Janus kinase 2 activation by the platelet-activating factor receptor (PAFR): roles of Tyk2 and PAFR C terminus

Platelet-activating factor (PAF) is a phospholipid with multiple physiological and pathological actions. The PAF receptor (PAFR) belongs to the G protein-coupled, heptahelical receptor superfamily. Recently, we have shown that PAF signals through the Janus kinase (Jak)/STAT pathway and that Tyk2 plays an essential role in PAF-induced PAFR promoter 1 activation. In the present study we found that PAF stimulated Jak2 tyrosine phosphorylation in the monocytic cell line MonoMac-1 as well as in COS-7 cells transfected with PAFR and Jak2 cDNAs. The use of a G protein-uncoupled PAFR (D289A) mutant indicated that Jak2 activation was G protein independent. Interestingly, following PAF stimulation, Jak2 coimmunoprecipitated with PAFR in the presence of active Tyk2, but not with a kinase-inactive Tyk2 mutant, K930I. Moreover, Tyk2-K930I completely blocked PAF-stimulated Jak2 phosphorylation. Gradual deletion of C-terminal residues of the PAFR resulted in progressively decreased Jak2 activation. Deletion of 12 C-terminal residues in mutant V330Stop diminished Jak2 tyrosine phosphorylation by 17%. Further deletions of 25-37 residues from the PAFR C-tail (C317Stop, M311Stop, and T305Stop) resulted in a 50% decrease in Jak2 phosphorylation compared with the wild-type receptor. Complete removal of the C tail resulted in a mutant (K298Stop) that failed to activate Jak2, suggesting that the receptor C-terminal region contains important domains for Jak2 activation. Finally, the coexpression of a minigene encoding the C terminus of PAFR partially inhibited PAF-induced kinase activation. Taken together, our results indicate that PAF activates Jak2 and that Tyk2 and the C-terminal tail of PAFR are of critical importance for PAF-induced Jak2 activation.     

Activation of ERK1/2 by platelet-activating factor receptor is independent of receptor internalisation and G-protein activation

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator involved in a broad range of physiological and pathophysiological processes. The receptor of PAF (PAFR) is a heptahelical G-protein-coupled receptor. We have shown previously that upon agonist stimulation, PAFR internalised through clathrin-coated vesicles in an arrestin-dependent, but G-protein-coupling-independent manner. In the current report, we demonstrate that PAF stimulates Erk1/2 phosphorylation and: (1). dominant negative mutants of arrestins and dynamin do not influence Erk1/2 activation, (2). hypertonic conditions do not decrease the extent of Erk1/2 phosphorylation, (3). internalisation-deficient and/or G-protein-coupling-deficient mutants of PAFR activate Erk1/2 as efficiently as the wild-type PAFR, and (4). inhibition of epidermal growth factor receptor (EGFR) does not block Erk1/2 activation. Taken together, our results suggest that PAFR-mediated activation of mitogen-activated protein kinases Erk1/2 does not require receptor endocytosis, receptor tyrosine kinase transactivation or G-protein activation. In addition, our studies reveal that PAFR-mediated signals of G-protein activation, receptor internalisation and MAPK activation are differentially regulated by receptor structure and/or conformation.     

H2O2-induced activation of transcription factors STAT1 and STAT3: the role of EGF receptor and tyrosine kinase JAK2

Reactive oxygen species initiate multiple signal transduction pathways including tyrosine kinase signaling. Here, we demonstrate tyrosine phosphorylation of EGF receptor, STAT3, and, to a lesser extent, STAT1 upon H2O2 treatment of HER14 cells (NIH3T3 fibroblasts transfected with full-length EGF receptor). Maximum phosphorylation levels were observed in 5 min of stimulation at 1-2 mM H2O2. It has been shown that the intrinsic EGF-receptor tyrosine kinase is responsible for the receptor phosphorylation upon H2O2 stimulation. STAT3 and STAT1 activation in HER14 cells was demonstrated to depend on EGF receptor kinase activity, rather than JAK2 activity, while in both K721A and CD126 cells (NIH3T3 transfected with kinase-dead EGF receptor, and EGF receptor lacking major autophosphorylation sites, respectively) STAT1 and STAT3 tyrosine phosphorylation requires JAK2 kinase activity. Furthermore, STAT3 is constitutively phosphorylated in K721A and CD126 cells, and STAT1 H2O2-stimulated activation in these cells is much more prominent than in HER14. In all the cell lines used, Src-kinase activity was demonstrated to be unnecessary for ROS-initiated phosphorylation of STATs. Herein, we postulate that EGF receptor plays a role in H2O2-induced STAT activation in HER14 cells. Our data also prompted a hypothesis of constitutive inhibition of JAK2-dependent STAT activation in this cell line.     

