Evidence for a physical association between the Shc-PTB domain and the beta c chain of the granulocyte-macrophage colony-stimulating factor receptor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates the growth and function of several myeloid cell types at different stages of maturation. The effects of GM-CSF are mediated through a high affinity receptor that is composed of two chains: a unique, ligand-specific alpha chain and a beta common chain (beta c) that is also a component of the receptors for interleukin 3 (IL-3) and IL-5. Beta c plays an essential role in the transduction of extra cellular signals to the nucleus through its recruitment of secondary messengers. Several downstream signaling events induced by GM-CSF stimulation have been described, including activation of tyrosine kinases and tyrosine phosphorylation of cellular proteins (including beta c) and activation of the Ras/mitogen-activated protein kinase and the JAK/STAT pathways. A region within the beta c cytoplasmic tail (amino acids 517-763) has been reported to be necessary for tyrosine phosphorylation of the adapter protein, Shc, and for the subsequent GM-CSF-induced activation of Ras. In this paper, we describe a physical association between the tyrosine phosphorylated GM-CSF receptor (GMR)-beta c chain and Shc in vivo. Using a series of cytoplasmic truncation mutants of beta c and various mutant Shc proteins, we demonstrate that the N-terminal phosphotyrosine-binding (PTB) domain of Shc binds to a short region of beta c (amino acids 549-656) that contains Tyr577. Addition of a specific phosphopeptide encoding amino acids surrounding this tyrosine inhibited the interaction between beta c and shc. Moreover, mutation of a key residue within the phosphotyrosine binding pocket of the Shc-PTB domain abrogated its association with beta c. These observations provide an explanation for the previously described requirement for Tyr577 of beta c for GM-CSF-induced tyrosine phosphorylation of Shc and have implications for Ras activation through the GM-CSF, IL-3, and IL-5 receptors.     

APS, an adaptor protein containing PH and SH2 domains, is associated with the PDGF receptor and c-Cbl and inhibits PDGF-induced mitogenesis

Previously we cloned a novel adaptor protein, APS (adaptor molecules containing PH and SH2 domains) which was tyrosine phosphorylated in response to c-kit or B cell receptor stimulation. Here we report that APS was expressed in some human osteosarcoma cell lines, markedly so in SaOS-2 cells, and was tyrosine-phosphorylated in response to several growth factors, including platelet derived growth factor (PDGF), insulin-like growth factor (IGF), and granulocyte-macrophage colony stimulating factor (GM-CSF). Ectopic expression of the wild type APS, but not C-terminal truncated APS, in NIH3T3 fibroblasts suppressed PDGF-induced MAP kinase (Erk2) activation, c-fos and c-myc induction as well as cell proliferation. In vitro binding experiments suggest that APS bound to the beta type PDGF receptor, mainly via phosphotyrosine 1021 (pY1021). Indeed, tyrosine phosphorylation of PLC-gamma, which has been demonstrated to bind to pY1021, but not that of PI3 kinase and associated proteins, was reduced in APS transformants. PDGF induced phosphorylation of the tyrosine residue of APS close to the C-terminal end. In vitro and in vivo binding experiments indicate that the tyrosine phosphorylated C-terminal region of APS bound to c-Cbl, which has been shown to be a negative regulator of tyrosine kinases. Since coexpression of c-Cbl with wild type APS, but not C-terminal truncated APS, synergistically inhibited PDGF-induced c-fos promoter activation, c-Cbl could be a mechanism of inhibitory action of APS on PDGF receptor signaling.     

The treatment of disseminated malignant melanoma with special reference to the role of interferons, vinca alkaloids and tamoxifen

Treatment of quiescent NIH3T3 cells with PDGF BB results in the transient activation and hyperphosphorylation of the protein-tyrosine kinase, c-Src. These effects correlate with novel serine and tyrosine phosphorylations in the N-terminal non-catalytic region of the molecule, which contains an SH3 and SH2 domain. In this study, a site of PDGF-induced tyrosine phosphorylation was mapped to Tyr 138 in the SH3 domain; Tyr 138 is exposed on the SH3 peptide binding surface. This same site is phosphorylated in vitro by the PDGF receptor when purified baculovirus-expressed c-Src is complexed with the activated receptor. Phosphorylation of Tyr 138 required association of c-Src with the PDGF receptor via its SH2 domain. When a c-Src Phe 138 mutant was stably expressed in Src- mouse fibroblasts, it was activated to the same extent as wild type c-Src following PDGF stimulation, indicating that phosphorylation of this site is not required for PDGF-mediated activation. However, Tyr 138 phosphorylation was found to diminish SH3 domain peptide ligand binding ability in vitro.     

