Tyrosine phosphorylation is an obligatory event in IL-2 secretion.

Activation of T lymphocytes leads to the production of the T cell growth factor IL-2 that regulates T cell proliferation. This activation is associated with several potential intracellular signalling events including increased activity of phospholipase C (PLC) and resultant increases in production of inositol phosphates and diacylglycerols. In addition, phosphorylation of specific intracellular proteins on serine, threonine, and tyrosine residues increases. The role of each of these events in IL-2 production is unclear. Using Western blotting with antiphosphotyrosine antibodies, we demonstrate that activation of murine T cells with mitogenic lectins or anti-CD3 antibodies leads to a rapid increase in tyrosine phosphorylation of proteins of 120, 72, 62, 55, and 40 kDa. Similar patterns of antiphosphotyrosine antibodies reactivity were observed in splenocytes, a T cell hybridoma, and a T lymphoma. Tyrosine phosphorylation was detectable within minutes of addition of mitogenic lectins and persisted for at least 6 h. Pretreatment of the cells with pertussis toxin did not inhibit tyrosine phosphorylation indicating that a pertussis toxin-sensitive G protein is not involved in signal transduction. Neither increasing cytosolic-free calcium nor activating protein kinase C mimicked the effects of mitogenic lectins suggesting that tyrosine phosphorylation was not a consequence of activation of PLC. This was confirmed by demonstrating that mitogenic lectins induced similar patterns of tyrosine phosphorylation in cells in which activation of the TCR leads to increased PLC activity and in cells in which PLC is not stimulated. To test whether tyrosine phosphorylation is linked to IL-2 secretion, we determined the effect of three specific tyrosine kinase inhibitors (tyrphostins) on tyrosine phosphorylation, IL-2 secretion, and cellular proliferation. The concentration dependence of inhibition of tyrosine phosphorylation and IL-2 production were similar. However, higher concentrations of the tyrphostins were required to inhibit constitutive proliferation of the T cell line indicating that inhibition of IL-2 secretion was not secondary to nonspecific toxic effects of the tyrphostins. Addition of the tyrphostins after mitogenic lectin decreased the amount of tyrosine phosphorylation and IL-2 secretion in parallel. This indicates that both tyrosine kinases and phosphatases are activated and that continuous tyrosine phosphorylation is likely required for IL-2 secretion. Therefore, tyrosine phosphorylation appears to represent an obligatory event in the transmembrane signaling processes that lead to IL-2 secretion.     

Phospholipase C-gamma 1 and phospholipase C-gamma 2 are substrates of the B cell antigen receptor associated protein tyrosine kinase.

Cross-linking the antigen receptor on B cells results in a rapid increase in protein tyrosine kinase activity as detected by increased phosphorylation on tyrosine residues of multiple proteins. Although the identity of most of this substrates remains unknown, some have been proposed. One possible substrate of the antigen receptor-associated kinase is phospholipase C (PLC). Since multiple isoforms of PLC have been identified, we have studied which isoforms are targets of the antigen receptor. PLC-gamma 1 and PLC-gamma 2 but not PLC-beta 1 or PLC-delta 1 were detected in human B cells. Immunoprecipitation with antibodies against PLC-gamma 1 or PLC-gamma 2 and subsequent Western blotting with anti-phosphotyrosine antibodies revealed that both PLC-gamma 1 and PLC-gamma 2 are tyrosine phosphorylated in stimulated but not in resting B cells. This was confirmed by experiments whereby B cell lysates were immunoprecipitated with anti-phosphotyrosine antibody and subsequently blotted with antibodies against PLC-gamma 1 or PLC-gamma 2. Further, the specific protein tyrosine kinase inhibitors, tyrphostins, which block phospholipase-C activation and proliferation of B cells also inhibited tyrosine phosphorylation on both PLC-gamma 1 and PLC-gamma 2. We conclude that both isoforms PLC-gamma 1 and PLC-gamma 2 are targets of the antigen receptor-associated protein tyrosine kinase.     