Evidence for association of the cloned liver growth hormone receptor with a tyrosine kinase.

The ability of the cloned liver growth hormone (GH) receptor, when expressed in mammalian cell lines, to copurify with tyrosine kinase activity and be tyrosyl phosphorylated was examined. 125I-human growth hormone-GH receptor complexes isolated from COS-7 cells transiently expressing high levels of the cloned liver GH receptor bound to anti-phosphotyrosine antibody, suggesting that the cloned GH receptor is tyrosyl phosphorylated in vivo. GH-GH receptor complexes purified from transfected COS-7 cells using anti-GH antibody incorporated 32P when incubated with [gamma-32P]ATP, indicating association of tyrosine kinase activity with cloned liver GH receptor. The level of phosphorylation of the GH receptor was very low, as compared with the endogenous GH receptor in 3T3-F442A cells, suggesting that tyrosine kinase activity is not intrinsic to the cloned GH receptor but rather resides with a kinase present at low levels in the COS-7 cells. To test whether a higher level of GH receptor phosphorylation would be observed when the GH receptor was expressed in a different cell line, GH receptor cDNAs were stably transfected into mouse L and CHO cells, which have few or no endogenous GH receptors, and RIN5-AH cells, which do express endogenous GH receptors. In vivo tyrosyl phosphorylation of the cloned GH receptor in mouse L cells and in vitro phosphorylation of the cloned GH receptor in both L and CHO cells were higher than in transfected COS-7 cells but still substantially lower than in untransfected 3T3-F442A cells. Significantly increased 32P incorporation into tyrosyl residues in GH receptors in the in vitro kinase assay was demonstrated for GH receptors isolated from the transfected RIN5-AH cells. These studies show that the cloned liver GH receptor can be tyrosyl phosphorylated when expressed in a variety of cell types. The finding that the level of phosphorylation of GH receptor appears to vary with cell type is consistent with the cloned liver GH receptor being a substrate for an associated tyrosine kinase and with the amount of such a GH receptor-associated tyrosine kinase being cell type-specific.     

Single nucleotide polymorphism of human platelet-activating factor receptor impairs G-protein activation

Various proinflammatory and vasoactive actions of platelet-activating factor (PAF) are mediated through a specific G-protein-coupled PAF receptor (PAFR). We identified a novel DNA variant in the human PAFR gene, which substitutes an aspartic acid for an alanine residue at position 224 (A224D) in the putative third cytoplasmic loop. This mutation was observed in a Japanese population at an allele frequency of 7.8%. To delineate the functional consequences of this structural alteration, Chinese hamster ovary cells were stably transfected with constructs encoding either wild-type or A224D mutated PAFR. No significant difference was observed in the expression level of the receptor or the affinity to PAF or to an antagonist, WEB2086, between the cells transfected with wild-type and mutant PAFR. Chinese hamster ovary cells expressing A224D mutant PAFR displayed partial but significant reduction of PAF-induced intracellular signals such as calcium mobilization, inositol phosphate production, inhibition of adenylyl cyclase, and chemotaxis. These findings suggest that this variant receptor produced by a naturally occurring mutation exhibits impaired coupling to G-proteins and may be a basis for interindividual variation in PAF-related physiological responses, disease predisposition or phenotypes, and drug responsiveness.     

