Cbl--a polyfunctional regulator of cellular processes

C-Cb1 protein is a protooncogene product that was initially identified as part of a murine retrovirus transforming protein. C-Cb1 is ubiquitously expressed in cells of different origin. A number of isoforms subsequently identified in vertebrates and invertebrates allows to consider the existence of a family of Cb1 proteins. These proteins contain a set of sequences providing interactions with a wide range of receptor and nonreceptor tyrosine kinases and signaling proteins with SH2- and SH3-domains (for example, EGF and PDGF receptors, Src-kinases, PI-3-kinase p85, Crk, GRB2, Vav, etc.). Cb1 proteins possess also multiple tyrosine, residues, which undergo phosphorylation upon stimulation of several surface receptors. These properties permit Cb1 to take part in many protein-protein interactions as an adaptor, which forms multimolecular signaling complexes, and coordinates the activity of its components. C-Cb1 and its mutant transforming forms can act as both positive and negative regulators of many signaling pathways. Negative action of C-Cb1 on signals stimulated by receptor tyrosine kinases is thought to result from accelerated receptor degradation caused by Cb1. This ability is attributed mostly to ubiquitin-ligase activity of Cb1 proteins, since the latest research evidence suggests that ubiquitination may be a signal of not only proteasomal, but also lysosomal degradation. Thus, Cb1 manifests itself as a many-sided protein working both as an adaptor and a regulator of endocytic trafficking. In spite of numerous studies in this area, the regulation of Cb1 functions, interrelations between these functions, physiological significance of Cb1-mediated interactions, and the place of Cb1 proteins in signaling coordinating still remain obscure. In the present review, an attempt is made to summarize the recent data, with special reference to Cb1 functioning as a regulator of tyrosine kinase receptor endocytosis.     

Regulation and roles of PI3Kβ, a major actor in platelet signaling and functions

Phosphoinositide 3-kinases (PI3Ks) are important signaling enzymes involved in the regulation of a number of critical cell functions. Significant progress has been made during the last few years in defining the implication of individual PI3K isoforms. The role of the class IA PI3Kβ in different cell types has only been recently uncovered by the use of isoform-selective inhibitors and the development of mouse models harboring p110β catalytic subunit knock-out or germline knock-in of a kinase-dead allele of p110β. Although it is classically admitted that class IA PI3Ks are activated by receptor tyrosine kinases through recruitment of the regulatory subunits to specific tyrosine phosphorylated motifs via their SH2 domains, PI3Kβ is activated downstream of G protein-coupled receptors, and by co-operation between heterotrimeric G proteins and tyrosine kinases. PI3Kβ has been extensively studied in platelets where it appears to play an important role downstream of ITAM signaling, G protein-coupled receptors and aIIbβ3 integrin. Accordingly, mouse exhibiting p110β inactivation selectively in megakaryocyte/platelets are resistant to thromboembolism induced by carotid injury. The present review summarizes recent data concerning the mechanisms of PI3Kβ regulation and the roles of this PI3K isoform in blood platelet functions and other cell types.     

Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.     

Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor.

The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have recently been elucidated biochemically and genetically. The present study was undertaken to determine whether common signaling components are used by these two distinct classes of receptors. Here we report that the adaptor protein Shc, is phosphorylated on tyrosine residues following stimulation of the thrombin receptor in growth-responsive CCL39 fibroblasts. Shc phosphorylation by thrombin or the thrombin receptor agonist peptide is maximal by 15 min and persists for > or = 2 h. Following thrombin stimulation, phosphorylated Shc is recruited to Grb2 complexes. One or more pertussis toxin-insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre-treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4-beta-phorbol-12,13-dibutyrate has no effect. Rather, thrombin-induced Shc phosphorylation is enhanced in cells depleted of phorbol ester-sensitive protein kinase C isoforms. Expression of mutant Shc proteins defective in Grb2 binding displays a dominant-negative effect on thrombin-stimulated p44 MAP kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein-coupled receptors.     

