Purification of the murine interleukin 3 receptor.

In this report we describe the purification of the murine interleukin 3 receptor (mIL-3R) to apparent homogeneity using a two-step procedure involving biotinylated mIL-3 (B-mIL-3) and affinity binding to immobilized antiphosphotyrosine and streptavidin agarose (SA). Purification was monitored using an assay for detergent solubilized-mIL-3Rs that utilized unglycosylated 125I-mIL-3 and concanavalin A (ConA)-Sepharose beads. The final material consisted of a 140-kDa tyrosine and serine phosphorylated protein that was greater than 98% pure as assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of either [35S]methionine-labeled, silver-stained, or radioiodinated preparations. Characterization of the purified receptor revealed that it migrated identically under reducing and nonreducing conditions in SDS gels, possessed 10 kDa of N-linked carbohydrate, and was cleaved upon storage at 4 degrees C to a 70-kDa form. These properties suggested that the purified mIL-3R was identical to that identified by cross-linking studies. The KD of the purified receptor was 1-5 nM, similar to estimates obtained using intact normal mouse bone marrow cells and mIL-3-dependent cell lines. The two-step purification procedure also isolated a 120-kDa serine phosphorylated but nontyrosine phosphorylated mIL-3R species. Apart from phosphorylation differences, the 140- and 120-kDa species were apparently identical, yielding, after alkaline phosphatase treatment, the same molecular mass on SDS gels and similar chymotryptic peptide maps. Amino acid sequences and composition data obtained from the more abundant and more stable serine phosphorylated 120-kDa mIL-3R, further purified by SDS-polyacrylamide gel electrophoresis, suggested that the purified mIL-3R may be identical to the predicted sequence of the recently isolated cDNA clone AIC2A. This was further suggested by comparing chymotryptic maps of the 120-kDa mIL-3R with the Aic2A protein and using antibodies corresponding to the amino and carboxyl termini of the AIC2A cDNA product. However, the Aic2A protein, when expressed on the surface of COS or 3T3 cells or following detergent solubilization and partial purification with biotinylated mIL-3 and SA, displayed a substantially lower affinity for mIL-3.     

Growth hormone stimulates the tyrosine phosphorylation of 42- and 45-kDa ERK-related proteins.

Growth hormone (GH) influences a number of tissue-specific biological activities in diverse cell types. However, little is known about the biochemical pathway by which the signal initiated by GH binding to its cell-surface receptor is transduced. The GH receptor has been reported to be phosphorylated on tyrosine in 3T3-F442A cells, a cell line in which GH promotes differentiation and inhibits mitogen-stimulated growth; however, it is not known whether tyrosine phosphorylation plays a role in GH signal transduction. We report that GH treatment of 3T3-F442A cells resulted in the rapid tyrosine phosphorylation of at least four proteins. These included 42- (pp42) and 45-kDa (pp45) proteins immunologically related to ERK1 (extracellular signal-regulated kinase 1), a member of a family of serine/threonine protein kinases that are phosphorylated on tyrosine in response to mitogens. Prolonged phorbol ester pretreatment attenuated the tyrosine phosphorylation of pp42 and pp45 in platelet-derived growth factor-treated cells, but not in GH-treated cells. Maximal GH-stimulated tyrosine phosphorylation of pp42 and pp45 coincided with peak levels of a 42-kDa renaturable MBP kinase activity in lysates of GH-treated cells resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The observation that multiple cellular proteins are rapidly phosphorylated on tyrosine in response to physiological concentrations of GH suggests that tyrosine phosphorylation plays a role in GH signal transduction. Moreover, the stimulation of tyrosine phosphorylation of ERK-related proteins by GH suggests that mitogens and nonmitogens may employ common phosphotyrosyl proteins in the activation of ultimately distinct cellular programs.     

