Fc gamma receptors differ in their structural requirements for interaction with the tyrosine kinase Syk in the initial steps of signaling for phagocytosis

Receptors for the constant region of IgG, Fc gamma receptors, are expressed on the surface of hematopoietic cells, where they mediate signaling events, such as phagocytosis, essential for host defense. Fc gamma receptors also play a role in the pathophysiology of autoimmune diseases. We have demonstrated that members of each of the three classes of human Fc gamma receptors, Fc gamma RI, Fc gamma RII, and Fc gamma RIII, mediate phagocytosis, but that important differences exist in their requirements for phagocytic signaling. For example, the Fc gamma receptors Fc gamma RI and Fc gamma RIIIA induce signaling largely by association with a gamma subunit containing a conserved cytoplasmic motif (ITAM) whose tyrosines are phosphorylated following receptor stimulation. Fc gamma RIIA contains a similar motif in its own cytoplasmic domain and does not require the gamma chain for phagocytic signaling. The tyrosine kinase Syk associates with the cytoplasmic domain of both the Fc gamma receptor gamma chain and Fc gamma RIIA and is required for phagocytosis by both Fc gamma receptor systems. To elucidate the differences in phagocytic signaling by the gamma chain and Fc gamma RIIA, we investigated the requirements for Fc gamma receptor/Syk co-immunoprecipitation, tyrosine phosphorylation, and phagocytosis. Both Fc gamma RIIA and the human gamma chain contain a tyrosine seven amino acids upstream of the ITAM motif. We observed that the upstream tyrosine plays a role in Fc gamma RIIA phagocytic signaling but is not involved in phagocytic signaling by the human gamma chain. Our data also indicate that the two ITAM tyrosines of the human gamma chain and Fc gamma RIIA do not contribute equally to Fc gamma receptor association with Syk kinase and phagocytic signaling. The data indicate that the carboxy-terminal tyrosine of the receptor cytoplasmic domain is especially important both for the interaction with Syk kinase and for phagocytosis. Elucidating such differences in gamma chain and Fc gamma RIIA signaling may be valuable in designing strategies for therapeutic intervention in hematopoietic and immunological disorders.     

alpha 2-macroglobulin stimulation of protein tyrosine phosphorylation in macrophages via the mannose receptor for Fc gamma receptor-mediated phagocytosis activation.

Macrophage phagocytic activity has previously been shown to be increased by binding of modified alpha 2-macroglobulin (alpha 2M) with mannose residues at termini of sugar chains to mannose receptors on macrophages, with a subsequent increase in the number of Fc gamma receptors at the cell surface. In the present study, an examination was made of the association of protein tyrosine kinase with the increase in number of Fc gamma receptors following binding of modified alpha 2M to mannose receptors. The phagocytosis of IgG-opsonized sheep red blood cells through the action of the Fc gamma receptor by modified alpha 2M was inhibited by mannose and herbimycin A and slightly so by genistein. The mannose receptor would thus appear to be associated with tyrosine kinase activity. By Western blotting, tyrosine phosphorylated proteins with molecular weights of 32000, 34000, 36000, 65000, 85000 and 110000 appeared or increased upon treating macrophages with modified alpha 2M. The degree of tyrosine phosphorylated proteins was the same for control macrophages following incubation in the presence of mannose and herbimycin A. Genistein treatment affected only tyrosine phosphorylated proteins of 65000 and 110000. The binding of modified alpha 2M to mannose receptors was demonstrated by the inducement of tyrosine kinase activation that was sensitive to herbimycin A, followed by an increase in Fc gamma receptors and consequently greater phagocytosis.     

Activation and tyrosine phosphorylation of protein kinase C delta in response to B cell antigen receptor stimulation

Activation of the B cell through antigen receptor (BCR) crosslinking is known to initiate a prominent tyrosine kinase cascade and lipid second messenger production through the activation of phospholipase Cgamma and phosphatidylinositol 3' kinase. In this study, we demonstrate that protein kinase C delta (PKCdelta) responds to crosslinking of the BCR by becoming activated and tyrosine phosphorylated within 30s of stimulation. PKC6 activation was dependent primarily on phosphatidylinositol 3' kinase, and this in turn was dependent on an upstream tyrosine phosphorylation event. Inhibition of PKCdelta activation by blocking phosphatidylinositol 3' kinase was also accompanied by a decrease in its tyrosine phosphorylation, suggesting that PKCdelta must be activated in order to become tyrosine phosphorylated. Inhibition of phospholipase C activation had an insignificant effect on the activation of PKCdelta, however it attenuated the tyrosine phosphorylation of PKCdelta. This suggests a distinct role for phospholipase C in the regulation of PKCdelta. This report describes a role for PKCdelta in response to the combined signals originated by the BCR.     