Vav2 activates Rac1, Cdc42, and RhoA downstream from growth factor receptors but not beta1 integrins

The Rho family of GTPases plays a major role in the organization of the actin cytoskeleton. These G proteins are activated by guanine nucleotide exchange factors that stimulate the exchange of bound GDP for GTP. In their GTP-bound state, these G proteins interact with downstream effectors. Vav2 is an exchange factor for Rho family GTPases. It is a ubiquitously expressed homologue of Vav1, and like Vav1, it has previously been shown to be activated by tyrosine phosphorylation. Because Vav1 becomes tyrosine phosphorylated and activated following integrin engagement in hematopoietic cells, we investigated the tyrosine phosphorylation of Vav2 in response to integrin-mediated adhesion in fibroblasts and epithelial cells. However, no tyrosine phosphorylation of Vav2 was detected in response to integrin engagement. In contrast, treating cells with either epidermal growth factor or platelet-derived growth factor stimulated tyrosine phosphorylation of Vav2. We have examined the effects of overexpressing either wild-type or amino-terminally truncated (constitutively active) forms of Vav2 as fusion proteins with green fluorescent protein. Overexpression of either wild-type or constitutively active Vav2 resulted in prominent membrane ruffles and enhanced stress fibers. These cells revealed elevated rates of cell migration that were inhibited by expression of dominant negative forms of Rac1 and Cdc42. Using a binding assay to measure the activity of Rac1, Cdc42, and RhoA, we found that overexpression of Vav2 resulted in increased activity of each of these G proteins. Expression of a carboxy-terminal fragment of Vav2 decreased the elevation of Rac1 activity induced by epidermal growth factor, consistent with Vav2 mediating activation of Rac1 downstream from growth factor receptors.     

Src family kinase-dependent phosphorylation of a 29-kDa caveolin-associated protein

PDGF receptors and Src family kinases are concentrated in caveolae, where signal transduction cascades involving these molecules are thought to be organized. The Src family tyrosine kinases are cotransducers of signals emanating from the activated PDGF receptor. However, the Src family kinase substrates that are involved in PDGF-induced signaling remain to be fully elucidated. We have identified a 29-kDa protein in caveolae that was phosphorylated in response to PDGF stimulation. This protein, pp29, was tightly bound to the caveolar coat protein caveolin-1. pp29 was among the most prominent phosphoproteins observed in cells overexpressing Fyn, suggesting that it may be a Fyn substrate. Consistent with this, pp29 was among a specific subset of proteins whose PDGF-stimulated phosphorylation was blocked by expression of kinase inactive Fyn. These data indicate that pp29 lies downstream of Fyn activation in a PDGF-stimulated signaling pathway, and that pp29 is an abundant site for nucleation of signal transduction cascades.     

Vav family proteins are required for optimal regulation of PLCgamma2 by integrin alphaIIbbeta3

Vav proteins belong to the family of guanine-nucleotide-exchange factors for the Rho/Rac family of small G-proteins. In addition, they serve as important adapter proteins for the activation of PLCgamma (phospholipase Cgamma) isoforms by ITAM (immunoreceptor tyrosine-based activation motif) receptors, including the platelet collagen receptor GPVI (glycoprotein VI). Vav proteins are also regulated downstream of integrins, including the major platelet integrin alphaIIbbeta3, which has recently been shown to regulate PLCgamma2. In the present study, we have investigated the role of Vav family proteins in filopodia and lamellipodia formation on fibrinogen using platelets deficient in Vav1 and Vav3. Wild-type mouse platelets undergo a limited degree of spreading on fibrinogen, characterized by the formation of numerous filopodia and limited lamellipodia structures. Platelets deficient in Vav1 and Vav3 exhibit reduced filopodia and lamellipodia formation during spreading on fibrinogen. This is accompanied by reduced alphaIIbbeta3-mediated PLCgamma2 tyrosine phosphorylation and reduced Ca(2+) mobilization. In contrast, the G-protein agonist thrombin stimulates full spreading of control and Vav1/3-deficient platelets. Consistent with this, stimulation of F-actin (filamentous actin) formation and Rac activation by thrombin is not altered in Vav-deficient cells. These results demonstrate that Vav1 and Vav3 are required for optimal spreading and regulation of PLCgamma2 by integrin alphaIIbbeta3, but that their requirement is by-passed upon G-protein receptor activation.     

