Anti-immunoglobulin and phorbol ester induce phosphorylation of proteins associated with the plasma membrane and cytoskeleton in murine B lymphocytes

Protein phosphorylations are rapidly induced in intact B cells by antibodies to surface immunoglobulin (anti-IgM) and by phorbol 12-myristate 13-acetate (PMA). A comparison of the molecular weight, isoelectric points, phosphopeptides, and phosphoamino acids of the phosphoproteins induced by anti-IgM and by PMA suggests that anti-IgM acts through the activation of protein kinase C. This conclusion is strengthened by the observation that prolonged treatment with PMA ablates the ability of anti-IgM to induce phosphorylation, presumably by depleting cellular protein kinase C. Furthermore, the effects of dibutyryl cyclic AMP on protein phosphorylation are quite distinct from the effects of anti-IgM. The six most prominent phosphoproteins induced by PMA, with approximate Mr values of 47, 55, 62, 68, 68, and 65-70 X 10(3), are associated with the plasma membrane. Of these, four are apparently associated with the cytoskeleton, suggesting that the phosphorylation of cytoskeletal proteins may be important events early in B cell activation. Examination of protein phosphorylation in cell lines derived from different tissues has identified one major B cell phosphoprotein (Mr 65-70 X 10(3), which is absent in T cells, and two phosphoproteins (Mr 55 and 68 X 10(3), which are observed in cells of hematopoietic origin but which are absent or uncommon in other cell types.     

Phosphorylations of the subcellular matrix in cells transformed by Rous' sarcoma virus

The transforming protein of Rous' sarcoma virus (RSV) is a phosphoprotein of Mr 60 000 (pp60src) which displays protein kinase activity specific for tyrosine residues; pp60src is associated with the plasma membrane and is recovered in the detergent-insoluble material which represents the subcellular matrix of the cell. After phosphorylation of this material of RSV-transformed cells with [gamma-32P]ATP, five phosphoproteins have been detected which are not seen in normal cells. These proteins (Mr = 135 000, 125 000, 75 000, 70 000, 60 000) contain phosphotyrosine. Their phosphorylation is strongly inhibited by anti-pp60src antibodies. In cells transformed by a temperature-sensitive mutant of RSV, these phosphoproteins, present at the permissive temperature, are no longer detected at the non-permissive temperature. It is concluded that these phosphorylations are mediated by pp60src protein kinase activity. This supports a possible role of the phosphorylation of cytoskeletal proteins in the transformation process.     

Phosphorylation of the erbB gene product from an avian erythroblastosis virus-transformed chick fibroblast cell line

Polyclonal antiserum prepared against the human epidermal growth factor receptor immunoprecipitated four proteins of Mr = 66,000, 68,000, 74,000, and 82,000 from avian erythroblastosis virus-transformed chick embryo fibroblasts (cell line AEV-C23) which seemed to be related to the erbB gene product. The Mr = 66,000 and 68,000 proteins chased into the Mr = 74,000 and 82,000 proteins in pulse-chase experiments. The Mr = 68,000 and 82,000 proteins were found to be phosphorylated primarily on serine and threonine residues and contained minor amounts of phosphotyrosine. Tryptic peptide analysis of these phosphoproteins revealed several major peptides, and treatment of cells with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate resulted in the appearance of an additional phosphopeptide. 12-O-Tetradecanoyl-phorbol-13-acetate also inhibited growth of AEV-C23 cells in soft agar and in monolayer culture. In vitro phosphorylation of Mr = 68,000 and 74,000 proteins in immunoprecipitates occurred on tyrosine with lesser amounts of phosphoserine and phosphothreonine detected.     

The uncovering of a novel regulatory mechanism for PLD2: formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2

The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2, and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase.     

Comparative study on the effect of phosphorylated TCR ζ chain ITAM sequences on early activation events in Jurkat T cells

One of the main dilemma in T cell receptor (TCR) signal transduction is whether the presence of multiple Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) within the TCR signaling module serves for signal amplification or signal distribution. To contribute to answer this question, we analyzed the effect of synthetic oligopeptides representing the three bi-phosphorylated ζ chain-ITAMs on the early signaling events in permeabilized leukemia T cells. Our main observations were as follows: 1/Stimulation of the cells with the bi-phosphorylated membrane proximal and central ITAMs (ζ (1)y^py^p and ζ (2)y^py^p, respectively) resulted in a strong phosphorylation of proteins with a similar pattern. In contrast, the membrane distal ITAM, ζ (3)y^py^p had a reduced ability to promote tyrosine phosphorylation and failed to induce the phosphorylation of a number of proteins. 2/ The phospho-peptide induced tyrosine phosphorylation events were at least partially mediated by p56^lck and Syk/ZAP70 protein tyrosine kinases as it was shown in p56^lck and Syk/ZAP70 deficient Jurkat variants. 3/The patterns of the association of the adaptor protein, Grb2 with tyrosine phosphorylated proteins following cell stimulation with the bi-phosphorylated membrane proximal or the central ITAMs were similar, while the membrane distal ITAM was unable to induce any of these associations.