Characterisation and specificity of two single-chain Fv antibodies directed to the protein tyrosine kinase Syk

In order to obtain single chain Fv fragments (scFv) specific for the protein tyrosine kinase Syk, we screened a human synthetic phage-display library. Two glutathione S-transferase (GST):Syk fusion proteins containing both SH2 domains of Syk were used to perform three rounds of selection of the library. Among the scFv fragments resulting from the third round of selection, the ones specific for the GST portion of the fusion proteins were eliminated by performing enzyme-linked immunosorbent assay tests on GST:Syk versus GST coated plates, and the monoclonal scFv fragments binding only to the GST:Syk coated plates with high affinities were further analysed. We report here the in vitro characterisation of G4G11 and G6G2 anti-Syk scFvs. G4G11 shows the best performance in immunoprecipitation and immunofluorescence experiments, and G6G2 is able to detect Syk in immunoprecipitation, immunofluorescence and on Western blots. Both scFvs are also able to detect the phosphorylated form of Syk, and neither of them binds to Zap-70, the other member of the Syk family of protein tyrosine kinases.     

HLA class-II-mediated homotypic aggregation: involvement of a protein tyrosine kinase and protein kinase C.

Homotypic aggregation of B-lymphocytes, B-cell lines and class-II-positive T cells via HLA class II molecules was examined. Signaling via DR antigens induced rapid aggregation in a dose- and time-dependent manner, maximum and stable aggregation was induced within 20 minutes. On the contrary, rapid signaling via DP or DQ required prestimulation with either PMA or anti-sIg. Aggregation was temperature and energy dependent. [Ca2+] and [Mg2+] concentrations and an intact cytoskeleton were required while neither mRNA or protein synthesis were required. Furthermore, FACS analysis revealed that aggregation was not directly correlated with cell surface expression of HLA class II molecules. Our results demonstrate that aggregation was mediated through a protein tyrosine kinase (PTK)-dependent pathway that preceded activation of protein kinase C (PKC) and failure to generate either the PTK signal or the PKC signal prevented aggregation. The contribution of a tyrosine kinase was further demonstrated by the total inhibition of aggregation following treatment with an anti-CD45 mAb.     

Protein tyrosine kinase activity is essential for Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes.

Our previous study revealed that the intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma receptor I (Fc gamma RI) or Fc gamma RII is a phospholipase C (PLC)-dependent process. The aim of the present study was to investigate whether protein tyrosine kinase (PTK) activity plays a role in the Fc gamma R-mediated intracellular killing of bacteria and activation of PLC in these cells. The results showed that phagocytosis of bacteria by monocytes was not affected by the PTK inhibitors genistein and tyrphostin-47. The intracellular killing of S. aureus by monocytes after cross-linking Fc gamma RII or Fc gamma RII with anti-Fc gamma R monoclonal antibody and a bridging antibody or with human immunoglobulin G (IgG) was inhibited by these compounds in a dose-dependent fashion. The production of O2- by monocytes after stimulation with IgG or IgG-opsonized S. aureus was almost completely blocked by the PTK inhibitor. These results indicate that inhibition of PTK impairs the oxygen-dependent bactericidal mechanisms of monocytes. Genistein and tyrphostin-47, which do not affect the enzymatic activity of purified PLC, prevented activation of PLC after cross-linking Fc gamma RI or Fc gamma RII, measured as an increase in the intracellular inositol 1,4,5-trisphosphate concentration. Cross-linking Fc gamma RI or Fc gamma RII induced rapid tyrosine phosphorylation of several proteins in monocytes, one of which was identified as PLC-gamma 1, and the phosphorylation could be completely blocked by PTK inhibitors, leading to the conclusion that activation of PLC after cross-linking Fc gamma R in monocytes is regulated by PTK activity. Together, these results demonstrate that PTK activity is essential for the activation of PLC which is involved in the Fc gamma R-mediated intracellular killing of S. aureus by human monocytes.     