Fibrillar beta-amyloid-stimulated intracellular signaling cascades require Vav for induction of respiratory burst and phagocytosis in monocytes and microglia

Microglial interaction with extracellular beta-amyloid fibrils (fAbeta) is mediated through an ensemble of cell surface receptors, including the B-class scavenger receptor CD36, the alpha(6)beta(1)-integrin, and the integrin-associated protein/CD47. The binding of fAbeta to this receptor complex has been shown to drive a tyrosine kinase-based signaling cascade leading to production of reactive oxygen species and stimulation of phagocytic activity; however, little is known about the intracellular signaling cascades governing the microglial response to fAbeta. This study reports a direct mechanistic link between the fAbeta cell surface receptor complex and downstream signaling events responsible for NADPH oxidase activation and phagosome formation. The Vav guanine nucleotide exchange factor is tyrosine-phosphorylated in response to fAbeta peptides as a result of the engagement of the microglia fAbeta cell surface receptor complex. Co-immunoprecipitation studies demonstrate an Abeta-dependent association between Vav and both Lyn and Syk kinases. The downstream target of Vav, the small GTPase Rac1, is GTP-loaded in an Abeta-dependent manner. Rac1 is both an essential component of the NADPH oxidase and a critical regulator of microglial phagocytosis. The direct role of Vav in fAbeta-stimulated intracellular signaling cascades was established using primary microglia obtained from Vav(-/-) mice. Stimulation of Vav(-/-) microglia with fAbeta failed to generate NADPH oxidase-derived reactive oxygen species and displayed a dramatically attenuated phagocytic response. These findings directly link Vav phosphorylation to the Abeta-receptor complex and demonstrate that Vav activity is required for fAbeta-stimulated intracellular signaling events upstream of reactive oxygen species production and phagosome formation.     

Tyrosine phosphorylation of p95Vav in myeloid cells is regulated by GM-CSF, IL-3 and steel factor and is constitutively increased by p210BCR/ABL.

Vav is a recently described proto-oncogene expressed only in hematopoietic cells which contains an SH2 and two SH3 domains and shares homology with the Dbl GDP-GTP exchange factor and BCR. p95Vav is phosphorylated on tyrosine residues in response to stimulation of the T cell antigen receptor, cross-linking of IgE or IgM receptors and stimulation of immature hematopoietic cells by Steel factor. Monoclonal antibodies to human Vav were generated and used to examine the events which regulate tyrosine phosphorylation of p95Vav in myeloid cells. In the factor-dependent MO7e cell line, p95Vav was rapidly phosphorylated on tyrosine residues in a dose- and time-dependent manner by GM-CSF, IL-3 and Steel factor. Introduction of the BCR/ABL oncogene into this cell line resulted in factor-independent proliferation and constitutive phosphorylation of p95Vav. Tyrosine phosphorylation of p95Vav was also substantially increased by treatment of cytokine-deprived cells with the tyrosine phosphatase inhibitor sodium vanadate. Since many of the cytokines known to induce tyrosine phosphorylation of p95Vav are also known to activate JAK family tyrosine kinases, we looked for an interaction of p95Vav with JAK kinases. p95Vav co-precipitated with JAK2 in MO7e cells stimulated with GM-CSF, but not in unstimulated cells. Also, JAK2 was found to be constitutively associated with p95Vav in vivo when expressed at high levels in insect cells using baculovirus vectors. A fusion protein consisting of glutathione-S-transferase and the SH2 domain of p95Vav (GST-Vav-SH2) precipitated JAK2, suggesting that this interaction is mediated by the SH2 domain of p95Vav.(ABSTRACT TRUNCATED AT 250 WORDS)     

The c-Kit/D816V mutation eliminates the differences in signal transduction and biological responses between two isoforms of c-Kit

