinases, is biosynthetically transported to the plasma membrane via the Golgi pool of caveolin along the secretory pathway. The trafficking of Lyn from the Golgi apparatus to the plasma membrane is inhibited by deletion of the kinase domain or Csk-induced "closed conformation" but not by kinase inactivation. Four residues (Asp346 and Glu353 on alphaE helix, and Asp498 and Asp499 on alphaI helix) present in the C-lobe of the kinase domain, which can be exposed to the molecular surface through an "open conformation," are identified as being involved in export of Lyn from the Golgi apparatus toward the plasma membrane but not targeting to the Golgi apparatus. Thus, the kinase domain of Lyn plays a role in Lyn trafficking besides catalysis of substrate phosphorylation.     

Differential involvement of Src family kinases in Fc gamma receptor-mediated phagocytosis

The tyrosine phosphorylation cascade originated from Fc gamma receptors (Fc gamma Rs) is essential for macrophage functions including phagocytosis. Although the initial step is ascribed to Src family tyrosine kinases, the role of individual kinases in phagocytosis signaling is still to be determined. In reconstitution experiments, we first showed that expression in the RAW 264.7 cell line of C-terminal Src kinase (Csk) inhibited and that of a membrane-anchored, gain-of-function Csk abolished the Fc gamma R-mediated signaling that leads to phagocytosis in a kinase-dependent manner. We next tested reconstruction of the signaling in the membrane-anchored, gain-of-function Csk-expressing cells by introducing Src family kinases the C-terminal negative regulatory sequence of which was replaced with a c-myc epitope. Those constructs derived from Lyn and Hck (a-Lyn and a-Hck) that associated with detergent-resistant membranes successfully reconstructed Fc gamma R-mediated Syk activation, filamentous actin rearrangement, and phagocytosis. In contrast, c-Src-derived construct (a-Src), that was excluded from detergent-resistant membranes, could not restore the series of phagocytosis signaling. Tyrosine phosphorylation of Vav and c-Cbl was restored in common by a-Lyn, a-Hck, and a-Src, but Fc gamma RIIB tyrosine phosphorylation, which is implicated in negative signaling, was reconstituted solely by a-Lyn and a-Hck. These findings suggest that Src family kinases are differentially involved in Fc gamma R-signaling and that selective kinases including Lyn and Hck are able to fully transduce phagocytotic signaling.     

Identification of UNC119 as a novel activator of SRC-type tyrosine kinases

Lyn, an Src-type tyrosine kinase, is associated with the interleukin (IL)-5 receptor in eosinophils. The mechanism of its activation is unknown. Through yeast two-hybrid screening we have cloned and characterized a new signaling molecule, Unc119, that associates with IL-5Ralpha and Src family tyrosine kinases. Unc119 induces the catalytic activity of these kinases through interaction with Src homology 2 and 3 domains. IL-5 stimulation of eosinophils increases Unc119 association with Lyn and induces its catalytic activity. Lyn is important for eosinophil survival. Eosinophils that are transduced with Unc119 have increased Lyn activity and demonstrate prolonged survival in the absence of IL-5. Inhibition of Unc119 down-regulates eosinophil survival. To our knowledge Unc119 is the first receptor-associated activator of Src family tyrosine kinases.     

Immunoseparation of sphingolipid-enriched membrane domains enriched in Src family protein tyrosine kinases and in the neuronal adhesion molecule TAG-1 by anti-GD3 ganglioside monoclonal antibody

Rat cerebellar granule cells differentiated in culture were fed [1-(3)H]sphingosine, allowing the metabolic radiolabelling of all cell sphingolipids and phosphatidylethanolamine. A detergent-insoluble sphingolipid-enriched membrane fraction, containing about 60% of cell sphingolipids, but only trace amounts of phosphatidylethanolamine, was prepared from [1-(3)H]sphingosine-fed cells by sucrose gradient centrifugation. This fraction was enriched in the Src family protein tyrosine kinases c-Src, Lyn and Fyn and in the GPI-anchored neuronal adhesion molecule TAG-1. The cell lysate and the sphingolipid-enriched membrane fraction were subjected to immunoprecipitation with anti-GD3 ganglioside monoclonal antibody R24, under experimental conditions designed to preserve the integrity of the domain. The radioactive lipid composition of the immunoprecipitates obtained from the cell lysate and from the sphingolipid-enriched fraction were very similar, and closely resembled the sphingolipid composition of the whole sphingolipid-enriched membrane fraction. In fact, the immunoprecipitates contained, together with GD3 ganglioside, all cell glycosphingolipids and sphingomyelin, whereas they did not contain phosphatidylethanolamine. Moreover, cholesterol and phosphatidylcholine were detected in the immunoprecipitates by qualitative TLC analysis followed by colourimetric visualization. c-Src, Lyn, Fyn and TAG-1 were associated with the anti-GD3 antibody immunoprecipitate. These proteins were not detected in the immunoprecipitates obtained under experimental conditions different from those designed to preserve the integrity of the domain. These data suggest that a membrane domain containing cholesterol, phosphatidylcholine, sphingolipids and proteins can be separated from the total cell membranes by anti-GD3 antibody immunoprecipitation, and that the association of c-Src, Fyn, Lyn, and TAG-1 with the sphingolipid-enriched domain is mediated by the interaction with a complex lipid environment, rather than by specific interactions with a single sphingolipid species.     

Real-time monitoring of hematopoietic cell interaction with fibronectin fragment

Real-time cell analysis (RTCA) system based on measurement of electrical microimpedance has been introduced to monitor adherent cell cultures. We describe its use for real-time analysis of hematopoietic cell adhesion to bone marrow stroma proteins. Cells growing in suspension do not generate any significant change in the microimpedance signal until the surface with embedded microelectrodes is coated with a cell-binding protein. We show that in this case, the microimpedance signal specifically reflects cell binding to the coated surface. The optimized method was used to monitor the effect of two histone deacetylase inhibitors, suberoylanilide hydroxamic acid (SAHA) and tubastatin A, on JURL-MK1 cell adhesion to cell-binding fragment of fibronectin (FNF). Both compounds were used in non-toxic concentrations and induced an increase in the cell adhesivity. The kinetics of this increase was markedly slower for SAHA although tubulin hyperacetylation occurred rapidly for any of the two drugs. The strengthening of cell binding to FNF was paralleled with a decrease of Lyn kinase activity monitored using an anti-phospho-Src family antibody. The inhibition of Src kinase activity with PP2 accordingly enhanced JURL-MK1 cell interaction with FNF. Actin filaments were present at the proximity of the plasma membrane and in numerous membrane protrusions. In some cells, F-actin formed clusters at membrane regions interacting with the coated surface and these clusters colocalized with active Lyn kinase. Our results indicate that the role of Src kinases in the regulation of hematopoetic cell adhesion signaling is similar to that of c-Src in adherent cells.