Entamoeba histolytica: tyrosine kinase activity induced by fibronectin through the beta1-integrin-like molecule

Previously, we characterized a 140-kDa protein from Entamoeba histolytica as a beta1-integrin-like molecule that binds fibronectin. In this work we present data showing that the amoebic receptor is associated with another surface molecule, the 220-kDa lectin, and with protein tyrosine kinase activity. By immunoprecipitation with the alphabeta1Eh antibody, we demonstrated by immune complex assays for tyrosine protein kinases that the amoebic fibronectin receptor was associated with two phosphorylated proteins of 50 and 70 kDa when internal membranes were used as the source of antigen. When cells were stimulated with fibronectin, two proteins of 55 and 90 kDa were tyrosine phosphorylated, as shown by Western blot with alphaPY20, its phosphorylation being time dependent after fibronectin stimulation. However, when the actin cytoskeleton of fibronectin-stimulated trophozoites was stabilized with phalloidin, the level and the pattern of phosphorylated proteins were different. In this case, a high-molecular-weight component, heavily phosphorylated, was present, which may include the 220-kDa lectin. We also present data confirming that the signaling pathway that is activated by fibronectin is specific. This was demonstrated by comparing the pattern of phosphoproteins obtained in immune complexes prepared with alphabeta1Eh, alphaL220, and alphaPY20 from total extracts obtained in the presence of phalloidin, from cells that had been exposed to fibronectin, soluble concanavalin A, or concanavalin-A-coated substrate. The presence of tyrosine kinases associated with the beta1-integrin-like amoebic molecule was confirmed by immunoprecipitation assays along with the combined use of a tyrosine kinase-specific substrate, the peptide RR-SRC, and a tyrosine kinase inhibitor, genistein.     

The tyrosine-phosphorylated hepatocyte growth factor/scatter factor receptor associates with phosphatidylinositol 3-kinase.

The receptor for hepatocyte growth factor, also known as scatter factor (HGF/SF), has recently been identified as the 190-kDa heterodimeric tyrosine kinase encoded by the MET proto-oncogene (p190MET). The signaling pathway(s) triggered by HGF/SF are unknown. In A549 cells, a lung epithelial cell line, nanomolar concentrations of HGF/SF induced tyrosine phosphorylation of the p190MET receptor. The autophosphorylated receptor coprecipitated with phosphatidylinositol 3-kinase (PI 3-kinase) activity. In GTL16 cells, a cell line derived from a gastric carcinoma, the p190MET receptor, overexpressed and constitutively phosphorylated on tyrosine, coprecipitated with PI 3-kinase activity and with the 85-kDa PI 3-kinase subunit. In these cells activation of protein kinase C or the increase of intracellular [Ca2+] inhibits tyrosine phosphorylation of the p190MET receptor as well as the association with both PI 3-kinase activity and the 85-kDa subunit of the enzyme. In an in vitro assay, tyrosine phosphorylation of the immobilized p190MET receptor was required for binding of PI 3-kinase from cell lysates. These data strongly suggest that the signaling pathway activated by the HGF/SF receptor includes generation of D-3-phosphorylated inositol phospholipids.     

Molecular basis of a high affinity murine interleukin-5 receptor.

The mouse interleukin-5 receptor (mIL-5R) consists of two components one of which, the mIL-5R alpha-chain, binds mIL-5 with low affinity. Recently we demonstrated that monoclonal antibodies (Mabs) recognizing the second mIL-5R beta-chain, immunoprecipitate a p130-140 protein doublet which corresponds to the mIL-3R and the mIL-3R-like protein, the latter chain for which so far no ligand has been identified. In this study we show that a high affinity mIL-5R can be reconstituted on COS1 cells by co-expression of the mIL-5R alpha-chain with the mIL-3R-like protein (beta-chain). Cross-linking of 125I-labeled mIL-5 to the COS1 cells co-transfected with both cDNAs revealed the same pattern as in B13 cells, i.e. two proteins of 60 and 130 kd which correspond to the low affinity mIL-5R alpha-chain and the mIL-3R-like protein, respectively. The dissociation rate of mIL-5 from this reconstituted high affinity site was lower than that of the low affinity site, whereas the association rate was unchanged. Nonetheless, the apparent dissociation constant (Kd) for this reconstituted receptor was still 10-fold higher than the Kd observed for B13 cells. Although the mIL-3R is greater than 90% homologous to the mIL-3R-like protein, no increase in affinity for mIL-5 was detected on COS1 cells co-transfected with the cDNAs for the mIL-5R alpha-chain and the mIL-3R protein.     

