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.     

Microfilament assembly is involved in B-cell apoptosis.

Crosslinking of the B-cell antigen receptor (BCR) initiates a chain of reactions which culminate in a number of biologic responses, including entry into the cell cycle or cell death. The signals and processes which lead to cell death are slowly being unraveled. Based on the dramatic changes in cell shape which occur during progression of the apoptotic response, activation of cytoskeletal assembly may be critical as this appears to be essential to the mitogenic response. In this study, we demonstrate that crosslinking of the human BCR with anti-IgM antibodies results in the rapid assembly of actin. Our data also suggest that this conversion of G- to F-actin may be a prerequisite for the apoptosis response, as prevention of this conversion by botulinum C2 toxin or cytochalasin D results in rescue of the cells from apoptosis. Prevention of tyrosine kinase activation, disruption of microfilament assembly, and rescue of B lymphocytes from apoptosis imply that tyrosine phosphorylation is needed for both microfilament assembly and apoptosis. In each instance where microfilament assembly is inhibited, anti-IgM-induced activation of the protease CPP32 (caspase) is also inhibited. Taken together, these results suggest that the microfilament system is actively involved in delivering signals for apoptosis.     

Protein tyrosine phosphorylation is induced in murine B lymphocytes in response to stimulation with anti-immunoglobulin.

Activation of both T and B lymphocytes through their membrane receptors for antigen is known to induce breakdown of inositol phospholipids. In addition, T cell activation by antigen is accompanied by increased protein tyrosine phosphorylation of components of the T cell antigen receptor. We now provide evidence that B cell activation through membrane immunoglobulin is also coupled to stimulation of protein tyrosine kinase activity. One potential candidate for a B lymphocyte protein tyrosine kinase is an 80 kd molecule that is itself phosphorylated at tyrosine residues in response to stimulation with anti-immunoglobulin antibodies.     

Association of the 72-kDa protein-tyrosine kinase PTK72 with the B cell antigen receptor.

The antigen receptors on B lymphocytes are cell-surface immunoglobulins. Antibodies against surface IgM (sIgM) coimmunoprecipitate several sIgM-associated proteins. Incubation of anti-IgM complexes with [gamma-32P]ATP leads to the phosphorylation on tyrosine of IgM-associated proteins including MB-1 and a protein of 72 kDa. Peptide mapping and reimmunoprecipitation experiments indicate that the 72-kDa phosphoprotein is PTK72, a protein-tyrosine kinase that is expressed at highest levels in B lymphocytes. MB-1 is also phosphorylated in immune complexes prepared with antibodies to PTK72, indicating that components of the IgM complex are associated with PTK72. In addition, PTK72 is associated with sIgD complexes isolated from spleen B lymphocytes. The cross-linking of sIgM antigen receptors on B lymphocytes leads to the rapid phosphorylation of PTK72 on tyrosine and to the activation of PTK72 as measured by autophosphorylation and by the phosphorylation of an exogenous substrate in anti-PTK72 immune complexes. These results suggest that the signaling cascade initiated by engagement of the B cell antigen receptor involves the increased enzymatic activity of PTK72, which is already present in a preformed antigen receptor complex.     

Tyrosine phosphorylation is an essential event in the stimulation of B lymphocytes by Staphylococcus aureus Cowan I.

