The intracellular signaling pathways activated upon ligation of the co-stimulatory receptor CD28 remain relatively ill-defined, although CD28 ligation does result in the strong association with, and ...activation of, phosphatidylinositol (PI) 3-kinase. The downstream effector targets of the CD28-activated PI 3-kinase-dependent signaling pathway remain poorly defined, but recent evidence from other systems has shown that Akt/protein kinase B (PKB) is a major target of PI 3-kinase and have indicated that a major function of PKB is the regulation of cell survival events. Given the strong coupling of CD28 to PI 3-kinase and the known protective effects of both CD28 and PI 3-kinase against apoptosis in different cell models, we investigated the effects of CD28 on PKB activation. We demonstrate that ligation of CD28 by either anti-CD28 monoclonal antibodies or the natural ligand B7.1, results in the marked activation of PKB in both the leukemic T cell line Jurkat and freshly isolated human peripheral blood-derived normal T lymphocytes. Our data suggest therefore, that PKB may be an important intracellular signal involved in CD28 signal transduction and demonstrate CD28 coupling to downstream elements of a signaling cascade known to promote cell survival.
Immunoglobulin family tyrosine-based activation motifs (ITAM), which define the conserved signaling sequence EX2YX2L/IX7YX2L/I, couple the T cell antigen receptor (TCR) to cellular proteins including ...protein tyrosine kinases (PTK) and adapter molecules. The TCR is a multichain complex with four invariant chains CD3 gamma, delta and epsilon that each contain a single ITAM and the TCR zeta chain that contains three ITAM. The present study explores the protein interactions of the doubly phosphorylated CD3 gamma, delta, epsilon ITAM to determine whether they have common or unique biochemical properties. The data show that the doubly phosphorylated ITAM all bind the PTK ZAP-70, but the ITAM also variably bind the PTK p59fyn and the adapters Shc, Grb-2 and the p85 regulatory subunit of phosphoinositol 3' kinase. The CD3 and zeta ITAM display a hierarchy of ZAP-70 binding: zeta 1 = gamma = delta > zeta 3 > zeta 2 = epsilon. Shc, Grb-2 and p85 could bind the zeta ITAM and the CD3 gamma and delta ITAM, but not the CD3 epsilon ITAM. There were also subtle differences in the hierarchy of reactivity of these adapters for the CD3 gamma, delta and zeta ITAM that show that the zeta, CD3 gamma, delta and epsilon ITAM have different binding properties. The present study thus shows that the different ITAM of the TCR/CD3 complex can interact with different cytosolic effectors, indicating that differential ITAM phosphorylation during T cell activation could be a mechanism to generate signaling diversity by the TCR complex.
Despite the established role for PI3Ks in cell migration, the PI3Ks involved in lymphocyte chemotaxis are poorly defined. In this study, we report that p110gamma-deficient T cells, but not B cells, ...show reduced chemotactic responses to the lymphoid chemokines, CCL19, CCL21, and CXCL12. As B cell and T cell chemotactic responses were both sensitive to the general PI3K inhibitors, wortmannin (WMN) and LY294002, we explored whether B cell responses were affected in mice lacking p110delta, a major PI3K isoform in lymphocytes. B cells deficient in p110delta showed diminished chemotactic responses, especially to CXCL13. Adoptive transfer experiments with WMN-treated wild-type B cells and with p110delta-deficient B cells revealed diminished homing to Peyer's patches and splenic white pulp cords. WMN selectively inhibited CXCR5-dependent B cell homing to Peyer's patches. These observations establish that p110gamma and p110delta function in lymphocyte chemotaxis, and show differential roles for PI3K family members in B and T cell migration.
Despite the established role for PI3Ks in cell migration, the PI3Ks involved in lymphocyte chemotaxis are poorly defined. In this study, we report that p110 gamma -deficient T cells, but not B cells, ...show reduced chemotactic responses to the lymphoid chemokines, CCL19, CCL21, and CXCL12. As B cell and T cell chemotactic responses were both sensitive to the general PI3K inhibitors, wortmannin (WMN) and LY294002, we explored whether B cell responses were affected in mice lacking p110 delta , a major PI3K isoform in lymphocytes. B cells deficient in p110 delta showed diminished chemotactic responses, especially to CXCL13. Adoptive transfer experiments with WMN-treated wild-type B cells and with p110 delta -deficient B cells revealed diminished homing to Peyer's patches and splenic white pulp cords. WMN selectively inhibited CXCR5-dependent B cell homing to Peyer's patches. These observations establish that p110 gamma and p110 delta function in lymphocyte chemotaxis, and show differential roles for PI3K family members in B and T cell migration.