Our data provide additional evidence that the three ζITAMs differ in their capacity to induce tyrosine phosphorylation of intracellular proteins in permeabilized T cells, depending to their primary sequence. The first and second ITAM sequences of the ζ chain may have similar but not totally overlapping functions. This conclusion results from their similar but not identical abilities to induce tyrosine phosphorylation and association of Grb-2 with intracellular phosphoproteins. In contrast, the third ITAM (ζ3) may have distinct functions since this peptide fails to induce tyrosine phosphorylation of a number of proteins compared to the other two ITAMs, and it is unable to induce either new association or the increase in the amount of Grb-2 associated phosphoproteins.     

Tyrosine phosphorylation in cells treated with transforming growth factor-beta

Cells stimulated with epidermal growth factor (EGF) or any one of a diverse group of other mitogenic agents display an increased tyrosine phosphorylation of a pair of 42,000 Mr proteins. Transforming Growth Factor-beta (TGF-beta) is able to potentiate the mitogenic effects of Epidermal Growth Factor on some fibroblastic cells (such as the NRK-49F cell line) and, in addition, permits the anchorage-independent growth of these cells. In this study we asked whether these growth-regulatory actions of Transforming Growth Factor-beta are associated with changes in tyrosine phosphorylation of cellular proteins, in particular the 42,000 Mr proteins. We found no effect of Transforming Growth Factor-beta on the extent or time-course of tyrosine phosphorylation, either by itself or in combination with Epidermal Growth Factor. Since the tyrosine phosphorylation of the 42,000 Mr proteins is stimulated both by receptors with tyrosine kinase activity and by diacylglycerol analogs (but not by Transforming Growth Factor-beta), we suggest that the activity of the receptor for Transforming Growth Factor-beta is linked neither to tyrosine phosphorylation nor to phosphatidyl inositol turnover.     

Bradykinin and bombesin rapidly stimulate tyrosine phosphorylation of a 120-kDa group of proteins in Swiss 3T3 cells

We have examined the effect of bradykinin (BK) and other peptide mediators with related cellular actions on tyrosine phosphorylation in confluent Swiss 3T3 fibroblast cells using an anti-phosphotyrosine antibody. Immunoblots of extracts from cells stimulated with BK showed a major heterogeneous band centered at Mr 120,000. Three phosphorylated protein species were present within this band. The lower of these three phosphoproteins was occasionally present under basal conditions. The detection of this group of phosphoproteins by the antibody was prevented by coincubation with an excess of phosphotyrosine but not with an excess of phosphoserine or phosphothreonine. The BK-promoted increase in phosphorylation was rapid and transient with the peak response apparent following BK exposure for 1 min. The response was dose-dependent with half-maximal effect occurring at 10-30 nM BK. The antagonist Arg0, Hyp3, Thi5,8, D-Phe7-BK completely inhibited the response indicating that BK was acting via a B2 kinin receptor. Bombesin, at 0.1 microM, stimulated an increase in phosphorylation of the 120-kDa group of proteins with the same efficacy as 0.1 microM BK. On the other hand, 1 microM vasopressin was considerably less efficaceous than either of the former agonists. Short-term preexposure to 0.1 microM 12-O-tetradecanoyl-phorbol-13-acetate (1 min), a protein kinase C stimulator, or 30 microM H7 (15 min), a protein kinase C inhibitor, had no significant effect either on the basal or BK-promoted increase in tyrosine phosphorylation of these proteins. BK also stimulated inositol phosphate formation in these cells. Genistein, a tyrosine kinase inhibitor, inhibited BK stimulation of tyrosine phosphorylation. In addition, genistein partially inhibited BK stimulation of inositol phosphate formation. These results show that an increase in tyrosine phosphorylation of a 120-kDa group of proteins is an early protein kinase C-independent cellular signal elicited by both bradykinin and bombesin.     

Protein tyrosine phosphorylation associated with activation of the interleukin 2 receptor.