A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine.

Platelet-derived growth factor (PDGF) stimulates autophosphorylation of the PDGF receptor and association of the receptor with several cytoplasmic molecules, including phosphatidylinositol-3 kinase (PI3 kinase). In this study we examined the association of PI3 kinase with immunoprecipitated autophosphorylated PDGF receptor in vitro. The PI3 kinase from cell lysates bound to the wild-type receptor but not to a mutant receptor that had a deletion of the kinase insert region. A protein of an apparent size of 85 kDa bound to the receptor, consistent with previous observations that a protein of this size is associated with PI3 kinase activity. In addition, 110- and 74-kDa proteins bound to the phosphorylated receptor. Dephosphorylated receptors lost the ability to bind PI3 kinase activity as well as the 85-kDa protein. A 20-amino-acid peptide composed of a sequence in the kinase insert region that included one of the autophosphorylation sites of the receptor (tyrosine 719) as well as a nearby tyrosine (Y708) blocked the binding of PI3 kinase to the receptor, but only when the peptide was phosphorylated on tyrosine residues. A scrambled version of the peptide did not block PI3 kinase binding to the receptor even when it was phosphorylated on tyrosine. These tyrosine-phosphorylated peptides did not block binding of phospholipase C-gamma or GTPase-activating protein to the receptor. In separate experiments (receptor blots), soluble radiolabeled receptor bound specifically to an 85-kDa protein present in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated 3T3 cell lysates that were transferred to nitrocellulose paper. The binding was blocked by the same tyrosine-phosphorylated peptides that prevented binding of PI3 kinase activity to immobilized receptors. These findings show that the PDGF receptor binds directly to an 85-kDa protein and to a PI3 kinase activity through specific sequences in the kinase insert region. The association of a 110-kDa protein with the receptor also involve these sequences, suggesting that this protein may be a subunit of the PI3 kinase. Phosphotyrosine is an essential structure required for the interactions of these proteins with the PDGF receptor.     

Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK

Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin alpha IIb beta 3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-beta 3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in alpha IIb beta3. Proteins of 50-68 KD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen- dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125FAK. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (c) alpha IIb beta 3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or gamma 400-411). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to alpha IIb beta 3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125FAK. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.     

Insulin stimulates phosphatidylinositol-3-kinase activity in rat adipocytes.

Phosphatidylinositol (PtdIns) 3-kinase is thought to participate in the signal transduction pathways initiated by the activation of receptor tyrosine kinases including the insulin receptor. To approach the physiological relevance of this enzyme in insulin signaling, we studied the activation of PtdIns-3-kinase in adipocytes, a major insulin target tissue for glucose transport and utilisation. To analyze possible interactions of the enzyme with cellular proteins, immunoprecipitations with the following antibodies were performed: (a) anti-phosphotyrosine antibodies, (b) two antibodies to the 85-kDa subunit of PtdIns-3-kinase (p85) and (c) an antibody to the 185-kDa major insulin receptor substrate (p185). We show that in cell extracts from adipocytes exposed to insulin, and after immunoprecipitation with an anti-phosphotyrosine antibody and an antibody to p85, we are able to detect a PtdIns-3-kinase activity stimulated by the hormone. Similarly, after immunoprecipitation with an antibody to p185, an increase in the PtdIns-3-kinase activity could be demonstrated. Taken together these results suggest that, upon insulin stimulation of fat cells, PtdIns-3-kinase itself is tyrosine phosphorylated and/or associated with an insulin receptor substrate, such as p185, which could function as a link between the insulin receptor and PtdIns-3-kinase. The PtdIns-3-kinase was activated within 1 min of exposure to insulin, and the half-maximal effect was reached at the same concentration, i.e. 3 nM, as for stimulation of the insulin receptor kinase. Subcellular fractionation showed that PtdIns-3-kinase activity was found both in the membranes and in the cytosol. Further, immunoprecipitation with an antibody to p85, which possesses the capacity to activate PtdIns-3-kinase, suggests that the presence of the enzyme in the membrane may be due to an insulin-induced recruitment of the PtdIns-3-kinase from the cytosol to the membrane. Finally, we used isoproterenol, which exerts antagonistic effects on insulin action. This drug was found to inhibit both the PtdIns-3-kinase and the insulin receptor activation by insulin, suggesting that the activation of the PtdIns-3-kinase was closely regulated by the insulin receptor tyrosine kinase. The occurrence of an insulin-stimulated PtdIns-3-kinase in adipocytes leads us to propose that this enzyme might be implicated in the generation of metabolic responses induced by insulin.     

