Src kinase modulates the activation, transport and signalling dynamics of fibroblast growth factor receptors

The non-receptor tyrosine kinase Src is recruited to activated fibroblast growth factor receptor (FGFR) complexes through the adaptor protein factor receptor substrate 2 (FRS2). Here, we show that Src kinase activity has a crucial role in the regulation of FGFR1 signalling dynamics. Following receptor activation by ligand binding, activated Src is colocalized with activated FGFR1 at the plasma membrane. This localization requires both active Src and FGFR1 kinases, which are inter-dependent. Internalization of activated FGFR1 is associated with release from complexes containing activated Src. Src-mediated transport and subsequent activation of FGFR1 require both RhoB endosomes and an intact actin cytoskeleton. Chemical and genetic inhibition studies showed strikingly different requirements for Src family kinases in FGFR1-mediated signalling; activation of the phosphoinositide-3 kinase-Akt pathway is severely attenuated, whereas activation of the extracellular signal-regulated kinase pathway is delayed in its initial phase and fails to attenuate.     

RhoB and the mammalian Diaphanous-related formin mDia2 in endosome trafficking

Rho GTPases and the dynamic assembly and disassembly of actin filaments have been shown to have critical roles in both the internalization and trafficking of growth factor receptors. While all three mammalian Diaphanous-related (mDia1/2/3) formin GTPase effector proteins have been localized on endosomes, a role for their actin nucleation, filament elongation, and/or bundling remains poorly understood in the context of intracellular trafficking. In a study of a functional relationship between RhoB, a GTPase known to associate with both early- and late-endosomes, and the formin mDia2, we show that 1) RhoB and mDia2 interact on endosomes; 2) GTPase activity-the ability to hydrolyze GTP to GDP-is required for the ability of RhoB to govern endosome dynamics; and 3) the actin dynamics controlled by RhoB and mDia2 is necessary for vesicle trafficking. These studies further suggest that Rho GTPases significantly influence the activity of mDia family formins in driving cellular membrane remodeling through the regulation of actin dynamics.     

CpG-induced tyrosine phosphorylation occurs via a TLR9-independent mechanism and is required for cytokine secretion

Toll-like receptors (TLRs) recognize molecular patterns preferentially expressed by pathogens. In endosomes, TLR9 is activated by unmethylated bacterial DNA, resulting in proinflammatory cytokine secretion via the adaptor protein MyD88. We demonstrate that CpG oligonucleotides activate a TLR9-independent pathway initiated by two Src family kinases, Hck and Lyn, which trigger a tyrosine phosphorylation–mediated signaling cascade. This cascade induces actin cytoskeleton reorganization, resulting in cell spreading, adhesion, and motility. CpG-induced actin polymerization originates at the plasma membrane, rather than in endosomes. Chloroquine, an inhibitor of CpG-triggered cytokine secretion, blocked TLR9/MyD88-dependent cytokine secretion as expected but failed to inhibit CpG-induced Src family kinase activation and its dependent cellular responses. Knock down of Src family kinase expression or the use of specific kinase inhibitors blocked MyD88-dependent signaling and cytokine secretion, providing evidence that tyrosine phosphorylation is both CpG induced and an upstream requirement for the engagement of TLR9. The Src family pathway intersects the TLR9–MyD88 pathway by promoting the tyrosine phosphorylation of TLR9 and the recruitment of Syk to this receptor.     

Differential subcellular membrane recruitment of Src may specify its downstream signalling

Most Src family members are diacylated and constitutively associate with membrane "lipid rafts" that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at "rafts" remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to "lipid rafts"; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 degrees C and floated into sucrose density gradients like caveolin-1 and flotillin-2, i.e. "lipid rafts". By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe ( approximately 70%) cholesterol extraction with methyl-beta-cyclodextrin (MbetaCD) did not abolish "rafts" floatation, but strongly decreased Src association with floating "rafts" and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to MbetaCD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at "non-raft" domains on endosomes, then via PI3-kinase-Akt on a distinct set of "rafts" at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined.     

Microtubule and Actin Interplay Drive Intracellular c-Src Trafficking

The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms.     