The addition of interleukin 2 (IL2) to the IL2-dependent murine cytotoxic T cell line CTTL-2 induced increased tyrosine phosphorylation of a protein with a molecular weight of 80,000 and, to a lesser extent, proteins with molecular weights of 130,000, 100,000, and 69,000. To correlate the stimulation of tyrosine phosphorylation with increased tyrosine kinase activity, cell-free phosphorylation assays were performed. Phosphotyrosine-containing proteins were purified from detergent-solubilized cell lysates by immunoprecipitation with anti-phosphotyrosine antibodies. The level of tyrosine kinase activity was determined by incorporation of [gamma-32P]ATP into the exogenous substrate histone H2B. IL2 treatment of cells increased H2B phosphorylation 10-fold when compared with nonstimulated cells. Phosphorylation was first detected after 2.5 min of incubation with physiologically relevant (100 pM) IL2 doses. To examine if tyrosine kinase activity was resident within the IL2 receptor complex, cell-free phosphorylation assays were performed with ligand-receptor complexes following cross-linking with IL2 and purification by immunoabsorption with an anti-IL2 antibody. Tyrosine kinase activity was found specifically associated with the IL2 receptor complex. These results indicate that the IL2 receptor complex contains a tyrosine kinase activity that is induced by IL2 binding and suggest that components of the complex may be a substrate of this activity.     

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.     

Signal Transmission through MHC Class II Molecules in a Human B Lymphoid Progenitor Cell Line: Different Signaling Pathways Depending on the Maturational Stages of B Cells

The function of MHC class II HLA-DR molecules expressed on a human B lymphoid progenitor cell line FL8.2.4.4 (abbreviated as FL4.4) was examined. FL4.4 cells expressed HLA-DR molecules and stimulation of the DR molecules by anti-DR mAb or by superantigen TSST-1 induced strong augmentation of homocytic aggregation and protein tyrosine phosphorylation in FL4.4 cells. Induced homocytic aggregation in FL4.4 consists both of LFA-1/ICAM-1-dependent and -independent pathways as revealed by mAb blocking experiments. Metabolic inhibitors, NaN3 and cytochalasin B, blocked the induced homocytic aggregation of FL4.4. Early mature Daudi B cell lines also showed a similar type of homocytic aggregation by stimulation with anti-DR mAb. Daudi cells are more sensitive to protein kinase inhibitors herbimycin A and H7 than FL4.4 cells in their blocking of induced homocytic aggregation, while W7 showed stronger inhibitory effects on FL4.4 cells than on Daudi cells. Western blotting analysis revealed that the stimulation of DR molecules induced protein tyrosine phosphorylation of 100-kDa, 90-kDa, 60-kDa and 55-kDa proteins in FL4.4 cells, while, in Daudi cells 110-kDa, 100-kDa and 80-kDa proteins were phosphorylated. These results suggest that different signaling pathways through class II molecules are employed depending on the maturational stage of B-cell differentiation.     

Regulation of mouse preimplantation development: inhibitory effect of genistein, an inhibitor of tyrosine protein phosphorylation, on cleavage of one-cell embryos

We investigated the effects of genistein, an inhibitor of tyrosine protein phosphorylation, on mouse 1-cell embryos, since in response to mitogenic stimuli tyrosine protein phosphorylation in somatic cells is implicated in initiation of DNA synthesis. Genistein inhibits cleavage of 1-cell embryos in a concentration-dependent and reversible manner; biochanin A, which is a less potent inhibitor of tyrosine protein phosphorylation, is a less potent inhibitor of cell cleavage. Genistein does not inhibit [35S]methionine incorporation, but does inhibit [3H]thymidine incorporation. Consistent with genistein's ability to inhibit cleavage by inhibiting DNA synthesis is that the loss of genistein's ability to inhibit cleavage corresponds with exit of the 1-cell embryos from S phase. Genistein is likely to inhibit tyrosine protein phosphorylation in situ, since it reduces by 80% the relative amount of [32P]phosphotyrosine present in 1-cell embryos; genistein does not inhibit either [32P]orthophosphate uptake or incorporation. As anticipated, genistein has little effect on inhibiting changes in the pattern of phosphoprotein synthesis during the first cell cycle, since tyrosine protein phosphorylation constitutes a small percentage of total protein phosphorylation. Alkalai treatment of [32P]radiolabeled phosphoproteins transferred to Immobilon reveals a base-resistant set of phosphoproteins of Mr = 32,000 that displays cell-cycle changes in phosphorylation. Although these properties suggest that these phosphoproteins may be related to the p34cdc2 protein kinase, phosphoamino acid analysis of [32P]radiolabeled phosphoproteins reveals that they are not enriched for phosphotyrosine; the inactive for p34cdc2 protein kinase contains a high level of phosphotyrosine. Results of these experiments suggest that tyrosine protein phosphorylation in response to the fertilizing sperm may be involved in initiating DNA synthesis in the 1-cell embryo, as well as converting a meiotic cell cycle to a mitotic one.     

