Anti-human very late antigen-α4 (CD49d) monoclonal antibody (BU49) cross-reacts with the canine B-cell leukemia cell line GL-1, resulting in the induction of homotypic cell aggregation

We have found that the anti-human very late antigen-α4 (VLA-α4) (CD49d) monoclonal antibody (mAb) BU49 cross-reacts with the canine B-cell leukemia cell line GL-1. Interestingly, the BU49 mAb specifically induced the homotypic cell aggregation of GL-1 cells accompanied by morphological changes. Homotypic cell aggregates induced by BU49 mAb were blocked by the presence of a protein kinase C inhibitor, a protein kinase A inhibitor, an actin filament formation inhibitor, and an EDTA. On the other hand, a protein tyrosine kinase inhibitor, a DNA-synthesis inhibitor, and an anti-canine CD45 mAb did not affect the GL-1 homotypic cell aggregation induced by BU49 mAb. The BU49 mAb immunoprecipitated at a molecular weight of about 150 kDa in the GL-1 cells, similar to the results in the human monocyte-like cell line U937. Taken together, our results provide the first evidence that human CD49d recognized by BU49 mAb has unique immunological functions against canine cells.     

A monoclonal antibody to beta 1 integrin (CD29) stimulates VLA- dependent adherence of leukocytes to human umbilical vein endothelial cells and matrix components

The leukocyte beta 1 integrin receptor very late activation antigen-4 (VLA-4) (alpha 4 beta 1, CD49d/CD29) binds to vascular cell adhesion molecule-1 (VCAM-1) expressed on cytokine-activated endothelium. A mAb designated 8A2 was identified that stimulated the binding of U937 cells to CHO cells transfected with VCAM-1 cDNA but not endothelial-leukocyte adhesion molecule or CD4 cDNA. mAb 8A2 also rapidly stimulated the adherence of peripheral blood lymphocytes (PBLs) to VCAM-1-transfected CHO cells or recombinant human tumor necrosis factor-treated human umbilical vein endothelial cells. mAb 8A2-stimulated binding of PBL was inhibited by mAbs to VLA-4 or VCAM-1. Surface expression of VLA-4 was not altered by mAb 8A2 treatment and monovalent Fab fragments of mAb 8A2 were active. Immunoprecipitation studies reveal that mAb 8A2 recognizes beta 1-subunit (CD29) of integrin receptors. In contrast to mAbs directed to VLA-4 alpha-subunit (alpha 4, CD49d), mAb 8A2 did not induce homotypic aggregation of PBL. Additionally, mAb 8A2 stimulated adherence of PBL and hematopoietic cell lines to purified matrix components laminin and fibronectin. This binding was blocked by mAbs to the VLA alpha-subunits alpha 6 (CD49f), or alpha 5 (CD49e) and alpha 4 (CD49d), respectively. We conclude that mAb 8A2 modulates the affinity of VLA-4 and other leukocyte beta 1 integrins, and should prove useful in studying the regulation of beta 1 integrin function.     

Regulation of CD93 cell surface expression by protein kinase C isoenzymes

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.     

Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent inhibition of IL-5 from human T lymphocytes is not mediated by the cAMP-dependent protein kinase A

IL-5 is implicated in the pathogenesis of asthma and is predominantly released from T lymphocytes of the Th2 phenotype. In anti-CD3 plus anti-CD28-stimulated PBMC, albuterol, isoproterenol, rolipram, PGE2, forskolin, cholera toxin, and the cAMP analog, 8-bromoadenosine cAMP (8-Br-cAMP) all inhibited the release of IL-5 and lymphocyte proliferation. Although all of the above compounds share the ability to increase intracellular cAMP levels and activate protein kinase (PK) A, the PKA inhibitor H-89 failed to ablate the inhibition of IL-5 production mediated by 8-Br-cAMP, rolipram, forskolin, or PGE2. Similarly, H-89 had no effect on the cAMP-mediated inhibition of lymphocyte proliferation. Significantly, these observations occurred at a concentration of H-89 (3 microM) that inhibited both PKA activity and CREB phosphorylation in intact cells. Additional studies showed that the PKA inhibitors H-8, 8-(4-chlorophenylthio) adenosine-3',5'-cyclic monophosphorothioate Rp isomer, and a myristolated PKA inhibitor peptide also failed to block the 8-Br-cAMP-mediated inhibition of IL-5 release from PBMC. Likewise, a role for PKG was considered unlikely because both activators and inhibitors of this enzyme had no effect on IL-5 release. Western blotting identified Rap1, a downstream target of the cAMP-binding proteins, exchange protein directly activated by cAMP/cAMP-guanine nucleotide exchange factors 1 and 2, in PBMC. However, Rap1 activation assays revealed that this pathway is also unlikely to be involved in the cAMP-mediated inhibition of IL-5. Taken together, these results indicate that cAMP-elevating agents inhibit IL-5 release from PBMC by a novel cAMP-dependent mechanism that does not involve the activation of PKA.     