Agonist-dependent phosphorylation of the G protein-coupled receptor kinase 2 (GRK2) by Src tyrosine kinase

GRK2 is a member of the G protein-coupled receptor kinase (GRK) family, which phosphorylates the activated form of a variety of G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. It has been recently reported that stimulation of the mitogen-activated protein kinase cascade by GPCRs involves tyrosine phosphorylation of docking proteins mediated by members of the Src tyrosine kinase family. In this report, we have investigated the possible role of c-Src in modulating GRK2 function. We demonstrate that c-Src can directly phosphorylate GRK2 on tyrosine residues, as shown by in vitro experiments with purified proteins. The phosphorylation reaction exhibits an apparent K(m) for GRK2 of 12 nM, thus suggesting a physiological relevance in living cells. Consistently, overexpression of the constitutively active c-Src Y527F mutant in COS-7 cells leads to tyrosine phosphorylation of co-expressed GRK2. In addition, GRK2 can be detected in phosphotyrosine immunoprecipitates from HEK-293 cells transiently transfected with this Src mutant. Interestingly, phosphotyrosine immunoblots reveal a rapid and transient increase in GRK2 phosphorylation upon agonist stimulation of beta(2)-adrenergic receptors co-transfected with GRK2 and wild type c-Src in COS-7 cells. This tyrosine phosphorylation is maximal within 5 min of isoproterenol stimulation and reaches values of approximately 5-fold over basal conditions. Furthermore, GRK2 phosphorylation on tyrosine residues promotes an increased kinase activity toward its substrates. Our results suggest that GRK2 phosphorylation by c-Src is inherent to GPCR activation and put forward a new mechanism for the regulation of GPCR signaling.     

GTK tyrosine kinase-induced alteration of IRS-protein signalling in insulin producing cells

BACKGROUND: Insulin receptor substrate proteins (IRS) mediate various effects of insulin, including regulation of glucose homeostasis, cell growth and survival. To understand the underlying mechanisms explaining the effects of the Src-related tyrosine kinase GTK on beta-cell proliferation and survival, insulin-signalling pathways involving IRS-1 and IRS-2 were studied in islet cells and RINm5F cells overexpressing wild-type and two different mutants of the SRC-related tyrosine kinase GTK. MATERIALS AND METHODS: Islets isolated from transgenic mice and RINm5F cells overexpressing wild-type and mutant GTK were analysed for IRS-1, IRS-2, SHB, AKT and ERK phosphorylation/activity by Western blot analysis. RESULTS: RINm5F cells expressing the kinase active mutant Y504F-GTK and islet cells from GTK(Y504F) -transgenic mice exhibited reduced insulin-induced tyrosine phosphorylation of IRS-1 and IRS-2. In RINm5F cells, the diminished IRS-phosphorylation was accompanied by a reduced insulin-stimulated activation of phosphatidylinositol 3-kinase (PI3K), AKT and Extracellular Signal-Regulated Kinase, partly due to an increased basal activity. In addition, increased tyrosine phosphorylation of the SHB SH2 domain-adaptor protein and its association with IRS-2, IRS-1 and focal adhesion kinase was observed in these cells. RINm5F cells overexpressing wild-type GTK also exhibited reduced activation of IRS-2, PI3K and AKT, whereas cells expressing a GTK mutant with lower kinase activity (GTK(Y394F)) exhibited insignificantly altered responses to insulin compared to the mock transfected cells. Moreover, GTK was shown to associate with and phosphorylate SHB in transiently transfected COS-7 cells, indicating that SHB is a specific substrate for GTK. CONCLUSIONS: The results suggest that GTK signals via SHB to modulate insulin-stimulated pathways in beta cells and this may explain previous results showing an increased beta-cell mass in GTK-transgenic mice.     

Lipoteichoic acid-induced nitric oxide production depends on the activation of platelet-activating factor receptor and Jak2

NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-alpha and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-alpha. Jak2 tyrosine kinase inhibition blocked LTA-induced production of NO but not TNF-alpha. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-beta expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-alpha production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-beta, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production.     

Epidermal growth factor receptor tyrosine kinase mediates Ras activation by gonadotropin-releasing hormone