Tyrosine phosphorylation of the alpha and beta subunits of the type I interferon receptor. Interferon-beta selectively induces tyrosine phosphorylation of an alpha subunit-associated protein

We studied the phosphorylation of the alpha and beta subunits of the Type I interferon (IFN) receptor induced by Type I IFNs in the human U-266 and MOLT-4 cell lines. Both IFN-alpha and IFN-beta induced tyrosine phosphorylation of the beta subunit of the receptor. The Type I IFN-induced tyrosine phosphorylation of the beta subunit was rapid and transient, being detectable within 1 min of Type I IFN treatment and gradually diminishing to almost base-line levels by 60 min. All Type I IFNs studied were found to induce tyrosine phosphorylation of the alpha subunit of the Type I IFN receptor, the p135tyk2 and JAK-1 tyrosine kinases, and the ISGF3 alpha components. Interestingly, IFN-beta, but not IFN-alpha or IFN-omega, induced tyrosine phosphorylation of an alpha subunit-associated protein with an apparent molecular mass of approximately 100 kDa (p100). These data suggest the existence of a common signaling pathway(s) for Type I IFNs involving the alpha and beta subunits of the receptor, the tyrosine kinases p135tyk2 and JAK-1, and the ISGF3 alpha components. However, differences between the signaling pathways of different Type I IFNs exist, as suggested by tyrosine phosphorylation of an alpha subunit-associated protein only in response to IFN-beta.     

Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production, maturation, and function of cells in multiple hematopoietic lineages. These effects are mediated by a cell-surface receptor (GM-R) composed of alpha and beta subunits, each containing 378 and 881 amino acids, respectively. Whereas the alpha subunit exists as several isoforms that bind GM-CSF with low affinity, the beta common subunit (beta c) does not bind GM-CSF itself, but acts as a high-affinity converter for GM-CSF, interleukin-3 (IL-3), and IL-5 receptor alpha subunits. The cytoplasmic region of GM-R alpha consists of a membrane-proximal conserved region shared by the alpha 1 and alpha 2 isoforms and a C-terminal variable region that is divergent between alpha 1 and alpha 2. The cytoplasmic region of beta c contains membrane proximal serine and acidic domains. To investigate the amino acid sequences that influence signal transduction by this receptor complex, we constructed a series of cytoplasmic truncation mutants of the alpha 2 and beta subunits. To study these truncations, we stably transfected the IL-3-dependent murine cell line Ba/F3 with wild-type or mutant cDNAs. We found that the wild-type and mutant alpha subunits conferred similar low-affinity binding sites for human GM-CSF to Ba/F3, and the wild-type or mutant beta subunit converted some of these sites to high-affinity; the cytoplasmic domain of beta was unnecessary for this high-affinity conversion. Proliferation assays showed that the membrane-proximal conserved region of GM-R alpha and the serine-acidic domain of beta c are required for both cell proliferation and ligand-dependent phosphorylation of a 93-kD cytoplasmic protein. We suggest that these regions may represent an important signal transduction motif present in several cytokine receptors.     