Activating mutations of codon 816 of the Kit gene have been implicated in malignant cell growth of acute myeloid leukemia (AML), systemic mastocytosis and germ cell tumors. Substitution of aspartic acid with valine (D816V) renders the receptor independent of ligand for activation and signaling. Wild-type c-Kit is a tyrosine kinase receptor that requires its ligand, stem cell factor (SCF), for activation. Several isoforms of c-Kit exist as a result of alternative mRNA splicing, of which two are characterized by the presence or absence of four amino acids (GNNK? and GNNK+, respectively) in the extracellular domain. The two isoforms show differences in signal transduction and biological activities and the shorter isoform seems to be highly expressed than the longer isoform in human malignancies. In this study we analysed the signal transduction downstream of the oncogenic c-Kit mutant D816V in an isoform specific context, using the hematopoietic cell line Ba/F3 stably transfected with the different versions of isoform and mutant receptor. Our data show that in contrast to the differences shown in the activation of wild-type c-Kit isoforms, both isoforms of c-Kit/D816V are constitutively phosphorylated to the same extent. By the use of Western blot analysis we investigated the activation of different signaling proteins and found that both D816V/GNNK? and D816V/GNNK+ constitutively phosphorylated Gab2, Shc, SHP-2 and Cbl to almost the same extent as c-Kit/GNNK?. In addition, both isoforms of c-Kit/D816V induced SCF-independent cell survival and proliferation equally well. This is in contrast to wild-type c-Kit, where c-Kit/GNNK? induced better cell survival and stronger proliferation than c-Kit/GNNK+, and both required stimulation with SCF. Taken together, these findings reveal that the differences in downstream signal transduction and biological responses between the two GNNK isoforms are eliminated by the D816V mutant.     

Evidence that Llck-mediated phosphorylation of p56dok and p62dok may play a role in CD2 signaling

The Lck tyrosine kinase is involved in signaling by T cell surface receptors such as TCR/CD3, CD2, and CD28. As other downstream protein-tyrosine kinases are activated upon stimulation of these receptors, it is difficult to assign which tyrosine-phosphorylated proteins represent bona fide Lck substrates and which are phosphorylated by other tyrosine kinases. We have developed a system in which Lck can be activated independently of TCR/CD3. We have shown that activation of an epidermal growth factor receptor/Lck chimera leads to the specific phosphorylation of Ras GTPase-activating protein (RasGAP) and two RasGAP-associated proteins, p56(dok) and p62(dok). Activation of the chimeric protein correlates with an increase in cellular Ca(2+) in the absence of ZAP-70 and phospholipase Cgamma1 phosphorylation. Furthermore, we have found that p62(dok) co-immunoprecipitates with the activated epidermal growth factor receptor/LckF505 and that phosphorylated Dok proteins bind to the Src homology 2 domain of Lck in vitro. In addition, we have shown that activation via the CD2 but not the TCR/CD3 receptor leads to the phosphorylation of p56(dok) and p62(dok). Using JCaM1.6 cells, we have demonstrated that Lck is required for CD2-mediated phosphorylation of Dok proteins. We propose that phosphorylation and Src homology 2-mediated association of p56(dok) and p62(dok) with Lck play a selective function in accessory receptor signal transduction mechanisms.     

DIP (mDia interacting protein) is a key molecule regulating Rho and Rac in a Src-dependent manner

Cell movement is driven by the coordinated regulation of cytoskeletal reorganization through Rho GTPases downstream of integrin and growth-factor receptor signaling. We have reported that mDia, a target protein of Rho, interacts with Src and DIP. Here we show that DIP binds to p190RhoGAP and Vav2, and that DIP is phosphorylated by Src and mediates the phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation. When endogenous DIP was inhibited by expressing dominant-negative mutants of DIP or siRNA, phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation was diminished, and EGF-induced actin organization, distribution of p190RhoGAP and Vav2, and cell movement were affected. Therefore, DIP seems to transfer the complex of the three proteins from cytosol to beneath the membrane, and the three proteins, in turn, can be phosphorylated by Src. DIP inactivated Rho and activated Rac following EGF stimulation in the membrane fraction. Thus, DIP acts as a regulatory molecule causing Src kinase-dependent feedback modulation of Rho GTPases downstream of Rho-mDia upon EGF stimulation, and plays an important role in cell motility.     

Amyloid precursor protein mediates proinflammatory activation of monocytic lineage cells

Alzheimer's disease is a progressive neurodegenerative disorder characterized by extracellular deposition of beta-amyloid (Abeta) peptide containing neuritic plaques. Abeta peptides are proteolytically derived from the membrane-bound amyloid precursor protein (APP). Although the function of APP is not entirely clear, previous studies demonstrate that neuronal APP colocalizes with beta(1) integrin receptors at sites of focal adhesion, suggesting that APP is involved in mediating neuronal process adhesion. Integrin-dependent adhesion is also a well-characterized component of immune cell proinflammatory activation. Using primary mouse microglia and the human monocytic cell line, THP-1, we have begun investigating the role of APP in integrin-dependent activation. Co-immunoprecipitation studies demonstrate that APP is recruited into a multi-receptor signaling complex during beta(1) integrin-mediated adhesion of monocytes. Stimulation induces a subsequent, specific recruitment of tyrosine phosphorylated proteins to APP, including Lyn and Syk. Antibody cross-linking of cell surface APP leads to a similar response characterized by activation and recruitment of tyrosine kinases to APP as well as subsequent activation of mitogen-activated protein kinases and increased proinflammatory protein levels. These data demonstrate that APP can act as a proinflammatory receptor in monocytic lineage cells and provide insight into the contribution of this protein to the inflammatory conditions described in Alzheimer's disease.     