Role of tyrosine phosphorylation of phospholipase C gamma1 in the signaling pathway of HMG-CoA reductase inhibitor-induced cell death of L6 myoblasts

Our previous studies have shown that the HMG-CoA reductase (HCR) inhibitor (HCRI), simvastatin, kills L6 myoblasts by involving Ca2+ mobilization from the Ca2+ pool in the cells but not by influx from extracellular space. More recently, we found that HCRI induced tyrosine phosphorylation of several cellular proteins, followed by apoptotic cell death of L6 myoblasts. The present study was aimed to elucidate the molecular target(s) of these tyrosine phosphorylations induced by HCRI and demonstrated that simvastatin induces tyrosine phosphorylation of phospholipase C (PLC) gamma1. This tyrosine phosphorylation of PLC-gamma1 caused the increment of the intracellular inositol triphosphate (IP3) levels in L6 myoblasts. Pretreatment of the cells with herbimycin A, a specific inhibitor of protein tyrosine kinase, inhibited a simvastatin-induced increase in IP3 level in the cells as well as tyrosine phosphorylation of PLC-gamma1. Interestingly, pretreatment of the cells with U-73122, a specific inhibitor of PLC, prevented simvastatin-induced cell death. Thus, these results strongly suggest that simvastatin-induced tyrosine phosphorylation of PLC-gamma1 plays, at least in part, an important role for the development of simvastatin-induced cell death.     

Diverse mitogenic agents induce the phosphorylation of two related 42,000-dalton proteins on tyrosine in quiescent chick cells.

Incubation of quiescent chicken embryo cells with platelet-derived growth factor, epidermal growth factor, or serum was found to stimulate phosphorylation of two proteins of ca. 42,000 daltons on tyrosine. These proteins are structurally related to each other and to two proteins phosphorylated on tyrosine under similar conditions in mitogen-treated mouse fibroblasts. Three other very different mitogenic agents, the protease trypsin and the chemically unrelated tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate and teleocidin, stimulated phosphorylation of the same proteins. In all cases, phosphotyrosine was detected in these phosphoproteins. Although additional changes in protein phosphorylation were evident, no other proteins were observed by two-dimensional gel electrophoresis which contained increased amounts of phosphotyrosine in mitogen-treated chicken embryo cells. One of these 42,000-dalton proteins was shown previously to be phosphorylated on tyrosine in chicken embryo cells transformed with various retroviruses whose transforming proteins possess tyrosine protein kinase activity. Phosphorylation of the 42,000-dalton proteins could be important in the regulation of cell division.     

Rapid phosphorylation of MAP-2-related cytoplasmic and nuclear Mr 300,000 protein by serine kinases after growth stimulation in quiescent cells

Antibody against brain microtubule-associated protein 2 (MAP-2) immunoprecipitated Mr 300,000 and 80,000 proteins of cultured fibroblasts and kidney cells. These proteins were not appreciably phosphorylated in quiescent cells, but were rapidly phosphorylated after growth stimulation by insulin, epidermal and fibroblast growth factors, transferrin, phorbol ester and diacylglycerol in the presence of Ca2+, in a manner similar to that of MAP-1-related Mr 350,000 protein (J. Cell Biol. 100, 748-753). A Ca2+ ionophore, which is known to make the quiescent cell competent but not to enter into the growth cycle, did not induce the phosphorylation. In a chase experiment, decay half lives of labeled phosphoproteins were 5 h for Mr 350,000 and 300,000 proteins, and 1.5 h for Mr 80,000 protein. On subcellular fractionation, phosphorylated Mr 350,000 and 300,000 proteins were detected first mainly in the cytoplasm and then in the nucleus, while Mr 80,000 phosphoprotein was consistently detected in the cytoplasm. The phosphorylation of these proteins occurred on serine residues after stimulation with various factors. Thus, the phosphorylation of cytoskeleton-associated Mr 350,000 and 300,000 proteins by serine kinases seems to be a common second process after growth stimulation and to link cytoplasmic and intranuclear events.     

Granulocyte-macrophage colony-stimulating factor and steel factor induce phosphorylation of both unique and overlapping signal transduction intermediates in a human factor-dependent hematopoietic cell line.