The reactivity of frozen B lymphocytes to B cell mitogens and human B cell growth factor: A study of step 1 and step 2 activators

Human B lymphocytes, purified from the peripheral blood of several different donors can be pooled, frozen, and stored in liquid nitrogen to provide an easy and reproducible source of cells for mitogenic assays. These B cell preparations did not show any reactivity to T cell mitogens, but responded to Staphylococcus aureus Cowan strain 1 (SAC) and anti-IgM antibodies to the same extent as freshly purified B cells. When stimulated with either anti-IgM antibodies or SAC, these B cells became responsive to B cell growth factor (BCGF), allowing a quantitative measurement of this important lymphokine activity. In addition, we have studied the reactivity of frozen B lymphocytes to various combinations of activators. We have confirmed that phorbol myristate acetate (PMA) was a very potent mitogenic agent for preactivated human B cells and shown that bacterial lipopolysaccharide (LPS), although not mitogenic by itself, could synergize with anti-IgM antibodies to yield increased levels of stimulation. Furthermore experiments using the lysosomotropic agent leucine methyl ester showed that the action of LPS on anti-IgM-stimulated B cells did not require the presence of functional monocytes. Neither PMA nor LPS could induce BCGF responsiveness and thus these two compounds can be considered exclusive step 2 activators for human peripheral blood B cells.     

Tyrosine phosphorylation is essential for microfilament assembly in B lymphocytes

The B cell antigen receptor regulates the tyrosine kinase signal transduction pathway and it mediates a variety of morphological changes such as capping and membrane ruffling. The relationship between these two events is unclear. We show here that cross-linking the antigen receptor on human B lymphocytes, in addition to increasing tyrosine phosphorylation of specific substrates, induces the conversion of G-actin to F-actin. Preincubation of B lymphocytes with two different tyrphostins blocked anti-IgM-induced tyrosine phosphorylation and actin polymerization. The ability of the tyrphostins to block anti-IgM induced conversion of G-actin to F-actin indicates that a tyrosine kinase acts as an essential link between the B cell antigen receptor the early changes in cytoskeletal reorganization.     

A cell-free Salmonella typhimurium extract induces inhibition of tyrosine phosphorylation in murine splenic T-lymphocytes

Abstract In a previous study, we observed that suppression of T-cell proliferation induced by Salmonella infection is associated with inhibition of tyrosine phosphorylation in T-cells, and that a cell-free Salmonella typhimurium LT2 extract (LT2 extract) also suppressed mitogen-induced T-cell proliferation. In the present study, therefore, we attempted to clarify whether the T-cell suppression induced by LT2 extract involved inhibition of tyrosine phosphorylation in T-cells. Western blotting using anti-phosphotyrosine antibodies showed that the mitogen-induced tyrosine phosphorylation of 120-, 106-, 94-, 76-, 68-, 57- and 36-kDa proteins in murine splenic T-cells was inhibited by treatment with LT2 extract. These results suggest that the suppression of T-cell proliferation induced by LT2 extract is also associated with inhibition of tyrosine phosphorylation in T-cells.     

Comparison of tyrosine kinase activation by mitogenic and nonmitogenic anti-IgD antibodies.

Low concentrations of anti-Ig dextran conjugates that stimulate very high levels of B cell proliferation and Ig secretion stimulate no detectable increases in tyrosine phosphorylation. To study this point further, we compared tyrosine phosphorylation patterns induced by mitogenic and nonmitogenic anti-Ig antibodies. Whereas the mitogenic, strongly cross-linking, antibody H delta a/1 induced greater levels of tyrosine phosphorylation than did the nonmitogenic antibody FF1-4D5, the pattern of substrate phosphorylation was equivalent. At lower concentrations of H delta a/1, which were still mitogenic, the degree of phosphorylation that was induced was similar to that induced by high concentrations of FF1-4D5. Both antibodies stimulated comparable increases in the kinase activity of the three src-related kinases present in normal B cells and linked to the IgR, i.e., Blk, Fyn, and Lyn. These results suggest that the extent of tyrosine kinase activation is proportional to mIg cross-linking, that induction of B cell DNA synthesis may require little tyrosine kinase activation, and that activation of tyrosine kinase per se does not necessarily lead to B cell DNA synthesis.     