p21ras is activated by the T cell antigen receptor (TCR) and then co-ordinates important signaling pathways for T lymphocyte activation. Effector pathways for this guanine nucleotide binding protein ...in T cells are mediated by the serine/threonine kinase Raf-1 and the Ras-related GTPase Rac-1. In fibroblasts, an important effector for the Ras oncogene is Phosphatidylinositol 3-kinase (PtdIns 3-kinase). Activation of this lipid kinase is able to induce critical Rac-1 signaling pathways and can couple p21ras to cell survival mechanisms via the serine/threonine kinase Akt/PKB. The role of PtdIns 3-kinase in Ras signaling in T cells has not been explored. In the present study, we examined the ability of PtdIns 3-kinase to initiate the Rac-1 signaling pathways important for T cell activation. We also examined the possibility that Akt/PKB is regulated by Ras signaling pathways in T lymphocytes. The results show that Ras can initiate a Rac-1 mediated pathway that regulates the transcriptional function of AP-1 complexes. PtdIns 3-kinase signals cannot mimic p21ras and induce the Rac mediated responses of AP-1 transcriptional activation. Moreover, neither TCR or Ras activation of AP-1 is dependent on PtdIns 3-kinase. PKB is activated in response to triggering of the T cell antigen receptor; PtdIns 3-kinase activity is both required and sufficient for this TCR response. In contrast, p21ras signals are unable to induce Akt/PKB activity in T cell nor is Ras function required for Akt/PKB activation in response to the TCR. The present data thus highlight that PtdIns 3-kinase and Akt/PKB are not universal Ras effector molecules. Ras can initiate Rac-1 regulated signaling pathways in the context of T cell antigen receptor function independently of PtdIns 3-kinase activity.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
In T lymphocytes activated via the T cell antigen receptor (TCR), the SH2- and SH3-containing adapter molecule Grb2 forms
a complex with the Ras guanine nucleotide exchange protein Sos and tyrosine ...phospho-proteins. The interaction of Sos with
Grb2 is mediated via the Grb2 SH3 domains. In this study, it is shown that a 75-kDa protein is also complexed with the Grb2
SH3 domains in T cells, but not in Rat-1 fibroblasts. The identity of the p75 protein is not known, but immunoblot analysis
with phosphotyrosine antibodies indicated that it is rapidly tyrosine-phosphorylated in TCR-activated T cells. This characteristic
clearly distinguishes p75 from Sos since Sos is not a phosphotyrosine protein. In vitro binding studies indicated that the
p75 phosphotyrosine protein binds to a glutathione S-transferase fusion protein of intact Grb2, but not to a Grb2 fusion protein
mutated in its SH3 domains. p75 can also bind to the single COOH-terminal Grb2 SH3 domain, whereas Sos has an in vitro binding
preference for the NH2-terminal Grb2 SH3 domain. Collectively, these data indicate that in T cells, two proteins can complex
with the Grb2 SH3 domains: Sos and a p75 molecule that is tyrosine-phosphorylated in TCR-activated cells. The significance
of p75 association with Grb2 is not clear, but by analogy with Sos, p75 is a potential candidate for a Grb2 effector protein.
Data are presented showing that the interaction of the Grb2 SH2 domains with tyrosine phosphoproteins may be regulated by
conformational restraints imposed by different molecules complexing with the Grb2 SH3 domains. It is thus possible to speculate
that the interaction of either p75 or Sos with the Grb2 SH3 domain may influence the interaction of the Grb2 SH2 domain with
tyrosine phosphoproteins.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Phosphatidylinositol (PtdIns) 3-kinase is composed of a catalytic p110 subunit and a regulatory p85 subunit. A synthetic phosphopeptide corresponding to the kinase insert of the human PDGF beta ...subunit receptor and monoclonal antibodies raised against the two described p85 isoforms, p85 alpha and p85 beta were used to isolate PtdIns 3-kinase from human T lymphocytes. We demonstrate that T cells express both p85 alpha and p85 beta proteins. Both isoforms tightly associate with a p110 protein and with PtdIns 3-kinase activity in T cells. Upon triggering of the T cell antigen receptor (TCR)/CD3 complex or activation of protein kinase C (PKC) the p110 protein complexed to p85 alpha becomes rapidly phosphorylated exclusively on serine residues. p85 alpha does not appear to undergo a change in its basal serine phosphorylation during T cell activation. In contrast, stimulation of the TCR/CD3 complex or PKC, results in a marked and rapid increase in phosphorylation of p85 beta on threonine residues. These data show that PtdIns 3-kinase can be a substrate for serine/threonine kinases in T cells. The differential phosphorylation of p85 alpha and p85 beta reveals the potential for divergent regulation and function of these two PtdIns 3-kinase isoforms during T cell activation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Antigenic cross-linking of the high affinity IgE receptor (Fc epsilon R1) on mast cells results in protein tyrosine kinase activation. The object of the present study was to explore the regulation of ...the SH2 and SH3 domain containing adapter molecule Grb2 by Fc epsilon R1-stimulated PTK signal transduction pathways. Affinity purification of in vivo Grb2 complexes together with in vitro experiments with Grb2 glutathione S-transferase fusion proteins were used to analyze Grb2 