Gonadotropin releasing hormone (GnRH) contributes to the maintenance of gonadotrope function by increasing extracellular signal-regulated kinase (ERK) activity subsequent to binding to its cognate G-protein-coupled receptor. As the GnRH receptor exclusively interacts with G(q/11) proteins and as receptor expression is regulated in a beta-arrestin-independent fashion, it represents a good model to systematically dissect underlying signaling pathways. In alphaT3-1 gonadotropes endogenously expressing the GnRH receptor, GnRH challenge resulted in a rapid increase in ERK activity which was attenuated by the epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitor AG1478. In COS-7 cells transiently expressing the human GnRH receptor, agonist-induced ERK activation was independent of free Gbetagamma subunits but could be mimicked by short-term phorbol ester treatment. Most notably, G(q/11)-induced ERK activation was sensitive to N17-Ras and to expression of the C-terminal Src kinase but also to other dominant negative mutants of signaling components localized upstream of Ras, like Shc and the EGFR. GnRH as well as phorbol esters led to Ras activation in COS-7 and alphaT3-1 cells, which was dependent on Src and EGFR tyrosine kinases, indicating that both tyrosine kinases act downstream of protein kinase C (PKC) and upstream of Ras. However, Src did not contribute to Shc tyrosine phosphorylation. GnRH or phorbol ester challenge resulted in PKC-dependent EGFR autophosphorylation. Furthermore, a 5-min phorbol ester treatment was sufficient to trigger tyrosine phosphorylation of the platelet-derived growth factor-beta receptor in L cells. Thus, in several cell systems PKC is able to stimulate Ras via activation of receptor tyrosine kinases.     

Distinct roles of receptor phosphorylation, G protein usage, and mitogen-activated protein kinase activation on platelet activating factor-induced leukotriene C(4) generation and chemokine production

Platelet activating factor (PAF) interacts with cell surface G protein-coupled receptors on leukocytes to induce degranulation, leukotriene C(4) (LTC(4)) generation, and chemokine CCL2 production. Using a basophilic leukemia RBL-2H3 cell line expressing wild-type PAF receptor (PAFR) and a phosphorylation-deficient mutant (mPAFR), we have previously demonstrated that receptor phosphorylation mediates desensitization of PAF-induced degranulation. Here, we sought to determine the role of receptor phosphorylation on PAF-induced LTC(4) generation and CCL2 production. We found that PAF caused a significantly enhanced LTC(4) generation in cells expressing mPAFR when compared with PAFR cells. In contrast, PAF-induced CCL2 production was greatly reduced in mPAFR cells. Pertussis toxin and U0126, which inhibit G(i) and p44/42 mitogen-activated protein kinase (ERK) activation, respectively, caused very little inhibition of PAF-induced CCL2 production (approximately 20% inhibition). In contrast, these inhibitors almost completely blocked both PAF-induced ERK phosphorylation and LTC(4) generation in PAFR cells. However, in mPAFR cells pertussis toxin only partially inhibited PAF-induced ERK phosphorylation. A Ca(2+)/calmodulin inhibitor had no effect on PAF-induced ERK phosphorylation in PAFR cells but completely blocked the response in mPAFR cells. These data demonstrate that receptor phosphorylation, which serves to desensitize PAF-induced LTC(4) generation, is required for chemokine CCL2 production. They also indicate a previously unrecognized selectivity in G protein usage and ERK activation for PAF-induced responses. Whereas PAF-induced CCL2 production is, in large part, mediated independently of G(i) activation or ERK phosphorylation, LTC(4) generation requires ERK phosphorylation, which is mediated by different G proteins depending on the phosphorylation status of the receptor.     

Phosphorylation of STAT-3 in response to basic fibroblast growth factor occurs through a mechanism involving platelet-activating factor, JAK-2, and Src in human umbilical vein endothelial cells. Evidence for a dual kinase mechanism

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid with multiple pathological and physiological effects. We have shown that basic fibroblast growth factor (bFGF) supplementation induces rapid proliferation of human umbilical vein endothelial cells (HUVEC), which is reduced upon removal of bFGF or by bFGF immunoneutralization. The PAF receptor antagonist LAU-8080 inhibited bFGF-stimulated HUVEC proliferation, indicating the involvement of PAF in the bFGF-mediated signaling of HUVEC. Although FGF receptor phosphorylation was not affected by LAU-8080, the bFGF-mediated prolonged phosphorylation, and activation of Erk-1 and -2 were attenuated. Phosphorylation of STAT-3 was observed in the presence of PAF or bFGF, which was attenuated by PAFR antagonists. PAF-induced STAT-3 phosphorylation observed in HUVEC pretreated with either Src inhibitor PP1 or JAK-2 inhibitor AG-490 indicated (i) immediate (1 min) phosphorylation of STAT-3 is dependent on Src, (ii) JAK-2-dependent STAT-3 phosphorylation occurs after the delayed (30 min) PAF exposure, and (iii) prolonged (60 min) STAT-3 phosphorylation may be either through Src and/or JAK-2. Attenuation of the STAT-3 phosphorylation by the PAFR antagonists indicated signaling through the PAF receptor. Taken together, these findings suggest the production of PAF is important for bFGF-mediated signaling and that a dual kinase mechanism is involved in the PAF-mediated signal transduction cascade.     