Immunosuppression induced by Salmonella involves inhibition of tyrosine phosphorylation in murine T lymphocytes

Abstract It is well known that facultative intracellular pathogens such as Salmonella suppress the host immune system. In the present study we attempted to clarify the mechanism responsible for the suppression of T-cell proliferation in mice infected with Salmonella typhimurium . The proliferation of murine spleen cells stimulated with a T-cell mitogen such as phytohemagglutinin (PHA) or concanavalin A (ConA) was significantly suppressed when the mice were infected with S. typhimurium , but not with Eschirichia coli . The suppression of T-cell proliferation did not necessarily parallel the level of interleukin-2 (IL-2) secretion, and was not restored by treatment with a calcium ionophore, indomethacin or IL-2. Only phorbol 12-myristate-13 acetate (PMA), an activator of protein kinase C (PKC), caused a slight recovery of cell proliferation with an augmentation of IL-2 secretion. Furthermore, Western blotting using anti-phosphotyrosine antibodies showed that the mitogen-induced tyrosine phosphorylation of 120-, 106-, 94-, 68- and 57-kDa proteins in murine splenic T-cells was inhibited by S. typhimurium infection. Also, the inhibition of tyrosine phosphorylation was not restored by treatment with PMA. These results suggest that the suppression of T-cell proliferation induced by Salmonella infection may be regulated by inhibition of tyrosine phosphorylation in T-cells, although the inhibition is not associated with PKC activation and subsequent IL-2 secretion of T cells.     

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.     

Interleukin 2-induced tyrosine phosphorylation. Interleukin 2 receptor beta is tyrosine phosphorylated

Interaction of interleukin 2 (IL2) with its high affinity membrane receptor complex (IL2R) is sufficient to induce proliferation of T lymphocytes. However, the biochemical mechanisms by which IL2 induces this process remain unresolved. The IL2R complex consists of at least two distinct polypeptides that bind IL2, a 75-kDa intermediate affinity subunit (IL2R beta) and a 55-kDa low affinity subunit (IL2R alpha). As indicated by Western blotting with anti-phosphotyrosine-specific antibodies and confirmed by phosphoamino acid analysis, we now demonstrate that interaction of the T cell growth factor interleukin 2 (IL2) with its high affinity receptor on IL2-sensitive human peripheral blood lymphoblasts induces tyrosine phosphorylation of proteins of 92, 80, 78, 70-75, and 57 kDa. IL2 induced tyrosine phosphorylation in YT 2C2 cells which express only the 75-kDa intermediate affinity IL2 binding molecule (IL2R beta) but not in cells which either express only the 55-kDa low affinity IL2 receptor molecule (IL2R alpha) or no IL2-binding sites. Therefore, IL2R beta, in the absence of IL2R alpha, appears sufficient to transduce the transmembrane signal leading to tyrosine phosphorylation. Two different antibodies reactive with phosphotyrosine specifically immunoprecipitated IL2R beta cross-linked to radiolabeled IL2. These findings suggest that IL2R beta is a substrate for the tyrosine kinase which is activated by IL2 binding to its receptor. Thus, like several other growth factor receptors, activation of the IL2R results in an increase in tyrosine phosphorylation with the receptor itself serving as one substrate.     

Lipid raft assembly and Lck recruitment in TRAIL costimulation mediates NF-κB activation and T cell proliferation

The TNF-related apoptosis-inducing ligand was shown to provide a costimulatory signal that cooperates with the TCR/CD3 complex to induce T cell proliferation and cytokine production. Although a number of signaling pathways were linked to the TCR/CD3 complex, it is not known how these two receptors cooperate to induce T cell activation. In this study, we show that TRAIL-induced costimulation of T cells depends on activation of the NF-κB pathway. TRAIL induced the NF-κB pathway by phosphorylation of inhibitor of κB factor kinase and protein kinase C in conjunction with anti-CD3. Furthermore, we demonstrated that TRAIL costimulation induced phosphorylation of the upstream TCR-proximal tyrosine kinases, Lck and ZAP70. Ligation of the TRAIL by its soluble receptor, DR4-Fc, alone was able to induce the phosphorylation of Lck and ZAP70 and to activate the NF-κB pathway; however, it was insufficient to fully activate T cells to support T cell proliferation. In contrast, TRAIL engagement in conjunction with anti-CD3, but not TRAIL ligation alone, induced lipid raft assembly and recruitment of Lck and PKC. These results demonstrate that TRAIL costimulation mediates NF-κB activation and T cell proliferation by lipid raft assembly and recruitment of Lck. Our results suggest that in TRAIL costimulation, lipid raft recruitment of Lck integrates mitogenic NF-κB-dependent signals from the TCR and TRAIL in T lymphocytes.     