Opposite effects of thrombospondin-1 via CD36 and CD47 on homotypic aggregation of monocytic cells.

Thrombospondin-1 (TSP-1), an extracellular matrix protein, has a multimodular structure and each domain specifies a distinct biological function through interaction with a specific ligand. In this study we found that exogenously added TSP-1 inhibits phorbol myristate acetate (PMA)/LPS-induced homotypic aggregation of human monocytic U937 cells, whereas the 70-kDa fragment of TSP-1 generated by the proteolytic cleavage of the intact molecule promotes the homotypic aggregation. The aggregation was also inhibited by anti-CD47 mAb or the 4N1K peptide, of which sequence is derived from the CD47-binding site of TSP-1 and absent in the 70-kDa fragment. In contrast, the augmented cell aggregation by the 70-kDa fragment was hampered by anti-CD36 mAb or antibody against the CD36-binding site of TSP-1. The cell aggregation of U937 cells was completely blocked, even in the presence of the 70-kDa fragment, by mAb against leukocyte function associated antigen-1 (LFA-1) or intercellular adhesion molecule-1 (ICAM-1). We therefore propose that TSP-1 may regulate LFA-1/ICAM-1-mediated cell adhesion of monocytes/macrophages by either the inhibitory effect through CD47 or the promoting effect through CD36 depending on which domain/fragment is functional in a given biological setting.     

Coordinate regulation of forskolin-induced cellular proliferation in macrophages by protein kinase A/cAMP-response element-binding protein (CREB) and Epac1-Rap1 signaling: effects of silencing CREB gene expression on Akt activation

In this study, we have examined the role of two cAMP downstream effectors protein kinase A (PKA) and Epac, in forskolin-induced macrophage proliferation. Treatment of macrophages with forskolin enhanced [(3)H]thymidine uptake and increased cell number, and both were profoundly reduced by prior treatment of cells with H-89, a specific PKA inhibitor. Incubation of macrophages with forskolin triggered the activation of Akt, predominantly by phosphorylation of Ser-473, as measured by Western blotting and assay of its kinase activity. Akt activation was significantly inhibited by LY294002 and wortmannin, specific inhibitors of phosphatidylinositol 3-kinase, but not by H-89. Incubation of macrophages with forskolin also increased Epac1 and Rap1.GTP. Immunoprecipitation of Epac1 in forskolin-stimulated cells co-immunoprecipitated Rap1, p-Akt(Thr-308), and p-Akt(Ser-473). Silencing of CREB gene expression by RNA interference prior to forskolin treatment not only decreased CREB protein and its phosphorylation at Ser-133, but also phosphorylation of Akt at Ser-473, and Thr-308. Concomitantly, this treatment inhibited [(3)H]thymidine uptake and reduced forskolin-induced proliferation of macrophages. Forskolin treatment also inhibited activation of the apoptotic mechanism while promoting up-regulation of the anti-apoptotic pathway. We conclude that forskolin mediates cellular proliferation via cAMP-dependent activation of both PKA and Epac.     