Platelet-activating factor-induced chemokine gene expression requires NF-kappaB activation and Ca2+/calcineurin signaling pathways. Inhibition by receptor phosphorylation and beta-arrestin recruitment

Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activation and a more robust Ca2+ mobilization in RBL-2H3 cells expressing carboxyl terminus deletion, phosphorylation-deficient mutant of PAF receptor (mPAFR) when compared with the wild-type receptor (PAFR). However, PAF did not provide sufficient signal for CC chemokine receptor ligand 2 (CCL2) production in cells expressing mPAFR. Based on these findings, we hypothesized that receptor phosphorylation provides a G protein-independent signal that synergizes with Ca2+ mobilization to induce CCL2 production. Here, we show that a mutant of PAFR (D289A), which does not couple to G proteins, was resistant to agonist-induced receptor phosphorylation. Unexpectedly, we found that when this mutant was coexpressed with mPAFR, it restored NF-kappaB activation and CCL2 production. PAF caused translocation of beta-arrestin from the cytoplasm to the membrane in cells expressing PAFR but not a phosphorylation-deficient mutant in which all Ser/Thr residues were replaced with Ala (DeltaST-PAFR). Interestingly, PAF induced significantly higher NF-kappaB and nuclear factor of activated T cells (NFAT)-luciferase activity as well as CCL2 production in cells expressing DeltaST-PAFR than those expressing PAFR. Furthermore, a Ca2+/calcineurin inhibitor completely inhibited PAF-induced NFAT activation and CCL2 production but not NF-kappaB activation. These findings suggest that the carboxyl terminus of PAFR provides a G protein-independent signal for NF-kappaB activation, which synergizes with G protein-mediated Ca2+/calcineurin activation to induce CCL2 production. However, receptor phosphorylation and beta-arrestin recruitment inhibit CCL2 production by blocking both NF-kappaB activation and Ca2+/calcineurin-dependent signaling pathways.     

Agonist-induced internalization of the platelet-activating factor receptor is dependent on arrestins but independent of G-protein activation. Role of the C terminus and the (D/N)PXXY motif.

As with most G-protein-coupled receptors, repeated agonist stimulation of the platelet-activating factor receptor (PAFR) results in its desensitization, sequestration, and internalization. In this report, we show that agonist-induced PAFR internalization is independent of G-protein activation but is dependent on arrestins and involves the interaction of arrestins with a limited region of the PAFR C terminus. In cotransfected COS-7 cells, both arrestin-2 and arrestin-3 could be coimmunoprecipitated with PAFR, and agonist stimulation of PAFR induced the translocation of both arrestin-2 and arrestin-3. Furthermore, coexpression of arrestin-2 with PAFR potentiated receptor internalization, whereas agonist-induced PAFR internalization was inhibited by a dominant negative mutant of arrestin-2. The coexpression of a minigene encoding the C-terminal segment of the receptor abolished PAF-induced arrestin translocation and inhibited PAFR internalization. Using C terminus deletion mutants, we determined that the association of arrestin-2 with the receptor was dependent on the region between threonine 305 and valine 330 because arrestin-2 could be immunoprecipitated with the mutant PAFRstop330 but not PAFRstop305. Consistently, stop330 could mediate agonist-induced arrestin-2 translocation, whereas stop305 could not. Two other deletion mutants with slightly longer regions of the C terminus, PAFRstop311 and PAFRstop317, also failed to induce arrestin-2 translocation. Finally, the PAFR mutant Y293A, containing a single substitution in the putative internalization motif DPXXY in the seventh transmembrane domain (which we had shown to be able to internalize but not to couple to G-proteins) could efficiently induce arrestin translocation. Taken together, our results indicate that ligand-induced PAFR internalization is dependent on arrestins, that PAFR can associate with both arrestin-2 and -3, and that their translocation involves interaction with the region of residues 318-330 in the PAFR C terminus but is independent of G-protein activation.