Estrogen stimulates protein tyrosine phosphorylation and Src kinase activity in avian osteoclasts

The estrogen, 17beta-estradiol, stimulated a profound increase in phosphotyrosine immunostaining of proteins that localized along the site of attachment in avian osteoclasts within 1 min of treatment. By 10 min, this rapidly occurring event had returned to basal levels. Pretreatment with 1 microM herbimycin A, a tyrosine kinase inhibitor, prevented the response. Immunoblotting revealed that Src kinase was one of the phosphorylated intermediates. Src kinase also appeared to translocate to the periphery of the cells during the 1 min 17beta-estradiol treatment and became dispersed by 10 min. Src kinase activity measurements indicated an increase in phosphotransferase activity after the 1 min estradiol treatment; this effect diminished with longer exposures to estrogen. Pretreatment of osteoclasts with 1 microg/ml cytochalasin B, an inhibitor of actin polymerization, delayed the appearance of increased phosphotyrosine immunostaining at attachment sites, possibly through inhibition of Src kinase translocation. These findings demonstrate that estrogen stimulates rapid tyrosine phosphorylation in osteoclasts, a process that involves activation and translocation of Src kinase to the plasma membrane.     

Prenylation of Rho G-Proteins: a Novel Mechanism Regulating Gene Expression and Protein Stability in Human Trabecular Meshwork Cells

Endogenous prenylation with sesquiterpene or diterpene isoprenoids facilitates membrane localization and functional activation of small monomeric GTP-binding proteins. A direct effect of isoprenoids on regulation of gene expression and protein stability has also been proposed. In this study, we determined the role of sesquiterpene or diterpene isoprenoids on the regulation of Rho G-protein expression, activation, and stability in human trabecular meshwork (TM) cells. In both primary and transformed human TM cells, limiting endogenous isoprenoid synthesis with lovastatin, a potent HMG-CoA reductase inhibitor, elicited marked increases in RhoA and RhoB mRNA and protein content. The effect of lovastatin was dose-dependent with newly synthesized inactive protein accumulating in the cytosol. Supplementation with geranylgeranyl pyrophosphate (GGPP) prevented, while inhibition of geranylgeranyl transferase-I mimicked, the effects of lovastatin on RhoA and RhoB protein content. Similarly, lovastatin-dependent increases in RhoA and RhoB mRNA expression were mimicked by geranylgeranyl transferase-I inhibition. Interestingly, GGPP supplementation selectively promoted the degradation of newly synthesized Rho proteins which was mediated, in part, through the 20S proteasome. Functionally, GGPP supplementation prevented lovastatin-dependent decreases in actin stress fiber organization while selectively facilitating the subcellular redistribution of accumulated Rho proteins from the cytosol to the membrane and increasing RhoA activation. Post-translational prenylation with geranylgeranyl diterpenes selectively facilitates the expression, membrane translocation, functional activation, and turnover of newly synthesized Rho proteins. Geranylgeranyl prenylation represents a novel mechanism by which active Rho proteins are targeted to the 20S proteasome for degradation in human TM cells.     

KIF13A‐regulated RhoB plasma membrane localization governs membrane blebbing and blebby amoeboid cell migration

Membrane blebbing‐dependent (blebby) amoeboid migration can be employed by lymphoid and cancer cells to invade 3D‐environments. Here, we reveal a mechanism by which the small GTPase RhoB controls membrane blebbing and blebby amoeboid migration. Interestingly, while all three Rho isoforms (RhoA, RhoB and RhoC) regulated amoeboid migration, each controlled motility in a distinct manner. In particular, RhoB depletion blocked membrane blebbing in ALL (acute lymphoblastic leukaemia), melanoma and lung cancer cells as well as ALL cell amoeboid migration in 3D‐collagen, while RhoB overexpression enhanced blebbing and 3D‐collagen migration in a manner dependent on its plasma membrane localization and down‐stream effectors ROCK and Myosin II. RhoB localization was controlled by endosomal trafficking, being internalized via Rab5 vesicles and then trafficked either to late endosomes/lysosomes or to Rab11‐positive recycling endosomes, as regulated by KIF13A. Importantly, KIF13A depletion not only inhibited RhoB plasma membrane localization, but also cell membrane blebbing and 3D‐migration of ALL cells. In conclusion, KIF13A‐mediated endosomal trafficking modulates RhoB plasma membrane localization to control membrane blebbing and blebby amoeboid migration.     