Steel factor (SF), the ligand for the proto-oncogene c-kit, acts synergistically with GM-CSF or IL-3 to support the growth of normal human hematopoietic progenitor cells. We examined the effects of SF on GM-CSF or IL-3 induced proliferation of a human factor-dependent cell line, MO7. SF supported MO7 cell proliferation as well as IL-3 or GM-CSF alone, and its addition dramatically enhanced (three- to sixfold) maximal GM-CSF or IL-3 stimulated proliferation. SF did not increase the number or affinity of cell surface GM-CSF receptors. We examined several early events of signal transduction in an effort to elucidate the biochemical mechanisms of synergy of these factors. Since each of these three cytokines is believed to function in part through activation of a tyrosine kinase, we examined their effects on cellular phosphotyrosine containing proteins. Each cytokine induced rapid, transient, and concentration dependent tyrosine phosphorylation of a number of substrates. For GM-CSF and IL-3, these phosphoproteins were indistinguishable (150, 125, 106, 93, 80, 79, 73, 44, 42, and 36 kDa), while SF induced major or minor tyrosine phosphorylation of 205, 140-150, 116, 106, 94, 90, 80, 79, 73, 44, 42, 39, 36, 32 kDa phosphoproteins. Two other signal transduction intermediates known to be phosphorylated and activated by GM-CSF and IL-3, the 70-75 kDa Raf-1 kinase, and p42 mitogen-activated protein kinase-2 (MAPK), were also phosphorylated by SF. Combinations of GM-CSF or IL-3 with SF did not further increase the phosphorylation of Raf-1 or p42 MAPK when compared to any of the factors alone. In contrast SF, but not GM-CSF or IL-3, induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma). These results indicate that SF and GM-CSF/IL-3 have partially overlapping effects on early signal transducing events, as well as striking differences, such as tyrosine phosphorylation of PLC-gamma. This cell line should provide a useful model system to investigate the complicated process of hematopoietic growth factor synergy.     

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.     

Interleukin-3 or erythropoietin induced nuclear localization of protein kinase C β isoforms in hematopoietic target cells

Protein kinase C (PKC) has been implicated in the signal transduction pathways for the biological effect of both interleukin-3 (IL-3) and erythropoietin (EPO) in hematopoietic target cells. The goal of this study was to identify specific classical isoforms of PKC and their localization in hematopoietic cells in response to the growth factors, IL-3 or EPO. In addition to murine fetal liver cells as a source of normal erythroid progenitor cells, we have utilized the B6SUt.EP cell line, a non-transformed hematopoietic cell line that requires IL-3 for proliferation, but for which EPO can substitute as a growth factor. With polyclonal antibodies prepared against peptide sequences specific for the α, βI, βII and γ isoforms of PKC, we have identified βI and βII as the predominant nuclear isoforms in target cells that proliferate in response to IL-3 or EPO.     

Insulin stimulates tyrosine phosphorylation of multiple high molecular weight substrates in Fao hepatoma cells.

Insulin rapidly stimulates tyrosine phosphorylation of cellular proteins which migrate between 165 and 190 kDa during SDS-PAGE. These proteins, collectively called pp185, were originally found in anti-phosphotyrosine antibody (alpha PY) immunoprecipitates from insulin-stimulated Fao rat hepatoma cells. Recently, we purified and cloned IRS-1, one of the phosphoproteins that binds to alpha PY and migrates near 180 kDa following insulin stimulation of rat liver [Sun, X. J., et al. (1991) Nature 352, 73-77]. IRS-1 and pp185 undergo tyrosine phosphorylation immediately after insulin stimulation and show an insulin dose response similar to that of insulin receptor autophosphorylation. However, IRS-1 was consistently 10 kDa smaller than the apparent molecular mass of pp185. The pp185 contained some immunoblottable IRS-1; however, cell lysates depleted of IRS-1 with anti-IRS-1 antibody still contained the high molecular weight forms of pp185 (HMW-pp185). Furthermore, the tryptic phosphopeptide map of IRS-1 was distinct from that of HMW-pp185, suggesting that at least two substrates migrate in this region during SDS-PAGE. Moreover, the phosphatidylinositol 3'-kinase and its 85-kDa associated protein (p85) bound to IRS-1 in Fao cells, but weakly or not at all to HMW-pp185. Our results show that Fao cells contain at least two insulin receptor substrates, IRS-1 and HMW-pp185, which may play unique roles in insulin signal transmission.