Staphylococcus aureus Cowan I (SAC) is a potent mitogen for purified human B cells. By using Western blotting with antiphosphotyrosine antibodies, we demonstrated that the mitogenic effect of SAC is associated with rapid tyrosine phosphorylation of proteins of 45, 68, 75, 97, and 145 kDa. This tyrosine phosphorylation was detected within 30 s of the addition of SAC; it reached a maximum within 10 min, after which it declined gradually. In contrast to SAC, most soluble anti-IgM antibodies do not induce proliferation of isolated human B cells. As indicated by Western blotting, soluble anti-IgM antibodies induced a similar pattern of tyrosine phosphorylation, with the exception of the 68-kDa protein, which was the most heavily phosphorylated protein in SAC-treated cells. A similar but less intense 68-kDa band was also induced by mitogenic anti-IgM bound to beads. This suggested that tyrosine phosphorylation, especially of p68, may play an important role in B cell mitogenesis. To test this hypothesis, we determined the effect of specific tyrosine kinase inhibitors (tyrphostins) on SAC-induced tyrosine phosphorylation, oncogene expression, and B cell proliferation. The concentration dependencies of inhibition of these processes suggested that they were linked. Nonspecific toxic effects of the tyrphostins were ruled out by the demonstration that the tyrphostins did not alter cell viability and did not inhibit B cell proliferation induced by phorbol esters, which do not induce tyrosine phosphorylation. For maximal inhibition of SAC-induced cell proliferation, the tyrophostins needed to be added before or shortly after addition of SAC. Taken together, these data indicate that tyrosine phosphorylation is an obligatory early signal in B cell proliferation.     

Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways.

Stimulation of B lymphocytes through their antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation on a number of proteins and induces both an increase of phosphatidylinositol and mobilization of cytoplasmic free calcium. The BCR associates with two classes of tyrosine kinase: Src-family kinase (Lyn, Fyn, Blk or Lck) and Syk kinase. To dissect the functional roles of these two types of kinase in BCR signaling, lyn-negative and syk-negative B cell lines were established. Syk-deficient B cells abolished the tyrosine phosphorylation of phospholipase C-gamma 2, resulting in the loss of both inositol 1,4,5-trisphosphate (IP3) generation and calcium mobilization upon receptor stimulation. Crosslinking of BCR on Lyn-deficient cells evoked a delayed and slow Ca2+ mobilization, despite the normal kinetics of IP3 turnover. These results demonstrate that Syk mediates IP3 generation, whereas Lyn regulates Ca2+ mobilization through a process independent of IP3 generation.     

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.     

Evidence that tyrphostins AG10 and AG18 are mitochondrial uncouplers that alter phosphorylation-dependent cell signaling

Receptor agonists that initiate fluid secretion in salivary gland epithelial cells also increase protein phosphorylation. To assess contributions of tyrosine phosphorylation to secretion, changes in muscarinic receptor-initiated secretion (estimated from sodium pump-dependent increases in oxygen consumption) were measured in parotid acinar cells exposed to tyrosine kinase inhibitors. However, like the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, tyrphostins AG10 and AG18 increased the rate of oxygen consumption and reduced cellular ATP by approximately 90% in the absence of the muscarinic agonist carbachol, indicating that these tyrphostins uncouple mitochondria. Exposure of isolated mitochondria to five structurally related tyrphostins demonstrated that their relative potencies as uncouplers differed from their in vitro kinase-inhibitory potencies due to different molecular requirements for the two effects. AG10 and AG18 blocked parotid phosphorylation events only at concentrations that reduced ATP content. The tyrosine kinase inhibitor genistein reduced ATP content by 15-20% and weakly uncoupled isolated mitochondria, but its inhibition of carbachol-mediated protein kinase Cdelta tyrosine phosphorylation and ERK1/2 activation appeared attributable to blocking tyrosine kinases directly. Carbachol itself rapidly reduced ATP content by 15-20%. Carbachol, 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (P2X(7) receptor agonist), AG10, AG18, and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone rapidly activated the fuel sensor AMP-activated protein kinase (AMPK); however, only AMPK activation by carbachol and BzATP was due to sodium pump stimulation. AG10 and AG18 also activated AMPK and/or uncoupled mitochondria in PC12, HeLa, and HEK293 cells. These studies demonstrate that some tyrosine kinase inhibitors produce cellular effects that are mechanistically different from their primary in vitro characterizations and, as do salivary secretory stimuli, promote rapid metabolic alterations that initiate secondary signaling events.     