RhoB-dependent modulation of postendocytic traffic in polarized Madin-Darby canine kidney cells

The Rho family of GTPases is implicated in the control of endocytic and biosynthetic traffic of many cell types; however, the cellular distribution of RhoB remains controversial and its function is not well understood. Using confocal microscopy, we found that endogenous RhoB and green fluorescent protein-tagged wild-type RhoB were localized to early endosomes, and to a much lesser extent to recycling endosomes, late endosomes or Golgi complex of fixed or live polarized Madin-Darby canine kidney cells. Consistent with RhoB localization to early endosomes, we observed that expression of dominant-negative RhoBN19 or dominant-active RhoBV14 altered postendocytic traffic of ligand-receptor complexes that undergo recycling, degradation or transcytosis. In vitro assays established that RhoB modulated the basolateral-to-apical transcytotic pathway by regulating cargo exit from basolateral early endosomes. Our results indicate that RhoB is localized, in part, to early endosomes where it regulates receptor egress through the early endocytic system.     

Regulation of contractility by Hsp27 and Hic-5 in rat mesenteric small arteries

The regulation of small artery contractility by vasoconstrictors is important for vascular function, and actin cytoskeleton remodeling is required for contraction. p38 MAPK and tyrosine kinases are implicated in actin polymerization and contraction through heat shock protein 27 (Hsp27) and the cytoskeletal protein paxillin, respectively. We evaluated the roles of downstream targets of p38 MAPK and tyrosine kinases in cytoskeletal reorganization and contraction and whether the two signaling pathways regulate contraction independent of each other. We identified the expression of the paxillin homologue hydrogen peroxide-inducible clone-5 (Hic-5) and showed its activation by norepinephrine (NE) in a Src-dependent manner. Furthermore, we demonstrated a NE-induced interaction of proline-rich tyrosine kinase-2 (PYK2) but not Src or p125 focal adhesion kinase with Hic-5. This interaction was Src dependent, suggesting that Hic-5 was a substrate for PYK2 downstream from Src. The activation of Hic-5 induced its relocalization to the cytosol. The parallel activation of Hsp27 by NE was p38 MAPK dependent and led to its dissociation from actin filaments and translocation from membrane to cytosol and increased actin polymerization. Both Hsp27 and Hic-5 activation resulted in their association within the same time frame as NE-induced contraction, and the inhibition of either p38 MAPK or Src inhibited the interaction between Hsp27 and Hic-5 and the contractile response. Furthermore, combined p38 MAPK and Src inhibition had no greater effect on contraction than individual inhibition, suggesting that the two pathways act through a common mechanism. These data show that NE-induced activation and the association of Hsp27 and Hic-5 are required for the reorganization of the actin cytoskeleton and force development in small arteries.     

Estrogen stimulates protein tyrosine phosphorylation and Src kinase activity in avian osteoclasts

The estrogen, 17β‐estradiol, stimulated a profound increase in phosphotyrosine immunostaining of proteins that localized along the site of attachment in avian osteoclasts within 1 min of treatment. By 10 min, this rapidly occurring event had returned to basal levels. Pretreatment with 1 μM herbimycin A, a tyrosine kinase inhibitor, prevented the response. Immunoblotting revealed that Src kinase was one of the phosphorylated intermediates. Src kinase also appeared to translocate to the periphery of the cells during the 1 min 17β‐estradiol treatment and became dispersed by 10 min. Src kinase activity measurements indicated an increase in phosphotransferase activity after the 1 min estradiol treatment; this effect diminished with longer exposures to estrogen. Pretreatment of osteoclasts with 1 μg/ml cytochalasin B, an inhibitor of actin polymerization, delayed the appearance of increased phosphotyrosine immunostaining at attachment sites, possibly through inhibition of Src kinase translocation. These findings demonstrate that estrogen stimulates rapid tyrosine phosphorylation in osteoclasts, a process that involves activation and translocation of Src kinase to the plasma membrane. J. Cell. Biochem. 76:206–216, 1999. © 1999 Wiley‐Liss, Inc.     

Regulation of epidermal growth factor receptor traffic by the small GTPase rhoB.