IgA-mediated inhibition of human leucocyte function by interference with Fc gamma and C3b receptors.

The inhibitory effects of IgA from human colostrum, and IgA1 and IgA2 from human serum on the chemiluminescence (CL) response and phagocytosis of polymorphonuclear leucocytes (PML) to Staphylococcus epidermidis and the CL response to formylmethionyl-leucyl-phenylalanine (FMLP) were studied. The dose-dependent inhibition of the luminol-mediated CL response of human PML to the bacteria was observed in the presence of more than 0.1 mg/ml IgA from both colostrum and serum. The preincubation of PML with a solution of IgA enhanced the suppressive effect of IgA on the cells. Removal of IgA from the reaction mixture after preincubation resulted in recovery, with time, of the response of PML to the bacteria. The bacteria treated with IgA did not give rise to any inhibition of the response. The CL response of PML to FMLP was not affected by the presence of IgA in the reaction mixture. The decrease of phagocytic activity of PML in the presence of IgA resulted in a decrease of NADPH oxidase activity of PML after stimulation with the bacteria as compared with the absence of IgA. The effect of IgA on the receptors of Fc and C3b (CR1) on the surface of PML was measured by monitoring erythrocyte-antibody (EA) or erythrocyte-antibody-complement (EAC) rosette formation and by direct and indirect immunofluorescence techniques using anti-CR1 antibody and Fc-specific antibodies. The presence of IgA in the reaction mixture led to a quantitative decrease in CR1 and the ability to bind IgG to the surface of PML.     

Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation.

Protein tyrosine phosphorylation is an important intracellular event accompanying the in-vitro capacitation of mouse, bovine and human spermatozoa. Here, we demonstrate that bovine serum albumin (BSA) and NaHCO(3) are required for protein tyrosine phosphorylation in ejaculated human spermatozoa. The absence of protein tyrosine phosphorylation in media minus these two constituents could be recovered by addition to the media of cAMP analogues and/or phosphodiesterase inhibitors. Since BSA is postulated to modulate capacitation by removal of cholesterol from the sperm plasma membrane, we determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Incubation of spermatozoa in media containing BSA resulted in the release of significant amounts of cholesterol when compared with media devoid of BSA. Preloading BSA with cholesterol-SO(4) inhibited protein tyrosine phosphorylation, as well as capacitation, and this inhibitory effect was overcome by the addition of dibutyryl cAMP plus isobutylmethylxanthine (IBMX). The functional significance of BSA-mediated cholesterol release, protein tyrosine phosphorylation and capacitation was confirmed by examining the effects of the cholesterol-binding heptasaccharides, methyl-beta-cyclodextrin or OH-propyl-beta-cyclodextrin. Both cyclodextrins caused cholesterol efflux from the spermatozoa, increased protein tyrosine phosphorylation, and stimulated capacitation. Therefore, cholesterol release is associated with the activation of a signal transduction pathway involving protein kinase A and tyrosine kinase second messenger systems, and resulting in protein tyrosine phosphorylation and capacitation.     

Serum-mediated inhibition of lymphocyte Fc gamma receptors in glomerulonephritis.

Non-complement-fixing immune complexes were detected by EA rosette inhibition in the sera of 47% of patients with primary glomerulonephritis (GN) and 60% with GN associated with systemic disease. Fifteen patients with minimal-change nephrotic syndrome (MCNS) in relapse produced significantly greater inhibition than the sera from 11 who were in remission.     