A monoclonal antibody to VLA-4 alpha-chain (CDw49d) induces homotypic lymphocyte aggregation

The complex processes of cellular adhesion involve a variety of receptor to ligand interactions that are extremely important during the development of immune function. Lymphocyte activation by Ag or mitogen, CTL- and NK-mediated cytolysis, homing to lymphoid-associated tissue, and the attachment of lymphocytes to extracellular matrix proteins are all governed, at least in part, by cell surface adhesion receptors. During the analysis of mAb for the ability to block human cytotoxic T lymphocyte-mediated killing an inhibitory mAb was noted that caused rapid and vigorous aggregation among the CTL. This antibody, mAb L25, also induced aggregation among human T and B tumor cell lines. mAb L25 binds to an epitope on the alpha 4 subunit of the integrin protein VLA-4 and induced an adhesion event requiring divalent cations, energy, a fluid plasma membrane, and an intact cytoskeleton. The Ag-independent homotypic adhesion induced by mAb L25 was not inhibited by mAb to the lymphocyte function associated Ag-1 (CD11a/CD18), CD2, CD4, and CD8, or to their ligands ICAM-1, LFA-3, MHC class I, or MHC class II. We believe that these experiments suggest a role for VLA-4 in a novel system of leukocyte adhesion.     

HT-29 human colon cancer cell proliferation is regulated by cytosolic phospholipase A(2)α dependent PGE(2)via both PKA and PKB pathways

Cytosolic phospholipase A(2)α (cPLA(2)α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA(2)α which coincided with a significant increase in cell proliferation. The inhibition of cPLA(2)α activity by pyrrophenone or by antisense oligonucleotide against cPLA(2)α (AS) or inhibition of prostaglandin E(2) (PGE(2)) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE(2). The secreted PGE(2) activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE(2). But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE(2). AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA(2)α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA(2)α-dependent PGE(2) production. PGE(2)via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway.     

A monoclonal antibody against a novel 20-kDa protein induces cell adhesion and cytoskeleton-dependent morphologic changes.

A new murine IgA mAb (JKT.M1), developed against Jurkat T cells chronically infected with HIV IIIB induces in vitro homotypic aggregation in several hemopoietic cell lines. The JKT.M1 Ag is expressed on a wide variety of cell types including human lymphocytes, monocytes, platelets, RBC, human umbilical vein endothelial cells, many T cell lines, myelomonocytic cell lines, and a primate kidney cell line. The JKT.M1 Ag shows differential expression on myelomonocytic cells; it is present on K562 and HL60 cell lines, which represent precursors of E and monocytes, respectively, but is not expressed on the surface of U937 and THP-1 cell lines, which appear to represent intermediate cell types of the monocytic cell lineage. However, the JKT.M1 Ag is expressed on mature peripheral blood monocytes and the MonoMac cell line. Immunoprecipitation from cell lysates (Jurkat, SupT1, PBMC, MonoMac) with the JKT.M1 mAb yields a 20-kDa Ag with few if any carbohydrate residues as determined by N-glycanase and neuraminidase treatments. The pI appears acidic by two-dimensional gel analysis, and the nonreduced form migrates more slowly than the reduced form when analyzed by SDS-PAGE suggesting the presence of intramolecular disulfide bridge(s). JKT.M1 mAb-induced cell adhesion is shown to be divalent cation- and temperature-dependent. The adhesion induced by JKT.M1 mAb is inhibited by 20 microM cytochalasin B and also by 2 mM 2-deoxyglucose plus 10 mM sodium azide suggesting that cytoskeletal changes and metabolic energy are required. Aggregation induced by JKT.M1 appears to be independent of CD43, CD44, and VLA4 (CD29/CD49d), mAb against which have also been shown to induce homotypic cell adhesion. Anti-CD18 mAb have been shown to inhibit homotypic aggregation in other studies but failed to do so in the present study. Thus JKT.M1-induced adhesion also appears to be independent of CD18, the beta-chain of leukocyte integrins. However, like mAb against LFA-1, immobilized JKT.M1 stimulates a T cell line to undergo dramatic morphologic changes which could be enhanced by the addition of phorbol ester. These data suggest that the novel 20-kDa molecule recognized by the JKT.M1 mAb may trigger cell adhesion through a previously undescribed mechanism.     