Members of the Rho family of small GTPases control cell adhesion and motility through dynamic regulation of the actin cytoskeleton. Although twelve family members have been identified, only three of these - RhoA, Rac and Cdc42 - have been studied in detail. RhoA regulates the formation of focal adhesions and the bundling of actin filaments into stress fibres. It is also involved in other cell signalling pathways including the regulation of gene expression and the generation of lipid second messengers [1] [2]. RhoA is very closely related to two other small GTPases about which much less is known: RhoB and RhoC (which are approximately 83% identical). Perhaps the most intriguing of these is RhoB. RhoA is largely cytosolic but translocates to the plasma membrane on activation. RhoB, however, is entirely localised to the cytosolic face of endocytic vesicles [3] [4]. This suggests a potential role for RhoB in regulating endocytic traffic; however, no evidence has been presented to support this. RhoA has been shown to act at the plasma membrane to regulate the clathrin-mediated internalisation of transferrin receptor [5] and of the muscarinic acetylcholine receptor [6]. We have recently demonstrated that RhoB binds the RhoA effector, PRK1 and targets it to the endosomal compartment [7]. We show here that RhoB acts through PRK1 to regulate the kinetics of epidermal growth factor receptor traffic.     

Acute ligand-independent Src activation mimics low EGF-induced EGFR surface signalling and redistribution into recycling endosomes

Src, a non-receptor tyrosine kinase, is a key signal transduction partner of epidermal growth factor (EGF) receptor (EGFR). In human breast cancer, EGFR and Src are frequently over-expressed and/or over-activated. Although reciprocal activation is documented, mechanisms underlying Src:EGFR interactions are incompletely understood. We here exploited ts/v-Src thermo-activation in MDCK monolayers to test whether acute Src activation impacts on signalling and trafficking of non-liganded EGFR. We found that thermo-activation caused rapid Src recruitment to the plasma membrane, concomitant association with EGFR, and its phosphorylation at Y845 and Y1173 predominantly at the cell surface. Like low EGF concentrations, activated Src (i) decreased EGF surface binding without affecting the total EGFR pool; (ii) triggered EGFR endocytosis via clathrin-coated vesicles; (iii) and led to its sequestration in perinuclear/recycling endosomes with avoidance of multivesicular bodies and lysosomal degradation. Combined Src activation and EGF were synergistic for EGFR-Y845 and -Y1173 phosphorylation at some endosomes. We conclude that acute effects of Src in MDCK cells may mimic those of low EGF on EGFR activation and redistribution. Src:EGFR interactions may be sufficient to trigger EGFR activation and might contribute to its local signalling, without requiring either soluble extracellular signal or receptor over-expression.     

Translocation of Src kinase to the cell periphery is mediated by the actin cytoskeleton under the control of the Rho family of small G proteins

We have isolated Swiss 3T3 subclones that are resistant to the mitogenic and morphological transforming effects of v-Src as a consequence of aberrant translocation of the oncoprotein under low serum conditions. In chicken embryo and NIH 3T3 fibroblasts under similar conditions, v-Src rapidly translocates from the perinuclear region to the focal adhesions upon activation of the tyrosine kinase, resulting in downstream activation of activator protein-1 and mitogen- activated protein kinase, which are required for the mitogenic and transforming activity of the oncoprotein. Since serum deprivation induces cytoskeletal disorganization in Swiss 3T3, we examined whether regulators of the cytoskeleton play a role in the translocation of v- Src, and also c-Src, in response to biological stimuli. Actin stress fibers and translocation of active v-Src to focal adhesions in quiescent Swiss 3T3 cells were restored by microinjection of activated Rho A and by serum. Double labeling with anti-Src and phalloidin demonstrated that v-Src localized along the reformed actin filaments in a pattern that would be consistent with trafficking in complexes along the stress fibers to focal adhesions. Furthermore, treatment with the actin-disrupting drug cytochalasin D, but not the microtubule- disrupting drug nocodazole, prevented v-Src translocation. In addition to v-Src, we observed that PDGF-induced, Rac-mediated membrane ruffling was accompanied by translocation of c-Src from the cytoplasm to the plasma membrane, an effect that was also blocked by cytochalasin D. Thus, we conclude that translocation of Src from its site of synthesis to its site of action at the cell membrane requires an intact cytoskeletal network and that the small G proteins of the Rho family may specify the peripheral localization in focal adhesions or along the membrane, mediated by their effects on the cytoskeleton.     