complexes in the mast cell line RBL2H3. The data show that in RBL2H3 cells several different proteins are complexed to the SH3 domains of Grb2. These include the p21ras guanine nucleotide exchange factor Sos, two basally tyrosine-phosphorylated 110- and 120-kDa molecules, and a 75-kDa protein that is a substrate for Fc epsilon R1-activated PTKs. By analogy with Sos, p75, p110 and p120 are candidates for Grb2 effector proteins which suggests that Grb2 may be a pleiotropic adapter. Two Grb2 SH2-binding proteins were also characterized in RBL2H3 cells; the adapter Shc and a 33-kDa molecule. Shc is constitutively tyrosine phosphorylated in unstimulated cells and Fc epsilon R1 ligation induces no changes in its phosphorylation or binding to Grb2. In contrast, p33 is a substrate for Fc epsilon R1-activated PTKs and binds to Grb2 SH2 domains in Fc epsilon R1 activated but not quiescent cells. The beta subunit of the Fc epsilon R1 is a 33-kDa tyrosine phosphoprotein, but the p33 Grb2-binding protein described in the present report is not the Fc epsilon R1 beta chain and its identity is unknown. The present report thus demonstrates that there are multiple Grb2 containing protein complexes in mast cells of which a subset are Fc epsilon R1-regulated. Two other of the Grb2-binding proteins described herein are tyrosine phosphorylated in response to Fc epsilon R1 ligation: the 75-kDa protein which binds to Grb2 SH3 domains and the 33-kDa protein that associates with the Grb2 SH2 domain. We propose that protein complex formation by Grb2 is an important consequence of Fc epsilon R1 cross-linking and that this may be a signal transduction pathway which acts synergistically with calcium/PKC signals to bring about optimal mast cell end function.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A phosphoinositide kinase that can phosphorylate phosphatidylinositol (PtdIns) is present in 4G10 monoclonal antibody (mAb)
phosphotyrosine immunoprecipitates isolated from T cells activated via the ...T cell antigen receptor (TCR).CD3 complex. This
PtdIns kinase is not the PtdIns 3-kinase that associates with activated protein tyrosine kinases in fibroblasts, since Western
blotting and immunoprecipitation experiments with antibodies specific for the p85 alpha subunit of the PtdIns 3-kinase indicate
that this polypeptide is not immunoprecipitated by the 4G10 mAb from TCR.CD3-activated Jurkat cells. Moreover, immunoprecipitated
PtdIns 3-kinase isolated from T cells with p85 antibodies is inhibited when PtdIns is presented in Nonidet P-40, whereas the
PtdIns kinase activity present in 4G10 mAb phosphotyrosine immunoprecipitates is enhanced in the presence of Nonidet P-40.
In vitro kinase assays of PtdIns 3-kinase immunoprecipitated with p85 antibodies from T cells indicate that it associates
with a serine kinase that can phosphorylate a p85 polypeptide. However, no protein tyrosine kinase activity capable of tyrosine
phosphorylating p85 in vitro associates with p85 alpha immunoprecipitates in quiescent or TCR.CD3-activated T cells. These
data suggest that the TCR.CD3 complex does not regulate PtdIns 3-kinase activity by a mechanism that involves protein tyrosine
kinases.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Antigenic cross-linking of the high affinity IgE receptor (Fc R1) on mast cells results in protein tyrosine kinase activation. The object of the present study was to explore the regulation
of the SH2 ...and SH3 domain containing adapter molecule Grb2 by Fc R1-stimulated PTK signal transduction pathways. Affinity purification of in vivo Grb2 complexes together with in vitro experiments with Grb2 glutathione S -transferase fusion proteins were used to analyze Grb2 complexes in the mast cell line RBL2H3. The data show that in RBL2H3
cells several different proteins are complexed to the SH3 domains of Grb2. These include the p21ras guanine nucleotide exchange
factor Sos, two basally tyrosine-phosphorylated 110- and 120-kDa molecules, and a 75-kDa protein that is a substrate for Fc R1-activated PTKs. By analogy with Sos, p75, p110 and p120 are candidates for Grb2 effector proteins which suggests that Grb2
may be a pleiotropic adapter. Two Grb2 SH2-binding proteins were also characterized in RBL2H3 cells; the adapter Shc and a
33-kDa molecule. Shc is constitutively tyrosine phosphorylated in unstimulated cells and Fc R1 ligation induces no changes in its phosphorylation or binding to Grb2. In contrast, p33 is a substrate for Fc R1-activated PTKs and binds to Grb2 SH2 domains in Fc R1 activated but not quiescent cells. The β subunit of the Fc R1 is a 33-kDa tyrosine phosphoprotein, but the p33 Grb2-binding protein described in the present report is not the Fc R1 β chain and its identity is unknown. The present report thus demonstrates that there are multiple Grb2 containing protein
complexes in mast cells of which a subset are Fc R1-regulated. Two other of the Grb2-binding proteins described herein are tyrosine phosphorylated in response to Fc R1 ligation: the 75-kDa protein which binds to Grb2 SH3 domains and the 33-kDa protein that associates with the Grb2 SH2 domain.
We propose that protein complex formation by Grb2 is an important consequence of Fc R1 cross-linking and that this may be a signal transduction pathway which acts synergistically with calcium/PKC signals to
bring about optimal mast cell end function.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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