Transmembrane signaling by the interleukin-2 receptor: progress and conundrums

Activation of the multicomponent interleukin-2 receptor (IL-2R) complex leads to a rapid increase in tyrosine phosphorylation of a number of cellular proteins including the IL-2R beta and IL-2R gamma chains of the IL-2R and the RAF-1 serine threonine kinase. In addition, phosphatidylinositol 3-kinase (PI-3K) protein and activity can be immunoprecipitated with anti-phosphotyrosine and anti-IL-2R beta antibodies from IL-2-activated but not resting T lymphocytes. We have demonstrated that the SH2 (SRC homology 2) domains of the 85 kDa subunit of PI-3K are sufficient to mediate binding of the PI-3K complex to tyrosine phosphorylated, but not non-phosphorylated IL-2R beta, suggesting that tyrosine phosphorylation is an integral component of the activation of PI-3K by the IL-2R. Since none of the members of the IL-2R complex contains an intrinsic tyrosine kinase domain, IL-2-induced tyrosine phosphorylation must be the consequence of activation of intracellular tyrosine kinases. SRC family members including lck, lyn and fyn have been demonstrated to associate with IL-2R beta through binding of the kinase domain to the acidic domain of IL-2R beta. However, we have demonstrated that the serine rich (SD) region of the cytosolic domain of IL-2R beta is also required for association of a tyrosine kinase with the IL-2R complex and that IL-2 can induce proliferation and tyrosine phosphorylation in cell lines which lack the known SRC family kinases expressed by T lymphocytes. Thus members of other kinase families besides SRC may also be involved in mediating IL-2 signal transduction. Biochemical studies and studies of cells expressing mutant IL-2 receptors indicate that IL-2-induced tyrosine kinase activation initiates a complex signaling cascade. The cascade includes SRC family kinase members such as lck, fyn, and lyn, activation of Raf-1 and PI-3K, and ras, and increased expression of the fos, fra-1, and jun protooncogenes. In addition, ligation of the IL-2R leads to rapid increases in myc expression and more delayed increases in the expression of the cdc2 and cdk2 kinases and the cyclins through a tyrosine phosphorylation independent pathway. Whether other biochemical processes initiated by IL-2R ligation, including activation of the MAP2, p70S6 and p90RSK serine threonine kinases, activation of NF-kappa B, and increased expression of Raf-1, Pim-1, bcl-2, IL-2R alpha and IL-2R beta, are consequences of the IL-2-induced tyrosine kinase cascade remains to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vivo induction of specific cytotoxic T lymphocytes in mice and rhesus macaques immunized with DNA vector encoding an HIV epitope fused with hepatitis B surface antigen

BACKGROUND: In human blood basophils, cross-linking the high-affinity IgE receptor Fc epsilonRI with multivalent antigen activates a signaling pathway leading to Ca2+ mobilization, actin polymerization, shape changes, secretion, and cytokine production. METHODS AND RESULTS: The role of tyrosine kinases in human Fc epsilonRI signaling was explored by using human basophils isolated by Percoll gradient centrifugation followed by negative and/or positive selection with antibody-coated magnetic beads. Fc epsilonRI cross-linking of more than 95% pure basophil preparations activates the protein-tyrosine kinases Lyn and Syk, previously linked to Fc epsilonRI-coupled rodent mast cell activation, as well as Zap-70, previously implicated in T-cell receptor signaling, and causes the tyrosine phosphorylation of multiple proteins. The presence of Lyn, Syk, and Zap-70 in basophils was confirmed by Western blotting in lysates of highly purified basophils and independently by confocal fluorescence microscopy in cells labeled simultaneously with kinase-specific antibodies and with the basophil-specific antibody 2D7. Comparable amounts of Lyn and Syk were found in basophils and B cells, whereas T cells appear to have greater amounts of Zap-70 than basophils. The tyrosine kinase inhibitor piceatannol spares IgE-mediated Lyn activation but inhibits IgE-induced Syk and Zap-70 activation as well as overall protein tyrosine phosphorylation and secretion. Overall protein-tyrosine phosphorylation increases steadily over a range of anti-IgE concentrations that are low to optimal for secretion. However, tyrosine phosphorylation continues to increase at high anti-IgE concentrations that elicit very little secretion (the characteristic high-dose inhibition of secretion). CONCLUSIONS: Our data demonstrate the association of anti-IgE-stimulated, protein-tyrosine phosphorylation by a cascade of tyrosine kinases, including Zap-70 as well as Lyn and Syk, with the initiation of Fc epsilonRI-mediated signaling in human basophils.     