Modulation of in vitro murine B-lymphocyte response by curcumin

Curcumin is a phenolic natural product isolated from the rhizome of Curcuma longa (tumeric). It was previously described that curcumin had a potent anti-inflammatory effect and inhibited the proliferation of a variety of tumor cells. In the present study, we investigated the inhibitory effects of curcumin on the response of normal murine splenic B cells. Curcumin inhibited the proliferative response of purified splenic B cells from BALB/c mice stimulated with the Toll-like receptor ligands LPS and CpG oligodeoxynucleotides. LPS-induced IgM secretion was also inhibited by curcumin. The proliferative response induced by either the T-independent type 2 stimuli anti-delta-dextran or anti-IgM antibodies was relatively resistant to the effect of curcumin. We investigated the intracellular signaling events involved in the inhibitory effects of curcumin on murine B cells. Curcumin did not inhibit the increase in calcium levels induced by anti-IgM antibody. Western blotting analysis showed that curcumin inhibited TLR ligands and anti-IgM-induced phosphorylation of ERK, IκB and p38. Curcumin also decreased the nuclear levels of NFκB. Our results suggested that curcumin is an important inhibitor of signaling pathways activated upon B cell stimulation by TLR ligands. These data indicate that curcumin could be a potent pharmacological inhibitor of B cell activation.     

Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes

The mechanisms responsible for initiating the conversion of globular to filamentous actin (assembly) after stimulation of B lymphocytes and the role of these cytoskeletal changes in cell activation are incompletely understood. We investigated the molecular basis of the signals leading to actin polymerization and concentrated on the involvement of guanosine triphosphate (GTP)-binding regulatory proteins, and protein kinase C (PKC). In addition, we related these early events to later events in B-cell activation, including cell proliferation. Cross-linking the Ag receptor with Staphylococcus aureus Cowan I (SAC) or anti-IgM antibodies, or stimulation of PKC with phorbol ester induced a time- and concentration-dependent increase in the filamentous actin content of B cells. Inhibition or depletion of PKC resulted in decreased actin assembly induced by anti-IgM, SAC, and PMA, suggesting that the signal for polymerization is generated distally to PKC activation. Pertussis toxin pretreatment inhibited the responses to anti-IgM and SAC but not PMA, and direct stimulation of permeabilized cells with GTP gamma S induced microfilament assembly, indicating the involvement of a GTP-binding protein for receptor-mediated events. Disruption of actin polymerization with botulinum C2 toxin or cytochalasin D inhibited the assembly of actin and [3H]TdR incorporation induced by all stimuli. We conclude that human B cell activation by receptor-mediated stimuli results in actin polymerization by signaling pathways coupled to GTP-binding proteins. These changes in the cytoskeleton may be involved in the transduction of messages leading to responses such as proliferation in B lymphocytes.     

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.     

Mechanism of Staphylococcus aureus exotoxin A inhibition of Ig production by human B cells

Staphylococcus enterotoxins and toxic shock syndrome toxin 1 are members of a family of exoproteins that are produced by staphylococci and bind specifically to MHC class II molecules. Upon binding to MHC class II molecules, these exoproteins are potent stimulators of T cell proliferation via interaction with specific TCR V-beta segments of both CD4+ and CD8+ T cells. These exoproteins also directly stimulate monocytes to secrete IL-1 and TNF-alpha. Furthermore, these exoproteins have a profound inhibitory effect on Ig production by PBMC. We examined the effects of Staphylococcus enterotoxin A (SEA) on proliferation and Ig production of highly purified human B cells. Our results demonstrated that the binding of SEA to MHC class II molecules on B cells does not alter their ability to proliferate in response to Staphylococcus aureus Cowan strain I (SAC) or to produce Ig in response to SAC plus rIL-2. In contrast, the anti-DR mAb L243 inhibited both B cell proliferation and Ig production. Unable to determine a direct effect of SEA on B cell function, we investigated whether the capacity of SEA to inhibit SAC-induced Ig production by PBMC was T cell-dependent. Our results demonstrated that in the presence of T cells, under appropriate conditions, SEA can either function as a nominal Ag for stimulation of B cell proliferation and Ig production or induce T cell-mediated suppression of Ig production. SEA-induced Ig production required T cell help, which was dependent on pretreatment of the T cells with irradiation or mitomycin C; Ig production was not induced by SEA in the absence of T cells or in the presence of untreated T cells. Furthermore, SEA inhibited Ig production in SAC-stimulated cultures of autologous B cells and untreated T cells; pretreatment of the T cells with irradiation or mitomycin C abrogated SEA-induced inhibition of Ig production. Thus, T cell suppression of SAC-induced Ig production was dependent on T cell proliferation. Similar results were observed with both SEA and toxic shock syndrome toxin 1.     