Nerve growth factor stimulates the activities of the raf-1 and the mitogen-activated protein kinases via the trk protooncogene.

Nerve growth factor (NGF) binds to two structurally unrelated transmembrane proteins on the surface of PC-12 cells, a 75-kDa glycoprotein with a short cytoplasmic sequence, and the trk protooncogene (pp140c-trk), a protein tyrosine kinase activated by NGF. Immediately after binding to cells, NGF induces changes in serine/threonine phosphorylation of several proteins. We have explored the relative roles of these two NGF binding proteins in mediating the activation of two intracellular kinases that may be responsible for some of these phosphorylations. The raf-1 protooncogene is a serine/threonine kinase activated by several growth factors and oncogenic proteins. Treatment of PC-12 cells with NGF increases the serine and threonine phosphorylation of raf-1 in an anti-raf-1 immunoprecipitate kinase assay. This increased phosphorylation observed in vitro is dose-dependent and transient and is accompanied by the NGF-dependent shift in the mobility of immunoblotted raf-1 on SDS sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an effect thought to reflect phosphorylation. NGF-dependent activation of raf-1 is not dependent on protein kinase C, since prolonged exposure to phorbol esters under conditions that cause down-regulation of cellular protein kinase C activity has no effect on the NGF response. Expression of pp140c-trk in 3T3 fibroblasts (3T3-c-trk), as evidenced by cross-linking of 125I-NGF to the 140-kDa protein, permits the NGF-dependent activation of raf-1 kinase, detected in the immunoprecipitate kinase assay, anti-raf immunoblot shift on gel electrophoresis, and incorporation of [32P]orthophosphate into the raf-1 protein. The concentration dependence of raf-1 activation is identical in 3T3-c-trk and PC-12 cells, despite the absence of the 75-kDa NGF binding protein in 3T3-c-trk cells. NGF is without effect in untransfected 3T3 cells or in Chinese hamster ovary cells overexpressing p75, although raf-1 is present in these cells. Similarly, the NGF-dependent activation of mitogen-activated protein (MAP) kinase is detected in 3T3-c-trk cells, but not in untransfected 3T3 or Chinese hamster ovary cells overexpressing p75. As described for raf-1 activation, the NGF dose responses for MAP kinase activation in 3T3-c-trk and PC-12 cells are virtually superimposable. These data indicate that the activation of these two serine/threonine kinases by NGF is mediated solely by binding to and activating the pp140c-trk receptor.     

Characterization of three related murine interleukin-3 surface receptor proteins

Iodinated interleukin-3 (IL-3) can be covalently cross-linked to three specific surface glycoproteins with net molecular masses of 170, 140, and 65-70 kDa under conditions in which ligand internalization and degradation do not occur. These three proteins plus two additional non-ligand-binding proteins of 90 and 55 kDa can be purified by IL-3 affinity chromatography. Comparative two-dimensional analysis of the tryptic digests of these five proteins indicates that the ligand-binding proteins are highly related at the peptide level. Incubation of cells with 125I-IL-3 at 37 degrees C results in rapid time- and energy-dependent internalization and degradation of ligand. Under these conditions only the 140- and 65-70-kDa binding proteins, which can recycle to the surface after internalization, can be identified. The lability of the 170-kDa protein indicates that it may not recycle. Thus, an energy-dependent mechanism is responsible for internalization and may be necessary for any potential interconversion of the higher 170- or 140-kDa proteins to the lower 140- and/or 65-70-kDa binding proteins.     