Positive and negative regulation of Fc epsilon receptor I-mediated signaling events by Lyn kinase C-terminal tyrosine phosphorylation

Although it is known that the Src family tyrosine kinase Lyn initiates Fc epsilon receptor I (Fc epsilon RI) signaling by phosphorylation of the receptor subunits, regulation of Lyn kinase activity and its consequences for receptor signaling are incompletely understood. Using a phospho-Lyn-specific antiserum, we show an increased phosphorylation of the Lyn C-terminal regulatory tyrosine and decreased Lyn kinase activity during Fc epsilon RI-mediated mast cell activation. Mutant Lyn, defective in the C-terminal tyrosine, constitutively phosphorylated several substrates in resting cells, but did not cause Fc epsilon RI internalization or spontaneous degranulation. Fc epsilon RI-induced signaling in the presence of constitutively active Lyn exhibited enhanced phosphorylation of the receptor subunits, Syk, LAT, Gab2, phospholipase C (PLC)gamma 1 and PLC gamma 2, and production of phosphatidylinositol 3,4,5-trisphosphate. Although enzymatic activities of PLC gamma 1 and PLC gamma 2 were also up-regulated, amounts of inositol 1,4,5-trisphosphate, mobilization of intracellular calcium and degranulation were suppressed. Additionally, constitutively active Lyn was strikingly less efficient than wild-type Lyn in restoring the receptor-mediated calcium responses in bone marrow mast cells derived from Lyn(-/-) mice. These findings pinpoint the tight regulation of Lyn kinase activity as a critical event in mast cell degranulation.     

Rottlerin-independent attenuation of pervanadate-induced tyrosine phosphorylation events by protein kinase C-delta in hemopoietic cells

The understanding and control of many pathophysiological conditions is based on knowledge of subtly regulated intracellular signaling networks. We have found that in pervanadate (PV)-treated J558L myeloma cells, amongst other signaling proteins, protein kinase C (PKC)-delta and src homology 2-containing inositol phosphatase (SHIP) are tyrosine phosphorylated on expression of the B cell receptor, suggesting a role for these proteins in the preformed B cell receptor transducer complex. Rottlerin, a widely used PKC-delta-specific inhibitor, efficiently blocks these PV-induced tyrosine phosphorylation events. Furthermore, PV treatment of bone marrow-derived mast cells (BMMC) also results in tyrosine phosphorylation of PKC-delta, SHIP, and additional proteins. Rottlerin also inhibits these responses, indicating that PKC-delta might play an important enhancing role in the propagation of phosphotyrosine signals in B cells and mast cells and hence in the regulation of function of both cell types. Therefore, BMMC from PKC-delta -/- mice were generated by in vitro differentiation and assayed for tyrosine phosphorylation events in response to PV. Intriguingly, and opposite to the Rottlerin data, PKC-delta -/- BMMC show a stronger response to PV than wild-type cells, suggesting an attenuating role for PKC-delta. This response can be inhibited equally well by Rottlerin, indicating clearly that Rottlerin is not specific for PKC-delta in vivo. A comparison between Rottlerin and the panspecific PKC inhibitor bisindolylmaleimide suggests that Rottlerin also targets kinases beyond the PKC family. Moreover, Ser473 phosphorylation of protein kinase B (PKB) after PV treatment is blocked by Rottlerin as efficiently as by the phosphatidylinositol 3-kinase inhibitor LY294002. In this report, we provide evidence that PKC-delta constitutes a crucial attenuating factor in B cell and mast cell signal transduction and suggest that PKC-delta is important for the regulation of physiological B and mast cell functions as well as for their pathophysiology. Furthermore, dominant PKC-delta-independent effects of Rottlerin are presented, indicating restrictions of this inhibitor for use in signal transduction research.     

Tyrosine phosphorylation of phospholipase C gamma 1 couples the Fc epsilon receptor mediated signal to mast cells secretion.

Mast cells respond to clustering of the type I Fc epsilon receptor (Fc epsilon RI) on their membranes by mediator secretion. Recently, a marked enhancement of tyrosine phosphorylation on several proteins has been observed as a result of antigen-induced Fc epsilon RI aggregation on these cells. We report here that the phosphatidyl inositide specific phospholipase C gamma 1 (PLC gamma 1) is one of the prime proteins that undergoes tyrosine phosphorylation as a result of this stimulus. This was determined by immunoprecipitation of phosphotyrosine containing proteins from detergent lysates of rat mucosal mast cells (rat basophilic leukemia cells, subline 2H3; RBL-2H3) and Western blotting analysis of the separated components. A fast appearance of phosphorylated tyrosine residues on PLC gamma 1 was observed, reaching its maximal intensity at approximately 1-3 min after stimulation and declined afterwards to basal levels. Moreover, the phosphorylation depended on maintaining the aggregated Fc epsilon RI as did other cellular responses (e.g. phosphatidyl inositides hydrolysis and secretion). The time course of both Fc epsilon RI induced phospholipase C gamma 1 activation, as monitored by the formation of inositol phosphates, and of the secretory response of the cells followed that of the PLC gamma 1 phosphorylation. Furthermore, the tyrphostin AG490, a protein tyrosine kinase inhibitor, caused similar inhibition of the Fc epsilon RI-induced PLC gamma 1 phosphorylation, inositol phosphates formation, and mediator secretion. Significantly, no tyrosine phosphorylation of PLC gamma 1 was induced by the Ca2+ ionophore, ionomycin, even at doses that cause optimal secretory response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of natural killer cells via the Fc gamma RIII (CD16) requires initial tyrosine phosphorylation.