Tyrphostins inhibit epidermal growth factor (EGF)-receptor tyrosine kinase activity in living cells and EGF-stimulated cell proliferation

Synthetic compounds called tyrphostins were examined for their effects on cells which are mitogenically responsive to epidermal growth factor (EGF). We studied in detail the effects of two tyrphostins on EGF binding, tyrosine phosphorylation in intact cells, EGF-receptor internalization, and mitogenesis. These compounds inhibited EGF-stimulated [3H]thymidine incorporation in a specific manner and the degree of selectivity varied. Both compounds inhibited EGF-stimulated receptor autophosphorylation and tyrosine phosphorylation of endogenous substrates in intact cells at doses that correlated with the IC50 for [3H] thymidine incorporation. These results are consistent with the notion that tyrosine phosphorylation is a crucial signal in transduction of the mitogenic message delivered by EGF. The compound RG50864 demonstrated specificity at inhibiting EGF-stimulated cell growth compared with stimulation with either platelet-derived growth factor or serum. For both compounds RG50864 and RG50810, long term exposure (16 h) of cells to tyrphostins was required for optimal inhibition because of the instability and slow action of these compounds. Tyrphostins did not alter cell surface display of EGF-receptor, EGF binding or EGF-induced internalization, degradation, and down-regulation of EGF receptors. These novel synthetic inhibitors, specific for EGF-receptor kinase, offer a new method to inhibit EGF-stimulated cell proliferation which may be useful in treating specific pathological conditions involving cellular proliferation, including different types of cancers.     

Respiratory burst in human B lymphocytes. Triggering of surface Ig receptors induces modulation of chemiluminescence signal.

B lymphocytes have been shown to proliferate and release oxygen metabolites when surface Ig is cross-linked and when stimulated with phorbol ester. Biochemical evidence has been provided for the presence of a superoxide generating system in B cells, which seems to be identical to the well-characterized NADPH-oxidase of phagocytes. In this report, we show that normal and EBV-transformed B cells produce superoxide anions after stimulation with phorbol ester and when surface Ig was cross-linked, as detected by lucigenin-dependent chemiluminescence. Anti-surface IgG antibodies induced a significant respiratory burst whereas those directed against surface IgM had no effect on B cell oxidative metabolism. Prestimulated B lymphocytes responded to further triggering by the same or another ligand. Pretreatment with Staphlococcus aureus Cowan I strain (SAC) or anti-IgM antibodies resulted in complete unresponsiveness to subsequent SAC or anti-IgG stimulation, but it did not affect PMA- and ionomycin-mediated B cell chemiluminescence. In contrast to preincubation with anti-IgM antibodies, the pretreatment of B cells with SAC induced a transient inhibitory effect on B cell signaling. In fact, SAC-pretreated B lymphocytes could be restimulated with the same ligand when blast cells were isolated. Furthermore, a 24-h incubation of the pretreated B cells in the absence of SAC completely restored the SAC-mediated respiratory burst. These results suggest that two distinct mechanisms may account for SAC- and anti-IgM-induced inhibition: a transient and reversible modulation of surface Ig, induced by SAC, and a long-lasting desensitization of the surface Ig receptors, respectively. These findings may have interesting implications for understanding the transduction of negative signals in B lymphocytes.     