Steel factor-induced tyrosine phosphorylation in murine mast cells. Common elements with IL-3-induced signal transduction pathways.

The c-kit/W gene encodes a transmembrane protein tyrosine kinase, which is the receptor for Steel factor (SLF). SLF shares many general characteristics of hemopoietic growth factors, stimulating the survival, proliferation, and differentiation of stem and progenitor cells. We have investigated the tyrosine phosphorylation events that ensue after SLF binding to the c-kit protein using primary cultures of murine mast cells as a model system and have compared the effects of SLF and IL-3. Proteins that became phosphorylated on tyrosine after treatment of cells with SLF included c-kit itself, and major protein substrates designated p130, p122, p118, p115, p112, p100, p77, p55, p44, and p42. The majority of these proteins were cytosolic and maximally phosphorylated within 2 min of growth factor treatment. Combinations of immunoprecipitation and immunoblotting with antibodies specific for proteins known to be associated with signaling pathways demonstrated that none of the major tyrosine-phosphorylated species correlated with phospholipase C-gamma 1, GTPase activating protein, or phosphatidylinositol 3' kinase. However, stimulation with SLF led to a modest increase in tyrosine phosphorylation of the 85-kDa subunit of the phosphatidylinositol 3' kinase and increased association with a 150-kDa phosphotyrosyl protein, likely to be c-kit. Two species that did correlate with known elements were the 44- and 42-kDa polypeptides, shown to be members of the mitogen-activated protein kinase family. A subset of these proteins (p130, p115/112, p100, p55, p44, p42) were also tyrosine-phosphorylated when cells were stimulated by IL-3. MonoQ ion-exchange chromatography and two dimensional gel analyses were used to demonstrate that at least the p55, p44, and p42 substrates were identical, as well as some more minor species of molecular weights 50, 38, and 36 kDa, thus indicating common pathways of signaling in hemopoietic cells. Whereas in the case of SLF the dose-response characteristics of the proliferative response and the induction of tyrosine phosphorylation were similar, in the case of IL-3, much lower concentrations were required for maximal proliferation than maximal tyrosine phosphorylation. These studies form the basis for further molecular characterization of common components of signal transduction pathways in hemopoietic cells.     

Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells.

The mutant c-fgr protein (p58c-fgr/F523) containing Phe-523 instead of Tyr-523 exhibited transforming activity in NIH 3T3 cells like other protein-tyrosine kinases of the src family, but normal p58c-fgr (p58c-fgr/wt) did not. The mutant protein showed tyrosine kinase activity threefold higher than that of the normal protein in vitro. Surprisingly, transfection of the normal c-fgr gene into NIH 3T3 cells resulted in induction of sodium fluoride (NaF)-sensitive alpha-naphthyl butyrate esterase (alpha-NBE), a marker enzyme of cells of monocytic origin, which was not induced in v-src-, v-fgr-, or lyn-transfected NIH 3T3 cells. The NaF-sensitive alpha-NBE induced in c-fgr transfectants was shown by isoelectric focusing to have a pI of 5.2 to 5.4, a range which was the same as those for thioglycolate-induced murine peritoneal macrophages and 1 alpha,25-dihydroxyvitamin D3-treated WEHI-3B cells. Immunoblotting studies with antiphosphotyrosine antibodies revealed that 58-, 62-, 75-, 120-, 200-, and 230-kDa proteins were commonly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in cells transfected with the mutated c-fgr. These findings suggest that tyrosine phosphorylation of specific cellular substrate proteins is important in induction of NaF-sensitive alpha-NBE and cell transformation by p58c-fgr.     