Detergent solubility defines an alternative itinerary for a subpopulation of PDGF beta receptors

Current models of platelet-derived growth factor (PDGF) beta receptor itinerary are based upon the properties of receptors recovered from nonionic detergent-solubilized cellular extracts. Comparing several commonly used cell extraction procedures, we have determined that up to 50% of immunoreactive PDGF beta receptors, reside in a Triton X-100 insoluble pool in a wide distribution of cultured cell lines, including Balb/c-3T3, NIH 3T3, and Swiss fibroblasts, primary murine and human fibroblasts, and primary human glial cells. Many properties of Triton insoluble receptors are distinct from the well-characterized PDGF beta receptors, including 1) delayed arrival of newly synthesized receptors into the Triton insoluble fraction, 2) prolonged half-life in the presence of PDGF, 3) increased abundance with increasing cell density, 4) inaccessibility to modification by extracellular compartment enzymes, 5) cofractionation with cytoskeletal proteins, and 6) a higher basal tyrosine phosphorylation state. PDGF stimulates accumulation of tyrosine phosphorylated PDGF beta receptors in the Triton X-100 insoluble fraction. Cell surface PDGF beta receptors modified by enzymatic desialylation redistribute to the insoluble fraction. These findings distinguish the itinerary of a large subpopulation of PDGF beta receptors from those characterized previously. Receptors in this fraction represent a long-lived tyrosine phosphorylated population that may effect responses for extended periods following ligand activation.     

Differences in interferon alpha and beta signaling. Interferon beta selectively induces the interaction of the alpha and betaL subunits of the type I interferon receptor

All Type I interferons (IFNalpha, IFNbeta, IFNomega) bind to the Type I IFN receptor (IFNR) and elicit a common set of signaling events, including activation of the Jak/Stat and IRS pathways. However, IFNbeta selectively induces the association of the alpha subunit of the Type I IFNR with p100, a tyrosyl phosphoprotein, to transduce IFNbeta-specific signals. Using antibodies raised against the different components of the Type I IFNR, we identified p100 as the long form of the beta subunit (betaL subunit) of the Type I IFNR. This was also confirmed in experiments with mouse L-929 cells transfected with truncated forms of betaL. Thus, IFNbeta stimulation of human cells or mouse L-929 transfectants expressing the human alpha and betaL subunits, selectively induces the formation of a signaling complex containing the alpha and betaL subunits of the receptor. The IFNbeta-regulated interaction of the alpha and betaL chains is rapid and transient and follows a similar time course with the tyrosine phosphorylation of these receptor components. These data demonstrate that the signaling specificity for different Type I IFNs is established early in the signaling cascade, at the receptor level, and results from distinct interactions between components of the Type I IFNR.     

Rapsyn clusters and activates the synapse-specific receptor tyrosine kinase MuSK

Nerve-induced clustering of the nicotinic acetylcholine receptor (AChR) requires rapsyn, a synaptic peripheral membrane protein, as well as protein-tyrosine kinase activity. Here, we show that rapsyn induces the clustering of the synapse-specific receptor-tyrosine kinase MuSK in transfected QT-6 fibroblasts. Furthermore, rapsyn stimulates the autophosphorylation of MuSK, leading to a subsequent MuSK-dependent increase in cellular tyrosine phosphorylation. Moreover, rapsyn-activated MuSK specifically phosphorylated the AChR beta subunit, the same subunit that is tyrosine phosphorylated during innervation or agrin treatment of muscle. These results suggest rapsyn may mediate the synaptic localization of MuSK in muscle and that MuSK may play an important role in the agrin-induced clustering of the AChR.     

Calmodulin dependence of NFkappaB activation

TF-1/TPO cells are derived from an erythroleukemia cell line, TF-1, and are absolutely dependent on either TPO or granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin-3 (IL3) for their continuous growth and survival. To gain insight into the molecular basis of hemopoietic activities shared by TPO and GM-CSF/IL3 in TF-1/TPO cells, we studied the cross-talk between signal transduction pathways elicited by these cytokines. Stimulation of TF-1/TPO cells with TPO resulted in tyrosine phosphorylation of the TPO receptor (c-Mpl) as well as the common beta subunit (beta c) of GM-CSF/IL3 receptor complex. GM-CSF, however, induced tyrosine phosphorylation of beta c but not c-Mpl. TPO-induced tyrosine phosphorylation of beta c was time- and dose-dependent. We next examined whether or not TPO-induced tyrosine phosphorylation of beta c led to recruitment of SH2-containing molecules such as Stat5 and Shc. While GM-CSF caused association of Stat5 and Shc with beta c, TPO caused association of Stat5, but not Shc, with beta c, suggesting that TPO and GM-CSF may not induce phosphorylation of the same sets of tyrosine residues in beta c. These results suggest that activation of c-Mpl affects the signaling pathway of GM-CSF/IL3 but not vice versa.     