Phosphodiesterase inhibitors control A172 human glioblastoma cell death through cAMP-mediated activation of protein kinase A and Epac1/Rap1 pathways

AimsWe investigated whether cAMP-mediated protein kinase A(PKA) and Epac1/Rap1 pathways differentially affect brain tumor cell death using 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone(rolipram), specific phosphodiesterase type IV(PDE IV) inhibitor.Main methodsA172 and U87MG human glioblastoma cells were used. Percentage of cell survival was determined by MTT assay. PKA and Epac1/Rap1 activation was determined by western blotting and pull-down assay, respectively. Cell cycle and hypodiploid cell formation were assessed by flow cytometry analysis.Key findingsNon-specific PDE inhibitors, isobutylmethylxanthine(IBMX) and theophylline reduce survival percentage of A172 and U87MG cells. The expression of PDE4A and PDE4B was detected in A172 and U87MG cells. Rolipram-treated A172 or U87MG cell survival was lower in the presence of forskolin, adenylate cyclase activator, than that in its absence. Co-treatment with rolipram and forskolin also enhanced CREB phosphorylation on serine 133 that was inhibited by H-89, PKA inhibitor and cAMP-responsive guanine nucleotide exchange factor 1(Epac1), a Rap GDP exchange factor-mediated Rap1 activity in A172 cells. When A172 cells were treated with cell-permeable dibutyryl-cAMP(dbcAMP), PKA activator or 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate(CPT), Epac1 activator, basal level of cell death was increased and cell cycle was arrested at the phase of G2/M. Rolipram-induced A172 cell death was also increased by the co-treatment with dbcAMP or CPT, but it was inhibited by the pre-treatment with H-89.SignificanceThese findings demonstrate that PKA and Epac1/Rap1 pathways could cooperatively play a role in rolipram-induced brain tumor cell death. It suggests that rolipram might regulate glioblastoma cell density through dual pathways of PKA- and Epac1/Rap1-mediated cell death and cell cycle arrest.     

Calcium-independent phospholipase A2 mediates CREB phosphorylation in double-stranded RNA-stimulated endothelial cells

One of the products of a calcium-independent phospholipase A2 (iPLA2) attack of plasmenylcholine, lysoplasmenylcholine, has previously been shown to activate cAMP-dependent protein kinase (PKA). Because endothelial cells respond to some agonists in part by the activation of iPLA2, the present study was designed to determine whether double-stranded RNA (dsRNA), the primary activator of the antiviral response in endothelial cells, elicits cAMP response element binding protein (CREB) phosphorylation through a mechanism mediated by iPLA2. dsRNA stimulated CREB phosphorylation in bovine pulmonary artery endothelial cells that was inhibited by the iPLA2 inhibitor, bromoenol lactone, and the PKA inhibitor, H-89. Additionally, the product of iPLA2 hydrolysis of plasmenylcholine and lysoplasmenylcholine elicited CREB phosphorylation in bovine pulmonary endothelial cells. Taken together, the present studies suggest that dsRNA as well as other agonists of endothelial cells elicit signaling mechanisms that include in part CREB phosphorylation mediated by iPLA2.     

PDE4 inhibitor, roflumilast protects cardiomyocytes against NO-induced apoptosis via activation of PKA and Epac dual pathways

Myocyte apoptosis plays an important role in myocardial infarction and cAMP is crucial in the regulation of myocyte apoptosis. Phosphodiesterase-4 (PDE4) inhibitor blocks the hydrolysis of cAMP via inhibition of PDE4 and is attractive candidate for novel anti-inflammatory drugs. However, its function in cardiovascular diseases and cardiomyocyte apoptosis is unclear. Therefore, we investigated whether roflumilast, a PDE4 inhibitor, exerts protective effect against NO-induced apoptosis in both of H9c2 cells and neonatal rat cardiomyocytes (NRCMs), focusing on cAMP downstream molecules such as protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). According to our data, intracellular cAMP was increased by roflumilast treatment in H9c2 cells and NRCMs. Roflumilast inhibited SNP-induced apoptosis and this effect was reversed by PKA specific inhibitor H-89 and KT-5720. In addition, PKA specific activator N(6)-benzoyladenosine 3',5-cyclic monophosphate (N(6)Bz-cAMP) mimicked the effects of roflumilast. CREB phosphorylation by roflumilast was also inhibited by H-89, indicating that roflumilast protects SNP-induced apoptosis via PKA-dependent pathway. Roflumilast increased Epac1/GTP-Rap1 and the protective effect was abolished by Epac1 siRNA transfection, demonstrating that Epac signaling was also involved in this protective response. In support, Epac specific activator 8-(4-chlrorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP) protected SNP-induced apoptosis. PI3K/Akt inhibitor LY294002 blocked roflumilast-induced Akt phosphorylation and protective effect. Furthermore, inhibition of Epac1 with siRNA had no effect on roflumilast-induced CREB phosphorylation, whereas inhibited Akt phosphorylation, implicating that Akt phosphorylation was regulated by Epac pathway. In addition, it was also observed that rolipram and cilomilast exert similar effects as roflumilast. In summary, our data indicate that roflumilast protects NO-induced apoptosis via both cAMP-PKA/CREB and Epac/Akt-dependent pathway. Our study suggests a possibility of PDE4 inhibitor roflumilast as a potential therapeutic agent against myocardial ischemia/reperfusion (I/R) injury.     