Interaction of matrix with integrin receptors is required for optimal LPS-induced MAP kinase activation

Exposure of macrophages to endotoxin [lipopolysaccharide (LPS)] results in a cascade of events resulting in the release of multiple inflammatory and anti-inflammatory mediators. The Toll-like receptor (TLR) 4 complex is the major receptor that mediates LPS signaling. However, there is evidence that other surface molecules may play a complementary role in the TLR-induced events. Integrin receptors are one class of receptors that have been linked to LPS signaling. This study investigates the role of macrophage integrin receptors in the activation of mitogen-activated protein (MAP) kinases by LPS. In conditions where macrophages were not permitted to adhere to matrix or a tissue culture surface, we found a decrease in LPS signaling as documented by a marked reduction in tyrosine phosphorylation of whole cell proteins. This was accompanied by a significant decrease in extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase MAP kinase activation. Inhibition of integrin signaling, with EDTA or RGD peptides, decreased LPS-induced MAP kinase activity. The functional consequence of blocking integrin signaling was demonstrated by decreased LPS-induced tumor necrosis factor-alpha production. These observations demonstrate that, in addition to the TLR receptor complex, optimal LPS signaling requires complementary signals from integrin receptors.     

Acetylcholine muscarinic receptor regulation of the Ras/Raf/MAP kinase pathway

Acetylcholine muscarinic m1 receptors and m2 receptors are predominantly coupled to the heterotrimeric G proteins Gq, 11 and Gi, respectively. Stimulation of the m1 and m2 receptors in different cell types activate the Ras/Raf/MAP kinase pathway. The ability of the m1 receptor to activate the MAP kinase pathway is dependent on the isoforms of adenylyl cyclase expressed in specific cell types. Specific adenylyl cyclases respond to different signals, including calcium and protein kinase C, with increased cAMP synthesis resulting in protein kinase A activation. Stimulation of protein kinase A inhibits Raf and subsequent MAP kinase activation by G protein-coupled receptors and growth factor receptor tyrosine kinases. G protein-coupled receptors can positively and negatively regulate the responsiveness of tyrosine kinase-stimulated response pathways.     

Proline-rich tyrosine kinase 2 and Rac activation by chemokine and integrin receptors controls NK cell transendothelial migration

Protein tyrosine kinase activation is an important requisite for leukocyte migration. Herein we demonstrate that NK cell binding to endothelium activates proline-rich tyrosine kinase 2 (Pyk-2) and the small GTP binding protein Rac that are coupled to integrin and chemokine receptors. Chemokine-mediated, but not integrin-mediated, Pyk-2 and Rac activation was sensitive to pretreatment of NK cells with pertussis toxin, a pharmacological inhibitor of G(i) protein-coupled receptors. Both Pyk-2 and Rac are functionally involved in chemokine-induced NK cell migration through endothelium or ICAM-1 or VCAM-1 adhesive proteins, as shown by the use of recombinant vaccinia viruses encoding dominant negative mutants of Pyk-2 and Rac. Moreover, we found that Pyk-2 is associated with the Rac guanine nucleotide exchange factor Vav, which undergoes tyrosine phosphorylation upon integrin triggering. Finally, we provide direct evidence for the involvement of Pyk-2 in the control of both chemokine- and integrin-mediated Rac activation. Collectively, our results indicate that Pyk-2 acts as a receptor-proximal link between integrin and chemokine receptor signaling, and the Pyk-2/Rac pathway plays a pivotal role in the control of NK cell transendothelial migration.     