Insulin treatment stimulates the tyrosine phosphorylation of the alpha-type 85-kDa subunit of phosphatidylinositol 3-kinase in vivo.

After adding insulin to cells overexpressing the insulin receptor, the activity of phosphatidylinositol (PI) 3-kinase in the anti-phosphotyrosine immunoprecipitates was rapidly and greatly increased. This enzyme may therefore be a substrate for the insulin receptor tyrosine kinase and may be one of the mediators of insulin signal transduction. However, it is unclear whether or not activated tyrosine kinase of the insulin receptor directly phosphorylates PI 3-kinase at tyrosine residue(s) and whether insulin stimulates the specific activity of PI 3-kinase. We reported previously that the 85-kDa subunit of purified PI 3-kinase was phosphorylated at tyrosine residue(s) by the insulin receptor in vitro. To examine the tyrosine phosphorylation of PI 3-kinase and change of its activity by insulin treatment in vivo, we used a specific antibody to the 85-kDa subunit of PI 3-kinase. The activity of PI 3-kinase in immunoprecipitates with the antibody against the p85 subunit of PI 3-kinase was increased about 3-fold by insulin treatment of cells overexpressing insulin receptors. Insulin treatment also stimulated the tyrosine, serine, and threonine phosphorylation of the alpha-type 85-kDa subunit of PI 3-kinase in vivo. Phosphatase treatment of the immunoprecipitates abolished the increase in PI 3-kinase activity. The phosphorylation(s) of the kinase itself, tyrosine phosphorylation(s) of associated protein(s), or the complex formation of the phosphorylated PI 3-kinase with associated proteins may increase the activity of PI 3-kinase.     

Differential modulation of two interferon-α binding proteins on a human lymphoblastoid cell line

The interaction between human interferon (IFN)-α or IFN-β with its receptor was originally described as the binding to a single class of high-affinity receptors. However, more recently, biphasic Scatchard plots as well as multiple IFN-α receptor cross-linked complexes have been reported. In this study using the Daudi B lymphoblastoid cell line, two primary IFN-α receptor cross-linked complexes with apparent M_r of 115 and 135 kilodaltons (kDa) were obtained. Both complexes were observed under a variety of cross-linking conditions, including the addition of a mixture of protease inhibitors throughout the binding reaction and solubilization of the cells. These two complexes appear to be caused by the binding and cross-linking of ¹²⁵I-rIFN-αA to two separate proteins because we also observed two IFN-α binding proteins using a ligand-blotting technique. At low concentrations of ¹²⁵I-rIFN-αA, it was found that the intensity of the signal in the 135-kDa cross-linked complex was greater than that of the 115-kDa complex. Addition of increasing concentrations of unlabeled rIFN-αA to a 4°C binding reaction reversed the ratio in intensities of the two complexes. Moreover, after pretreatment of the cells at 37°C with low concentrations of unlabeled rIFN-αA, there was preferential down-regulation of both the 135-kDa complex and the higher affinity binding component of the biphasic Scatchard plot. These results suggest that the 135-kDa complex represents the binding of ¹²⁵I-rIFN-αA to a protein having higher affinity for IFN than the protein that gives rise to the 115-kDa complex. These two proteins also appear to have different half lives in the plasma membrane in the absence of IFN because treatment with cycloheximide also caused a preferential decrease in the subsequent formation of the 135-kDa complex.     