HT-29 human colon cancer cell proliferation is regulated by cytosolic phospholipase A2α dependent PGE2via both PKA and PKB pathways

Cytosolic phospholipase A₂α (cPLA₂α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA₂α which coincided with a significant increase in cell proliferation. The inhibition of cPLA₂α activity by pyrrophenone or by antisense oligonucleotide against cPLA₂α (AS) or inhibition of prostaglandin E₂ (PGE₂) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE₂. The secreted PGE₂ activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE₂. But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE₂. AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA₂α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA₂α-dependent PGE₂ production. PGE₂via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway.     

Regulation of CD43-induced U937 homotypic aggregation

CD43 (leukosialin, sialophorin), a prominent component of the hemopoietic cell surface, has an enigmatic role in cell-cell interaction. The observation that CD43 ligation triggers homotypic aggregation of monoblastoid U937 cells has permitted analysis of this: CD43-induced aggregation was distinguishable from CD29- (also known as beta1 integrin) or CD98- (also known as 4F2, or fusion-related protein 1) induced aggregation, with different energy requirements and with partial dependence on beta2 integrins. Previous studies have focused on the role of CD43 ligation in tyrosine phosphorylation. However, in the homotypic adhesion assay, although there is initial tyrosine phosphorylation, protein tyrosine kinase inhibitors did not block aggregation. Therefore, other signaling pathways were examined. CD43 ligation induced protein tyrosine dephosphorylation, and protein tyrosine phosphatase inhibitors blocked aggregation. Activation of MAP kinases was not necessary. Cytoskeletal inhibitors amplified aggregation. Protein kinase C (PKC) inhibitors amplified aggregation, implicating PKC as a negative regulator. CD43 ligation up-regulated surface adhesion molecules and enhanced CD29- and CD98-induced aggregation. Thus, CD43 participation in cell-cell adhesion is under stringent control, involving both surface events and several different intracellular signaling pathways, acting together to regulate the process. These mechanisms add a further dimension to the potential role of CD43 in tissue immune responses.     

CD98-dependent homotypic aggregation is associated with translocation of protein kinase Cdelta and activation of mitogen-activated protein kinases

CD98 is a protein found on the surface of many activated cell types, and is implicated in the regulation of cellular differentiation, adhesion, growth, and apoptosis. Despite many studies addressing CD98 function, there is little information on the intracellular signalling pathways that mediate its activity. In this study, we examine protein kinase pathways that are activated following ligation by the CD98 antibody AHN-18, an antibody that induces U937 homotypic aggregation and inhibits antigen presenting activity and T-cell activation. Ligation by CD98 antibody AHN-18 induces tyrosine kinase activity, but inhibition of this activity does not affect U937 aggregation. Ligation also induces membrane translocation of the serine/threonine kinase novel PKCdelta, but not other members of the PKC family. Translocation is blocked by rottlerin, and this inhibitor also blocks aggregation. PKCdelta activation in turn mediates activation of ERK1/2 and p38, as well as tyrosine phosphorylation of multiple proteins, and MAPK activation is essential for cellular aggregation. One of the targets of CD98-induced tyrosine phosphorylation is itself PKCdelta, suggesting that this phosphorylation may act as a negative feedback to limit the overall activation of the CD98 pathway.