Triggering of the Fc gamma RIII (CD16) on natural killer (NK) cells by monoclonal antibodies or antibody-coated target cells stimulates a rapid phospholipase C (PLC)-mediated hydrolysis of inositol phospholipids and results in subsequent delivery of the lytic hit. The role of initial tyrosine phosphorylation in these events was investigated with a tyrosine protein kinase (TPK) inhibitor, genistein. At doses that inhibited CD16-triggered tyrosine phosphorylation of substrates in intact cells, genistein did not influence serine/threonine phosphorylation or target cell binding but prevented PLC activation, cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity. These findings indicate that tyrosine phosphorylation is an early and critical event during receptor-mediated activation of the lytic machinery.     

Role of activatory Fc gamma RI and Fc gamma RIII and inhibitory Fc gamma RII in inflammation and cartilage destruction during experimental antigen-induced arthritis.

IgG-containing immune complexes, which are found in most RA joints, communicate with hematopoietic cells using three classes of Fc receptors(Fc gamma RI, -II, -III). In a previous study we found that if a chronic T-cell-mediated antigen-induced arthritis (AIA) was elicited in knee joints of FcR gamma-chain-deficient mice that lack functional Fc gamma RI and Fc gamma RIII, joint inflammation was comparable but severe cartilage destruction was absent. We now examined the individual role of the stimulatory Fc gamma RI and Fc gamma RIII and inhibitory Fc gamma RII in inflammation and functional cartilage damage in knee joints with AIA using Fc gamma RI-, Fc gamma RII-, and Fc gamma RIII-deficient mice. Three weeks after immunization with the antigen-methylated bovine serum albumin (BSA), cellular (T-cell responses as measured by lymphocyte proliferation) immunity raised against mBSA was comparable in all groups examined. Humoral (total IgG, IgG1, IgG2a, and IgG2b levels) immunity against mBSA was comparable in Fc gamma RI-/- and Fc gamma RIII-/- but higher in Fc gamma RII-/- if compared to controls. Joint swelling as measured by (99m)Tc uptake at days 1, 3, and 7 was similar in Fc gamma RI-/- and Fc gamma RIII-/- mice and significantly higher in Fc gamma RII-/-. Chronic inflammation and cartilage damage (depletion of proteoglycans, metalloproteinase (MMP)-induced neoepitopes, and matrix erosion) was studied histologically in total knee joint sections stained with hematoxylin or safranin-O. Histologically, at day 7 after AIA induction, exudate and infiltrate in the knee joint was similar in Fc gamma RI-/- and Fc gamma RIII-/- and significantly higher (230% and 340%) in Fc gamma RII-/- mice if compared to controls. Aggrecan breakdown in cartilage caused by MMPs and, which is related to severe irreversible cartilage erosion, was further studied by immunolocalization of MMP-mediated neoepitopes (VDIPEN) and image analysis. MMP-induced neoepitopes determined in various cartilage layers (tibia and femur) were primarily inhibited in Fc gamma RI-/- (79 to 87% and 87 to 88%, respectively) and comparable in Fc gamma RIII-/-. VDIPEN neoepitopes were much higher (82 to 122% and 200 to 250%, respectively) in Fc gamma RII-/- mice. Initial depletion of proteoglycans was similar (60 to 100%) in all groups. In the chronic phase, cartilage matrix erosion in the lateral and medial tibia was significantly elevated in Fc gamma RII-/- (222% and 186%, respectively) but not in Fc gamma RI-/- or Fc gamma RIII-/- mice. These results suggest that during T-cell-mediated AIA, Fc gamma RI and Fc gamma RIII act in concert in acute and chronic inflammation whereas Fc gamma RI is the dominant FcR involved in severe cartilage destruction. Fc gamma RII is a crucial inhibiting factor in acute and chronic inflammation and cartilage erosion.     