Hepatocyte growth factor rapidly induces the tyrosine phosphorylation of 41-kDa and 43-kDa proteins in mouse keratinocytes.

We have examined the hepatocyte growth factor (HGF)-mediated changes in protein-tyrosine phosphorylation in mouse keratinocytes (PAM-212) and canine kidney epithelial cells (MDCK). In PAM-212 cells HGF and epidermal growth factor, both of which stimulated the DNA synthesis, rapidly induced the tyrosine phosphorylation of two 41-kDa and two 43-kDa proteins: increased tyrosine phosphorylation of those proteins has been commonly observed when quiescent fibroblasts are stimulated with a variety of mitogenic agents. In contrast, HGF did not stimulate the DNA synthesis but induced cell dissociation in MDCK cells; under this condition, increased tyrosine phosphorylation of the 41-kDa and 43-kDa protein was not observed. A possible role of the increased tyrosine phosphorylation of 41-kDa and 43-kDa protein in the signaling pathway of HGF is discussed.     

Tyrosine phosphorylation of phospholipase C-gamma 2 upon cross-linking of membrane Ig on murine B lymphocytes.

When membrane Ig (mIg) on the surface of B lymphocytes is cross-linked using anti-Ig antibodies, the enzyme phospholipase C (PLC) is activated to cleave inositol phospholipids. Tyrosine kinase inhibitors have been reported to inhibit this event. Therefore, we investigated the effect of cross-linking of mIg on the state of tyrosine phosphorylation of PLC activity in two murine B cell lines and in normal resting mouse B cells. Proteins from lysates of stimulated or unstimulated cells were immunoprecipitated with an antiphosphotyrosine antibody and subsequently assayed for PLC activity. Treatment of the B cell line WEHI-231 with anti-IgM led within 15 to 30 s to a 10- to 20-fold increase in tyrosine-phosphorylated PLC activity. Inositol trisphosphate generation by WEHI-231 cells stimulated under the same conditions demonstrated similar kinetics. Normal resting B cells treated with anti-IgM or anti-IgD demonstrated 2.5- and 4-fold increases, respectively, of tyrosine-phosphorylated PLC activity. To identify the isozyme of PLC that was phosphorylated, we immunoprecipitated PLC-gamma 1 or PLC-gamma 2 with specific antibodies and assessed the amount of tyrosine phosphorylation of these proteins by antiphosphotyrosine immunoblotting. Treatment of WEHI-231 or Bal17 cells with anti-IgM induced an increase in PLC-gamma 2 tyrosine phosphorylation over background levels. There was no detectable tyrosine phosphorylation of PLC-gamma 1 in treated or untreated WEHI-231 cells, whereas anti-IgM-treated Bal17 cells did exhibit low but detectable levels of tyrosine phosphorylation of PLC-gamma 1. In normal resting mouse B cells, there was no detectable PLC-gamma 1, but PLC-gamma 2 was abundant. These observations suggest that PLC-gamma 2 is a significant substrate for the mIg-activated protein tyrosine kinase and may be responsible for mediating mIg stimulation of inositol phospholipid hydrolysis in murine B cells.     

B lymphocyte activation is accompanied by phosphorylation of a 72-kDa protein-tyrosine kinase

The cross-linking of membrane IgM on the surface of splenic B lymphocytes or WEHI 231 cells leads to the rapid phosphorylation on tyrosine of a 72-kDa protein as detected in Western blotting experiments using anti-phosphotyrosine antibodies. The 72-kDa phosphoprotein detected in this manner comigrates, in both one- and two-dimensional polyacrylamide gel electrophoresis systems, with PTK72, a 72-kDa protein-tyrosine kinase characterized previously in this laboratory (Zioncheck, T. F., Harrison, M. L., Isaacson, C. C., and Geahlen, R. L. (1988) J. Biol. Chem. 263, 19195-19202). Anti-phosphotyrosine antibodies and anti-PTK72 antibodies immunoprecipitate the same protein-tyrosine kinase from extracts of anti-IgM-activated cells as determined by immune complex kinase assays and one-dimensional phosphopeptide mapping. These results indicate that the tyrosine phosphorylation of a 72-kDa protein-tyrosine kinase is an early event in the activation of B lymphocytes via the antigen receptor.