Affinity labeling of the sialoglycopeptide antimitogen receptor

An 18-kDa 125I-sialoglycopeptide growth inhibitor was covalently cross-linked to its binding site on intact cultured Swiss 3T3 cells by three bifunctional cross-linkers with short (dimethyl adipimate), medium (disuccinimidyl suberate), and long (bis(2-succinimidooxycarbonyloxyethyl)sulfone) chain lengths. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated a band of Mr approximately 168,000 regardless of which cross-linker was used. The labeling of this band was specific in that it was prevented by excess unlabeled inhibitor and the apparent molecular weight of the cross-linked receptor-ligand complex was unchanged by treatment with reducing agent. The efficiency of the cross-linking was increased by increasing pH, and the extent of covalent cross-linking was dependent on the concentration of the bifunctional reagent. Octyl glucoside and sodium dodecyl sulfate were effective in solubilizing the receptor while Triton X-100 did not extract the receptor from the plasma membrane. These observations suggest that the 168-kDa binding species represents the 125I-sialoglycopeptide cross-linked to a specific plasma membrane receptor and that the receptor does not appear to contain interchain disulfide bonds.     

Interactions of high affinity insulin-like growth factor-binding proteins with the type V transforming growth factor-beta receptor in mink lung epithelial cells

High affinity insulin-like growth factor-binding proteins (IGFBP-1 to -6) are a family of structurally homologous proteins that induce cellular responses by insulin-like growth factor (IGF)-dependent and -independent mechanisms. The IGFBP-3 receptor, which mediates the IGF-independent growth inhibitory response, has recently been identified as the type V transforming growth factor-beta receptor (TbetaR-V) (Leal, S. M., Liu, Q. L., Huang, S. S., and Huang, J. S. (1997) J. Biol. Chem. 272, 20572-20576). To characterize the interactions of high affinity IGFBPs with TbetaR-V, mink lung epithelial cells (Mv1Lu cells) were incubated with 125I-labeled recombinant human IGFBPs (125I-IGFBP-1 to -6) in the presence of the cross-linking agent disuccinimidyl suberate and analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography. 125I-IGFBP-3, -4, and -5 but not 125I-IGFBP-1, -2, and -6 bound to TbetaR-V as demonstrated by the detection of the approximately 400-kDa 125I-IGFBP.TbetaR-V cross-linked complex in the cell lysates and immunoprecipitates. The analyses of 125I-labeled ligand binding competition and DNA synthesis inhibition revealed that IGFBP-3 was a more potent ligand for TbetaR-V than IGFBP-4 or -5. Most of the high affinity 125I-IGFBPs formed dimers at the cell surface. The cell-surface dimer of 125I-IGFBP-3 preferentially bound to and was cross-linked to TbetaR-V in the presence of disuccinimidyl suberate. IGFBP-3 did not stimulate the cellular phosphorylation of Smad2 and Smad3, key transducers of the transforming growth factor-beta type I/type II receptor (TbetaR-I.TbetaR-II) heterocomplex-mediated signaling. These results suggest that IGFBP-3, -4, and -5 are specific ligands for TbetaR-V, which mediates the growth inhibitory response through a signaling pathway(s) distinct from that mediated by the TbetaR-I and TbetaR-II heterocomplex.     

Zinc causes tyrosine phosphorylation of hippocampal p60c-src.

Zinc cations at concentrations of 0.2 mM and greater catalyzed specific phosphorylation, by ATP, of two membrane-associated proteins from rat hippocampus. These proteins, corresponding to molecular weights of 60 and 49 kDa, were phosphorylated primarily at tyrosine residues. The 60-kDa protein was identified as p60c-src by immunoprecipitation using two different p60src-specific monoclonal antibodies. The 49-kDa protein co-immunoprecipitated with p60c-src. Cyanogen bromide cleavage of p60c-src and the 49-kDa protein phosphorylated in the presence of Zn2+ gave different patterns of phosphopeptides. It is suggested that tyrosine phosphorylation of p60c-src and the p60c-src-associated 49-kDa protein may be a way of zinc participation in hippocampal neurotransmission.     

Tyrosine phosphorylation events during coxsackievirus B3 replication.