Cross-linking of CD26 by antibody induces tyrosine phosphorylation and activation of mitogen-activated protein kinase.

CD26, a T-cell activation antigen that has dipeptidyl peptidase IV activity in its extracellular domain and has also been shown to play an important role in T-cell activation. The earliest biochemical events seen in stimulated T lymphocytes activated through the engagement of the T-cell receptor (TCR) is the tyrosine phosphorylation of a panel of cellular proteins. In this study we demonstrate that antibody-induced cross-linking of CD26-in CD26-transfected Jurkat cells induced tyrosine phosphorylation of several intracellular proteins with a similar pattern to that seen after TCR/CD3 stimulation. Herbimycin A, an inhibitor of the src family protein tyrosine kinases dramatically inhibited this CD26-mediated effect on tyrosine phosphorylation. Major tyrosine phosphorylated proteins were identified by immunoblotting, and included p56lck, p59fyn, zeta associated protein-tyrosine kinase of 70,000 MW (ZAP-70), mitogen-activated protein (MAP) kinase, c-Cb1, and phospholipase C gamma. CD26-induced tyrosine phosphorylation of MAP kinase correlated with increased MAP kinase activity. In addition, CD26 was costimulatory to CD3 signal transduction since co-cross-linking of CD26 and CD3 antigens induced prolonged and increased tyrosine phosphorylation in comparison with CD3 activation alone. We therefore conclude that CD26 is a true costimulatory entity that can up-regulate the signal transducing properties of the TCR.     

CD28 ligation induces rapid tyrosine phosphorylation of the linker molecule LAT in the absence of Syk and ZAP-70 tyrosine phosphorylation.

CD28 is a T cell surface molecule that is important for T cell activation. CD28-triggered T cell stimulation involves protein tyrosine phosphorylation, a process that is critical for CD28 function. Recently, a linker molecule has been identified as LAT (Linker for Activation of T cells). Studies involving LAT mutants and reconstitution experiments strongly implicate LAT in playing a critical role in T cell activation. We show in the present report that CD28 ligation induces tyrosine phosphorylation of LAT. CD28-induced tyrosine phosphorylation of LAT was rapid, as it was apparent within 1 min of CD28 ligation, reached a peak by 5 min, and declined thereafter. Previous studies implicated the protein tyrosine kinases ZAP-70 and Syk in the TCR-induced tyrosine phosphorylation of LAT. Here, tyrosine phosphorylation of Syk and ZAP-70 was detected after TCR but not after CD28 ligation. Thus, CD28 ligation appears to induce tyrosine phosphorylation of LAT by mechanisms that are independent of ZAP-70 and Syk. The concurrent ligation of CD28 and TCR increased tyrosine phosphorylation of LAT. These results implicate LAT in CD28 signal transduction pathways and in the co-stimulatory process in T cells.     

Amyloid precursor protein processing is separately regulated by protein kinase C and tyrosine kinase in human astrocytes

Regulation of amyloid precursor protein (APP) processing by protein kinase C (PKC) and phosphotyrosine pathways was investigated in cultured human astrocytes. Phorbol 12, 13-dibutyrate (PDBu), a PKC activator, increased secretion of APPalpha 2-3-fold over control values, and GF109203X, a PKC inhibitor, blocked this effect. Similarly, platelet derived growth factor (PDGF) increased the secreted form of APPalpha (sAPPalpha) level two-fold, and genistein, a tyrosine kinase inhibitor, blocked the stimulatory effect of PDGF. Co-treatment of PDGF and PDBu resulted in a five-fold increase in the sAPPalpha production, and genistein and GF109203X did not block the stimulatory effects of PDBu and PDGF, respectively. These results indicate that both PKC and phosphotyrosine pathways are involved in APP processing in human astrocytes, but they act independently. The two pathways appear to converge to mitogen-activated protein kinase (MAPK) because PD98059, a MAPK inhibitor, blocked the effects of PDBu and PDGF on APPalpha secretion.