A transmembrane-anchored chimeric focal adhesion kinase is constitutively activated and phosphorylated at tyrosine residues identical to pp125FAK

Focal adhesion kinase, pp125FAK, is a nonmyristylated cytosolic tyrosine kinase unrelated to protein-tyrosine kinase families categorized to date. The kinase activity and tyrosine phosphorylation of pp125FAK are induced by beta 1 and beta 3 integrin-mediated cell adherence or aggregation. pp125FAK is also a tyrosine phosphorylation substrate in v-src-transformed cells and is localized to focal adhesion contracts of adherent fibroblasts and carcinoma cells. In this report, we have transiently expressed in COS cells a transmembrane-anchored chimeric receptor kinase, CD2FAK, consisting of CD2 and pp125FAK. We analyzed its kinase activity and tyrosine phosphorylation and compared to those of pp125FAK. We found that CD2FAK exhibited constitutive kinase activity and a high basal tyrosine phosphorylation level when COS transfectants were suspended in serum-free media. The kinase activity of CD2FAK was similarly up-regulated upon beta 1 integrin-mediated cell adherence as the endogenous pp125FAK. Both CD2FAK and pp125FAK appeared to be active as autophosphorylating kinases as shown by mutation of the ATP binding site. We determined the major tyrosine phosphorylation site, Tyr397, identical for both the constitutively activated CD2FAK and pp125FAK in response to beta 1 integrin-mediated cell adherence by site-directed mutagenesis. Deletions of the NH2- or the COOH-terminal noncatalytic domain of FAK, including Tyr397 did not lead to abolition of the kinase activity of pp125FAK or CD2FAK. Taken together, CD2FAK exhibits properties of an activated pp125FAK and the kinase activity does not appear to require tyrosine phosphorylation in vitro or in vivo.     

Involvement of the Src homology 2-containing tyrosine phosphatase SHP-2 in growth hormone signaling

Growth hormone (GH) signaling requires activation of the GH receptor (GHR)-associated tyrosine kinase, JAK2. JAK2 activation by GH is believed to facilitate initiation of various pathways including the Ras, mitogen-activated protein kinase, STAT, insulin receptor substrate (IRS), and phosphatidylinositol 3-kinase systems. In the present study, we explore the biochemical and functional involvement of the Src homology 2 (SH2)-containing protein-tyrosine phosphatase, SHP-2, in GH signaling. GH stimulation of murine NIH 3T3-F442A fibroblasts, cells that homologously express GHRs, resulted in tyrosine phosphorylation of SHP-2. As assessed specifically by anti-SHP-2 coimmunoprecipitation and by affinity precipitation with a glutathione S-transferase fusion protein incorporating the SH2 domains of SHP-2, GH induced formation of a complex of tyrosine phosphoproteins including SHP-2, GHR, JAK2, and a glycoprotein with properties consistent with being a SIRP-alpha-like molecule. A reciprocal binding assay using IM-9 cells as a source of SHP-1 and SHP-2 revealed specific association of SHP-2 (but not SHP-1) with a glutathione S-transferase fusion incorporating GHR cytoplasmic domain residues 485-620, but only if the fusion was first rendered tyrosine-phosphorylated. GH-dependent tyrosine phosphorylation of SHP-2 was also observed in murine 32D cells (which lack IRS-1 and -2) stably transfected with the GHR. Further, GH-dependent anti-SHP-2 coimmunoprecipitation of the Grb2 adapter protein was detected in both 3T3-F442A and 32D-rGHR cells, indicating that biochemical involvement of SHP-2 in GH signaling may not require IRS-1 or -2. Finally, GH-induced transactivation of a c-Fos enhancer-driven luciferase reporter in GHR- and JAK2-transfected COS-7 cells was significantly reduced when a catalytically inactive SHP-2 mutant (but not wild-type SHP-2) was coexpressed; in contrast, expression of a catalytically inactive SHP-1 mutant allowed modestly enhanced GH-induced transactivation of the reporter in comparison with that found with expression of wild-type SHP-1. Collectively, these biochemical and functional data imply a positive role for SHP-2 in GH signaling.     