The SH2 domain containing tyrosine phosphatase-1 down-regulates activation of Lyn and Lyn-induced tyrosine phosphorylation of the CD19 receptor in B cells

SHP-1 is a cytosolic tyrosine phosphatase implicated in down-regulation of B cell antigen receptor signaling. SHP-1 effects on the antigen receptor reflect its capacity to dephosphorylate this receptor as well as several inhibitory comodulators. In view of our observation that antigen receptor-induced CD19 tyrosine phosphorylation is constitutively increased in B cells from SHP-l-deficient motheaten mice, we investigated the possibility that CD19, a positive modulator of antigen receptor signaling, represents another substrate for SHP-1. However, analysis of CD19 coimmunoprecipitable tyrosine phosphatase activity in CD19 immunoprecipitates from SHP-1-deficient and wild-type B cells revealed that SHP-1 accounts for only a minor portion of CD19-associated tyrosine phosphatase activity. As CD19 tyrosine phosphorylation is modulated by the Lyn protein-tyrosine kinase, Lyn activity was evaluated in wild-type and motheaten B cells. The results revealed both Lyn as well as CD19-associated Lyn kinase activity to be constitutively and inducibly increased in SHP-1-deficient compared with wild-type B cells. The data also demonstrated SHP-1 to be associated with Lyn in stimulated but not in resting B cells and indicated this interaction to be mediated via Lyn binding to the SHP-1 N-terminal SH2 domain. These findings, together with cyanogen bromide cleavage data revealing that SHP-1 dephosphorylates the Lyn autophosphorylation site, identify Lyn deactivation/dephosphorylation as a likely mechanism whereby SHP-1 exerts its influence on CD19 tyrosine phosphorylation and, by extension, its inhibitory effect on B cell antigen receptor signaling.     

Receptor-mediated activation of human B lymphocytes in a nonphosphotyrosine-dependent manner.

The B cell AgR regulates two signal transduction pathways: the tyrosine kinase and the phosphatidylinositol (PtdIns) pathways. Stimulation of B cells with Ag or anti-Ig antibody results in a rapid increase in tyrosine phosphorylation of multiple substrates. The AgR also mediates the activation of phospholipase C-gamma 1 (PLC-gamma 1) thus producing the second messengers, inositol trisphosphate and diacylglycerol. Although the detailed relationship between these two signaling pathways remains unclear, it has recently become apparent that PLC-gamma 1 might be a target for the AgR-associated protein tyrosine kinase. To address the question of whether tyrosine kinase activity is essential for B cell activation, we studied early biochemical changes and later cellular events induced by ligation of the purinoceptor (P2R). Ligation of ATP to its receptor on B cells has been previously shown to elicit increases in cytosolic free Ca2+ and inositol phosphate production as well as induction of c-fos mRNA expression and increased expression of IL-2 and transferrin receptors. We show here that ATP in a wide range of concentrations did not increase protein tyrosine kinase activity. In contrast with the AgR, P2R did not mediate tyrosine phosphorylation of PLC-gamma 1, thus suggesting that it may use another phosphoinositide-specific PLC that does not require phosphorylation on tyrosine residues for its activation. The results were supported by experiments with a specific tyrosine kinase inhibitor, tyrphostin AG-126. Preincubation with this inhibitor blocked AgR but not P2R-mediated inositol phosphate production, cytosolic free Ca2+ changes, and IL-2 and transferrin receptor expression. The results indicate that the PtdIns pathway may be sufficient to induce activation of B cells and that the tyrosine phosphorylation pathway is not necessary for nonantigenic B cell activation.     

Multiple kinases and signal transduction. Phosphorylation of the T cell antigen receptor complex

Multiple kinases interact at the multicomponent murine T cell antigen receptor. Antigen induces serine phosphorylation of the 21-kDa gamma glycoprotein and tyrosine phosphorylation of p21, a distinct 21-kDa chain. We demonstrate that tyrosine phosphorylation is due to kinase activation, and that all phosphorylated p21 is associated with the antigen receptor. We also show that antigen leads to polyphosphoinositide metabolism and subsequent protein kinase C activation. The two phosphorylation events can be dissociated by protein kinase C depletion, which eliminates phorbol 12-myristate 13-acetate-induced serine but not tyrosine phosphorylation. Activation of a third kinase, cyclic AMP-dependent protein kinase, inhibits both serine and tyrosine events, yet this inhibition can be modulated by addition of the protein kinase C activator, phorbol 12-myristate 13-acetate. Receptor-mediated signal transduction may be understood as the interaction of multiple stimulatory and inhibitory kinase activities.     