Regulation of MHC class II signal transduction by the B cell coreceptors CD19 and CD22

The major histocompatability class II heterodimer (class II) is expressed on the surface of both resting and activated B cells. Although it is clear that class II expression is required for Ag presentation to CD4(+) T cells, substantial evidence suggests that class II serves as a signal transducing receptor that regulates B cell function. In ex vivo B cells primed by Ag receptor (BCR) cross-linking and incubation with IL-4, or B cell lines such as K46-17 micromlambda, class II ligation leads to the activation of protein tyrosine kinases, including Lyn and Syk and subsequent phospholipase Cgamma-dependent mobilization of Ca(2+). In this study, experiments demonstrated reciprocal desensitization of class II and BCR signaling upon cross-linking of either receptor, suggesting that the two receptors transduce signals via common processes and/or effector proteins. Because class II and BCR signal transduction pathways exhibit functional similarities, additional studies were conducted to evaluate whether class II signaling is regulated by BCR coreceptors. Upon cross-linking of class II, the BCR coreceptors CD19 and CD22 were inducibly phosphorylated on tyrosine residues. Phosphorylation of CD22 was associated with increased recruitment and binding of the protein tyrosine phosphatase SHP-1. Similarly, tyrosine phosphorylation of CD19 resulted in recruitment and binding of Vav and phosphatidylinositol 3-kinase. Finally, co-cross-linking studies demonstrated that signaling via class II was either attenuated (CD22/SHP-1) or enhanced (CD19/Vav and phosphatidylinositol 3-kinase), depending on the coreceptor that was brought into close proximity. Collectively, these results suggest that CD19 and CD22 modulate class II signaling in a manner similar to that for the BCR.     

Molecular cloning of the mouse APS as a member of the Lnk family adaptor proteins

Engagement of cell-surface receptors leads to activation of protein tyrosine kinases, which in turn phosphorylate various downstream enzymes and adaptor proteins. Lnk is an adaptor protein that appears to be involved in signal transduction in lymphocytes, and forms an adaptor protein family with SH2-B. We tried to identify another member of the adaptor protein family and isolated the mouse APS (adaptor molecule containing PH and SH2 domains). APS contains a proline-rich region, PH and SH2 domains, and a putative tyrosine phosphorylation site at the C-terminal, and the overall structure resembles those of Lnk and SH2-B. APS is expressed in brain, kidney, muscle, and mature B cells in spleen. Mouse APS gene consists of 8 coding exons and is deduced to map to chromosome 5. APS is tyrosine phosphorylated at the C-terminal phosphorylation site conserved among the Lnk family adaptor proteins by stimulation of IL-5 or IL-3 as well as by crosslinking of B cell receptor complex. These results suggest that APS is a member of the Lnk family adaptor protein and likely plays a role in signaling in B cells.     

Dopamine D2 receptor stimulation of mitogen-activated protein kinases mediated by cell type-dependent transactivation of receptor tyrosine kinases

Dopamine D2 receptor activation of extracellular signal-regulated kinases (ERKs) in non-neuronal human embryonic kidney 293 cells was dependent on transactivation of the platelet-derived growth factor (PDGF) receptor, as demonstrated by the effect of the PDGF receptor inhibitors tyrphostin A9 and AG 370 on quinpirole-induced phosphorylation of ERKs and by quinpirole-induced tyrosine phosphorylation of the PDGF receptor. In contrast, ectopically expressed D2 receptor or endogenous D2-like receptor activation of ERKs in NS20Y neuroblastoma cells, which express little or no PDGF receptor, or in rat neostriatal neurons was largely dependent on transactivation of the epidermal growth factor (EGF) receptor, as demonstrated using the EGF receptor inhibitor AG 1478 and by quinpirole-induced phosphorylation of the EGF receptor. The D2 receptor agonist quinpirole enhanced the coprecipitation of D2 and EGF receptors in NS20Y cells, suggesting that D2 receptor activation induced the formation of a macromolecular signaling complex that includes both receptors. Transactivation of the EGF receptor also involved the activity of a matrix metalloproteinase. Thus, although D2 receptor stimulation of ERKs in both cell lines was decreased by inhibitors of ERK kinase, Src-family protein tyrosine kinases, and serine/threonine protein kinases, D2-like receptors activated ERKs via transactivation of the EGF receptor in NS20Y neuroblastoma cells and rat embryonic neostriatal neurons, but via transactivation of the PDGF receptor in 293 cells.