Epidermal growth factor-induced activation and translocation of c-Src to the cytoskeleton depends on the actin binding domain of the EGF-receptor

In the epidermal growth factor (EGF)-receptor signal transduction cascade, the non-receptor tyrosine kinase c-Src has been demonstrated to become activated upon EGF stimulation. In this paper we show that c-Src associates with the cytoskeleton and co-isolates with actin filaments upon EGF treatment of NIH-3T3 cells transfected with the EGF receptor. Immunofluorescence studies using CLSM show colocalization of F-actin and endogenous c-Src predominantly around endosomes and not on stress fibers and cell–cell contacts. Stimulation of EGF receptor-transfected NIH-3T3 cells with EGF induces an activation and translocation of c-Src to the cytoskeleton. These processes depend upon the presence of the actin binding domain of the EGF-receptor since in cells that express EGF-receptors lacking this domain, EGF fails to induce an activation and translocation to the cytoskeleton of c-Src. These data suggest a role for the actin binding domain of the EGF-receptor in the translocation of c-Src.     

The IpaC Carboxyterminal Effector Domain Mediates Src-Dependent Actin Polymerization during Shigella Invasion of Epithelial Cells

Shigella, the causative agent of bacillary dysentery, invades epithelial cells by locally reorganizing the actin cytoskeleton. Shigella invasion requires actin polymerization dependent on the Src tyrosine kinase and a functional bacterial type III secretion (T3S) apparatus. Using dynamic as well as immunofluorescence microscopy, we show that the T3S translocon component IpaC allows the recruitment of the Src kinase required for actin polymerization at bacterial entry sites during the initial stages of Shigella entry. Src recruitment occurred at bacterial-cell contact sites independent of actin polymerization at the onset of the invasive process and was still observed in Shigella strains mutated for translocated T3S effectors of invasion. A Shigella strain with a polar mutation that expressed low levels of the translocator components IpaB and IpaC was fully proficient for Src recruitment and bacterial invasion. In contrast, a Shigella strain mutated in the IpaC carboxyterminal effector domain that was proficient for T3S effector translocation did not induce Src recruitment. Consistent with a direct role for IpaC in Src activation, cell incubation with the IpaC last 72 carboxyterminal residues fused to the Iota toxin Ia (IaC) component that translocates into the cell cytosol upon binding to the Ib component led to Src-dependent ruffle formation. Strikingly, IaC also induced actin structures resembling bacterial entry foci that were enriched in activated Src and were inhibited by the Src inhibitor PP2. These results indicate that the IpaC effector domain determines Src-dependent actin polymerization and ruffle formation during bacterial invasion.     

Re-distribution of phospholipase C gamma 2 in macrophage precursors is mediated by the actin cytoskeleton under the control of the Src kinases

Macrophage colony-stimulating factor (M-CSF) is a growth factor that is known to trigger several signalling pathways through receptor tyrosine kinase activation. We investigated the specific requirements for the activation of phospholipase C gamma 2 (PLC-γ2) during the differentiation of mouse bone marrow-derived macrophage precursors. M-CSF stimulation induced rapid PLC-γ2 translocation and phosphorylation from the cytosolic compartment to the cell periphery. Both events were dependent on cytoskeleton integrity and Src kinase activity, but only PLC-γ2 phosphorylation did not require PI3-kinase activity. Biochemical experiments as well as confocal microscopy analyses indicate that the translocation of PLC-γ2 is mediated by the direct association of this protein with the actin cytoskeleton. Using GST-fusion proteins containing various deletions of the PLC-γ2 Src homology region, it was found that PLC-γ2 binds to F-actin via its SH2 domains, a feature that has equally been found in a co-sedimentation assay. This association, which is increased during actin reorganisation and disrupted by cytoskeleton inhibitors, seems to be a primary means to recruit this enzyme to the cell periphery. These results indicate that, upon M-CSF stimulation, PLC-γ2 cellular localisation and phosphorylation are strongly dependent on cytoskeleton architecture of the macrophage precursor as well as the PI3-kinase and the Src kinases.     

Activation of Rho GTPases by synaptic transmission in the hippocampus

Ras-related GTPases of the Rho family, such as RhoA and RhoB, are well-characterised mediators of morphological change in peripheral tissues via their effects on the actin cytoskeleton. We tested the hypothesis that Rho family GTPases are involved in synaptic transmission in the CA1 region of the hippocampus. We show that GTPases are activated by synaptic transmission. RhoA and RhoB were activated by low frequency stimulation, while the induction of long-term potentiation (LTP) by high frequency stimulation was associated with specific activation of RhoB via NMDA receptor stimulation. This illustrates that these GTPases are potential mediators of synaptic transmission in the hippocampus, and raises the possibility that RhoB may play a role in plasticity at hippocampal synapses during LTP.