T cell receptor tyrosine phosphorylation. Variable coupling for different activating ligands

We have previously reported (Samelson, L.E., Patel, M.D., Weissman, A.M., Harford, J.B., and Klausner, R.D. (1986) Cell 46, 1083-1090) that T cell activation by antigen is associated with activation of two biochemical pathways. In this scheme two protein kinases are activated by stimulation of the T cell antigen receptor (TCR). These kinases phosphorylate two different chains of the TCR complex. Protein kinase C is responsible for the phosphorylation of the gamma, and, to a lesser extent, the epsilon chains of the receptor on serine residues while the activation of an unidentified tyrosine kinase leads to phosphorylation of the p21 subunit of the receptor on tyrosine residues. In addition to activation by specific antigens, T cells can be functionally activated in vitro by the addition of antibodies that bind either the antigen receptor or the Thy-1 molecule, an entity independent of the receptor. We have used antibodies directed against these molecules and show that they result in the same dual kinase activation observed with antigen stimulation. In addition we have compared the three ligands, antigen, and antibodies directed against the epsilon chain of the TCR or against Thy-1, in terms of how they couple to the two kinase pathways. Activation of phosphatidylinositol breakdown and TCR phosphorylation on serine by all three stimuli are sensitive to cAMP inhibition. In contrast, only antigen-stimulated tyrosine kinase activation is sensitive to cAMP while the two antibody reagents activate the tyrosine kinase in a manner that is entirely insensitive to cAMP inhibition.     

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.     

Lymphocyte lineage-restricted tyrosine-phosphorylated proteins that bind PLC gamma 1 SH2 domains.

Epidermal growth factor (EGF) or platelet-derived growth factor binding to their receptor on fibroblasts induces tyrosine phosphorylation of PLC gamma 1 and stable association of PLC gamma 1 with the receptor protein tyrosine kinase. Similarly in lymphocytes, cross-linking of antigen receptors induces the formation of molecular complexes incorporating PLC gamma 1; however, associated kinase activity is thought to be mediated through cytoplasmic protein tyrosine kinase(s). In this report, we generated a fusion protein containing the SH2 domains of human PLC gamma 1 and human IgG1 heavy chain constant region to identify lymphocyte phosphoprotein-binding PLC gamma 1 SH2 domains following cellular activation. As in EGF- or platelet-derived growth factor-stimulated fibroblasts, PLC gamma 1 is coprecipitated in activated lymphocytes, complexed with associated tyrosine-phosphorylated proteins. One of these, a 35/36-kDa protein found prominently in T cells and at lower levels in B cells, bound to the fusion protein in immunoprecipitation experiments. The fusion protein showed lineage restricted association with a 74-kDa phosphoprotein in T cells and a 93-kDa phosphoprotein in B cells. It bound to activated EGF receptor in fibroblasts as expected, and protein tyrosine kinase activity was precipitated from EGF-stimulated cells. However, PLC gamma 1-associated protein tyrosine kinase activity was not detected in activated lymphocytes. These data suggest that lymphocyte PLC gamma 1 SH2-binding proteins are cell lineage specific and may be transiently associated with activated PLC gamma 1.     

Induction of cell shape changes through activation of the interleukin-3 common beta chain receptor by the RON receptor-type tyrosine kinase.