In order to study cellular and viral determinants of pathogenicity, interactions between coxsackievirus B3 (CVB3) replication and cellular protein tyrosine phosphorylation were investigated. During CVB3 infection of HeLa cells, distinct proteins become phosphorylated on tyrosine residues, as detected by the use of antiphosphotyrosine Western blotting. Two proteins of 48 and 200 kDa showed enhanced tyrosine phosphorylation 4 to 5 h postinfection (p.i.), although virus-induced inhibition of cellular protein synthesis had already occurred 3 to 4 h p.i. Subcellular fractionation experiments revealed distinct localization of tyrosine-phosphorylated proteins of 48 and 200 kDa in the cytosol and membrane fractions of infected cells, respectively. In addition, in Vero cells infected with CVB3, echovirus (EV)11, or EV12, increased tyrosine phosphorylation of a 200-kDa protein was detected 6 h p.i. Herbimycin A, a specific inhibitor of Src-like protein tyrosine kinases, was shown to inhibit virus-induced tyrosine phosphorylations and to reduce the production of progeny virions. In contrast, in cells treated with the inhibitors staurosporine and calphostin C, the synthesis of progeny virions was not affected. Immunoprecipitation experiments suggested that the tyrosine-phosphorylated 200-kDa protein in CVB3-infected cells is of cellular origin. In summary, these investigations have begun to unravel the effect of CVB3 as well as EV11 and EV12 replication on cellular tyrosine phosphorylation and support the importance of tyrosine phosphorylation events for effective virus replication. Such cellular phosphorylation events triggered in the course of enterovirus infection may enhance virus replication.     

Stimulation of factor-dependent myeloid cell lines with interleukin 3 induces tyrosine phosphorylation of several cellular substrates

Interleukin 3 (IL-3) is required for the proliferation of growth factor-dependent myeloid cell lines. To determine the possible signal transduction mechanisms involved in IL-3 growth regulation, we have examined the effects of IL-3 on tyrosine phosphorylation. Using a monoclonal antibody against phosphotyrosine, IL-3 was found to specifically and rapidly induce tyrosine phosphorylation of cytoplasmic proteins of 70, 56, and 38 kDa and a membrane-associated glycoprotein of 140 kDa. Minor and/or variable detected phosphoproteins of 120, 85, 51, and 28 kDa were also seen. Oncogenes encoding tyrosine protein kinases abrogate the requirement of factor-dependent myeloid cells for IL-3. We therefore compared the phosphoprotein profiles of a transformed, IL-3-independent cell line with the IL-3-induced profile. In cells transformed with trk, the 56-, 51-, and 38-kDa cytoplasmic phosphoproteins were constitutively phosphorylated, whereas the 140-kDa phosphoprotein was only phosphorylated in the presence of IL-3. Taken together, these results support a role for tyrosine phosphorylation in the IL-3 signal transduction pathway and suggest that growth factor abrogation by oncogenes encoding tyrosine protein kinases may be due to the phosphorylation of substrates which are normally phosphorylated in response to IL-3.     

Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells.

Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130,000, and Mr-90,000 proteins was approximately 0.2 to 0.5 ng/ml, which was consistent with the half-maximal concentration required for stimulation of DNA synthesis in NIH 3T3 cells. HBGF-1-induced tyrosine phosphorylation of the Mr-150,000 and Mr-130,000 proteins was detected within 30 s, whereas phosphorylation of the Mr-90,000 protein was not detected until 3 min after HBGF-1 stimulation. All three proteins were phosphorylated maximally after 15 to 30 min. Phosphoamino acid analysis of the Mr-150,000 and Mr-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the Mr-150,000 and Mr-90,000 proteins occurred when cells were exposed to HBGF-1 at 37 degrees C but not at 4 degrees C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the Mr-150,000, Mr-130,000, and Mr-90,000 proteins by anti-P-Tyr antibodies. Anti-P-Tyr affinity-based chromatography was used to adsorb the HBGF-1 receptor affinity labeled with 125I-HBGF-1. The cross-linked HBGF-1 receptor-ligand complex was eluded with phenyl phosphate as two components: Mr 170,000 and 150,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the (125)I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked (125)I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the (125)I-HBGF-1-receptor complex is phosphorylated on tyrosine residues and (ii) HBGF-1-induced signal transduction involves, in part, the tyrosine phosphorylation of at least three polypeptides.