Growth hormone, interferon-gamma, and leukemia inhibitory factor utilize insulin receptor substrate-2 in intracellular signaling

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is tyrosyl-phosphorylated following stimulation of 3T3-F442A fibroblasts with growth hormone (GH), leukemia inhibitory factor and interferon-gamma. In response to GH and leukemia inhibitory factor, IRS-2 is immediately phosphorylated, with maximal phosphorylation detected at 15 min; the signal is substantially diminished by 60 min. In response to interferon-gamma, tyrosine phosphorylation of IRS-2 was prolonged, with substantial signal still detected at 60 min. Characterization of the mechanism of signaling utilized by GH indicated that tyrosine residues in GH receptor are not necessary for tyrosyl phosphorylation of IRS-2; however, the regions of GH receptor necessary for IRS-2 tyrosyl phosphorylation are the same as those required for JAK2 association and tyrosyl phosphorylation. The role of IRS-2 as a signaling molecule for GH is further demonstrated by the finding that GH stimulates association of IRS-2 with the 85-kDa regulatory subunit of phosphatidylinositol 3'-kinase and with the protein-tyrosine phosphatase SHP2. These results are consistent with the possibility that IRS-2 is a downstream signaling partner of multiple members of the cytokine family of receptors that activate JAK kinases.     

A model for the interaction of the GM-CSF, IL-3 and IL-5 receptors with their ligands

The high affinity receptors for GM-CSF, IL-3 and IL-5 are heterodimers consisting of a ligand-specific alpha chain and a common beta chain. These proteins are members of a family of proteins known as the "cytokine receptor family" which is characterized by the presence of a 200-residue ligand-binding module. The GM-CSF, IL-3 and IL-5 receptor alpha chains constitute a distinct subgroup and share features not found in other members of the cytokine receptor family, features which we propose to be important for their interaction with the common beta chain and for their binding of the structurally-related ligands. The growth hormone receptor is a well-characterized member of the cytokine receptor family. Based on the structure of the complex between growth hormone and its receptor, we have proposed sites of contact between the GM-CSF, IL-3 and IL-5 receptors and their cognate ligands.     

Regulation of radiation-induced apoptosis in oncogene-transfected fibroblasts: influence of H-ras on the G2 delay

The high-affinity receptor (R) for IL-5 consists of a unique alpha chain (IL-5R alpha) and a beta chain (beta c) that is shared with the receptors for IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF). We defined two regions of IL-5R alpha for the IL-5-induced proliferative response, the expression of nuclear proto-oncogenes, and the tyrosine phosphorylation of cellular proteins including beta c, SH2/SH3-containing proteins and JAK2 kinase. In the studies described here, we demonstrate that IL-5, IL-3 or GM-CSF stimulation induces the tyrosine phosphorylation of JAK2, and to a lesser extent JAK1, and of STAT5. Mutational analysis revealed that one of the proline residues, particularly Pro352 and Pro355, in the membrane-proximal proline-rich sequence (Pro352-Pro353-X-Pro355) of the cytoplasmic domain of IL-5R alpha is required for cell proliferation, and for both JAK1 and JAK2 activation. In addition, transfectants expressing chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c responded to IL-5 for proliferation and tyrosine phosphorylation of JAK1. Intriguingly, electrophoretic mobility shift assay analysis revealed that STAT5 was activated in cells showing either JAK1 or JAK2 tyrosine phosphorylation. These results indicate that activation of JAK1, JAK2 and STAT5 is critical to coupling IL-5-induced tyrosine phosphorylation and ultimately mitogenesis, and that Pro352 and Pro355 in the proline-rich sequence appear to play more essential roles in cell growth and in both JAK1/STAT5 and JAK2/STAT5 activation than Pro353 does.