The RON receptor-type tyrosine kinase, a member of the hepatocyte growth factor receptor family, is a receptor for macrophage-stimulating protein (MSP). Recently, we observed that MSP induces morphological changes in interleukin (IL)-3-dependent Ba/F3 cells ectopically expressing RON. We show here that stimulation of those cells with either MSP or IL-3 increases tyrosine phosphorylation of proteins of 130, 110, 90, 62, and 58 kDa and induces similar morphological changes, accompanied by unique nuclear shape and redistribution of F-actin. A tyrosine kinase inhibitor, genistein, blocked both the increase in tyrosine phosphorylation and morphological changes. Upon stimulation with either MSP or IL-3, prominent tyrosine-phosphorylated pp90 was similarly co-immunoprecipitated with the common beta chain of IL-3 receptor (betac). Unlike IL-3, stimulation with MSP increased tyrosine phosphorylation of betac without activation of JAK2, resulting in morphological changes with modest cell growth. Confocal immunofluorescence analyses showed colocalization of RON, betac, and tyrosine-phosphorylated proteins. In vitro kinase assays revealed that autophosphorylated RON phosphorylated betac. These results suggest that the signaling pathway for morphological changes through betac and its associated protein pp90 is distinct from the pathway for cell growth in the IL-3 signal transduction system.     

Signal transduction mediated by antigen receptors on teleost lymphocytes

To generate an adaptive response from the mammalian immune system requires that antigen bind to cognate receptors on T and B cells, a process which activates intracellular signaling pathways. Crosslinking the B cell antigen receptor (BCR) ultimately activates cell proliferation in both higher and lower vertebrates. Recent studies suggest that many functional components of these intracellular pathways were evolutionarily conserved among the vertebrates. Antibody-mediated crosslinking of surface immunoglobulin leads to tyrosine phosphorylation on presumptive accessory molecules of the teleost BCR as well as several intracellular proteins. Crosslinking the teleost BCR also triggers calcium influx and activation of protein kinase C (PKC) which are hallmark components of the phosphatidyl inositol signal transduction pathway in mammalian lymphocytes. The activation of teleost PKC ultimately generates dually-phosphorylated forms of mitogen activated protein kinase. The latter enzyme is viewed as a key cytoplasmic control point for integrating signals arriving from several kinase/phosphatase pathways in mammalian cells. Preliminary evidence suggests that intracellular signaling mediated through antigen receptor complexes may be very sensitive to external factors, including heavy metals such as mercuric chloride which can alter calcium flux and tyrosine phosphorylation patterns in teleost leukocytes. As the process of lymphocyte activation in teleost fish is better understood, it may be possible to provide aquaculturists, environmental regulators and fisheries managers with better information on those natural and man-made conditions which interfere with the development of protective immune responses in natural and captive finfish populations.     

Novel association of the src family kinases, hck and c-fgr, with CCR3 receptor stimulation: A possible mechanism for eotaxin-induced human eosinophil chemotaxis.

The chemokine eotaxin is a potent and relatively eosinophil-specific chemoattractant implicated in the cell migration to inflammatory sites in allergic diseases. Eotaxin exerts its activity solely through the CCR3 receptor, but the signaling pathways are poorly defined. In this study, we show that eotaxin induces an increase in tyrosine phosphorylation of multiple cellular proteins in normal human eosinophils. Eotaxin-dependent tyrosine phosphorylation was detected 1 min after stimulation and increased for at least 15 min with kinetics similar to those of eotaxin-induced cell shape changes. Herbimycin A, a tyrosine kinase inhibitor, blocked both eotaxin-induced tyrosine phosphorylation and cell shape changes as well as chemotaxis. Immunofluorescence microscopy analyses showed that eotaxin-induced cell shape changes were accompanied by redistribution of tyrosine-phosphorylated proteins and F-actin reorganization that were sensitive to herbimycin A. Coimmunoprecipitation studies revealed that binding of eotaxin to CCR3 greatly enhanced association of the Src family kinases, Hck and c-Fgr, with CCR3 after internalization of CCR3. These results may indicate that recruitment of Hck and c-Fgr to CCR3 in a compartment triggers tyrosine phosphorylation, leading to rapid cell shape changes required for cell migration.     

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.