Role of associated gamma-chain in tyrosine kinase activation via murine Fc gamma RIII.

Type III receptors for the Fc portion of IgG (Fc gamma RIII), initially characterized on macrophages and NK cells, are also expressed on several pre-B cell lines. Surface expression of Fc gamma RIII requires the association of the ligand binding alpha-chain with homodimeric gamma-chains. Type II Fc gamma R is homologous to Fc gamma RIII alpha-chain in the extracellular portion and differs in the transmembrane and cytoplasmic domains. The role of Fc gamma R in cell activation was investigated by expressing Fc gamma RIII and the lymphocyte-specific b1 isoform of Fc gamma RII (Fc gamma RIIb1) in an Fc gamma R-negative, sIgG-positive B-cell line. We found that, in contrast to Fc gamma RIIb1, Fc gamma RIII triggers the same events of cell activation as sIG i.e. Ca2+ mobilization, tyrosine phosphorylation and IL-2 secretion. By expressing cytoplasmic domain-lacking Fc gamma RIII alpha-chain in the absence or in the presence of gamma-chains, we demonstrated that cell activation via Fc gamma RIII requires the co-expression of gamma-chains, and is independent of the cytoplasmic portion of the alpha-chain. Furthermore, the cytoplasmic portion of the gamma-chain, fused to the extracellular and transmembrane domains of Fc gamma RII confers on the chimeric receptor the ability to trigger cell activation. Mutation of one tyrosine residue in the cytoplasmic domain of the gamma-chain prevented triggering of cytoplasmic signals. We therefore demonstrate that a tyrosine-containing motif, present in the cytoplasmic domain of the associated gamma-chain, is necessary and sufficient to trigger cell activation via Fc gamma RIII.     

Syk and Bruton's tyrosine kinase are required for B cell antigen receptor-mediated activation of the kinase Akt.

Activation of Akt by multiple stimuli including B cell antigen receptor (BCR) engagement requires phosphatidylinositol 3-kinase and regulates processes including cell survival, proliferation, and metabolism. BCR cross-linking activates three families of non-receptor protein tyrosine kinases (PTKs) and these are transducers of signaling events including phospholipase C and mitogen-activated protein kinase activation; however, the relative roles of PTKs in BCR-mediated Akt activation are unknown. We examined Akt activation in Lyn-, Syk- and Btk-deficient DT40 cells and B cells from Lyn(-/-) mice. BCR-mediated Akt activation required Syk and was partially dependent upon Btk. Increased BCR-induced Akt phosphorylation was observed in Lyn-deficient DT40 cells and Lyn(-/-) mice compared with wild-type cells suggesting that Lyn may negatively regulate Akt function. BCR-induced tyrosine phosphorylation of the phosphatidylinositol 3-kinase catalytic subunit was abolished in Syk-deficient cells consistent with a receptor-proximal role for Syk in BCR-mediated phosphatidylinositol 3-kinase activation; in contrast, it was maintained in Btk-deficient cells, suggesting Btk functions downstream of phosphatidylinositol 3-kinase. Calcium depletion did not influence BCR-induced Akt phosphorylation/activation, showing that neither Syk nor Btk mediates its effects via changes in calcium levels. Thus, BCR-mediated Akt stimulation is regulated by multiple non-receptor PTK families which regulate Akt both proximal and distal to phosphatidylinositol 3-kinase activation.     

Evidence that phospholipase C-gamma2 interacts with SLP-76, Syk, Lyn, LAT and the Fc receptor gamma-chain after stimulation of the collagen receptor glycoprotein VI in human platelets.

Platelet activation by collagen is mediated by the sequential tyrosine phosphorylation of the Fc receptor gamma-chain (FcR gamma-chain), which is part of the collagen receptor glycoprotein VI, the tyrosine kinase Syk and phospholipase C-gamma2 (PLC-gamma2). In this study tyrosine-phosphorylated proteins that associate with PLC-gamma2 after stimulation by a collagen-related peptide (CRP) were characterized using glutathione S-transferase fusion proteins of PLC-gamma2 Src homology (SH) domains and by immunoprecipitation of endogenous PLC-gamma2. The majority of the tyrosine-phosphorylated proteins that associate with PLC-gamma2 bind to its C-terminal SH2 domain. These were found to include PLC-gamma2, Syk, SH2-domain-containing leucocyte protein of 76 kDa (SLP-76), Lyn, linker for activation of T cells (LAT) and the FcR gamma-chain. Direct association was detected between PLC-gamma2 and SLP-76, and between PLC-gamma2 and LAT upon CRP stimulation of platelets by far-Western blotting. FcR gamma-chain and Lyn were found to co-immunoprecipitate with PLC-gamma2 as well as with unidentified 110-kDa and 75-kDa phosphoproteins. The absence of an in vivo association between Syk and PLC-gamma2 in platelets is in contrast with that for PLC-gamma1 and Syk in B cells. The in vivo function of PLC-gamma2 SH2 domains was examined through measurement of Ca2+ increases in mouse megakaryocytes that had been microinjected with recombinant proteins. This revealed that the C-terminal SH2 domain is involved in the regulation of PLC-gamma2. These data indicate that the C-terminal SH2 domain of PLC-gamma2 is important for PLC-gamma2 regulation through possible interactions with SLP-76, Syk, Lyn, LAT and the FcR gamma-chain.     

Pre-T cell receptor signals are responsible for the down-regulation of Syk protein tyrosine kinase expression.

Thymocyte development proceeds through two critical checkpoints that involve signaling events through two different receptors, the TCR and the pre-TCR. These receptors employ two families of protein tyrosine kinases to propagate their signals, the Src and Syk families. Genetic and biochemical evidence has shown that the Src family kinases are critical for normal T cell maturation. ZAP-70, a Syk family kinase, has similarly been implicated as a critical component in thymocyte development. Although genetic evidence has suggested that Syk is involved during thymocyte development, a definitive study of Syk expression has not been performed. In this paper we report our reanalysis of Syk expression in subpopulations of murine and human thymocytes by intracellular staining and flow cytometry using anti-Syk mAbs. Syk is expressed at increased levels during the stages in which pre-TCR signaling occurs. Furthermore, Syk is down-regulated after the pre-TCR checkpoint has been passed. Syk may play an important role in thymic development during pre-TCR signal transduction. Finally, incomplete down-regulation of Syk expression was noted in human thymocytes, offering a possible explanation for the distinct phenotypes of mice and humans deficient in ZAP-70.     

The tyrosine kinase Syk regulates TPL2 activation signals

Tpl2/Cot is a serine/threonine kinase that plays a key physiological role in the regulation of immune responses to pro-inflammatory stimuli, including tumor necrosis factor-alpha (TNF-alpha). TNF-alpha stimulates the JNK, ERK, and p38 mitogen-activated protein kinases and the NF-kappaB pathway by recruiting RIP1 and TRAF2 to the TNF receptor 1. Here we showed that Tpl2 activation by TNF-alpha signals depends on the integrity of the Tpl2-interacting proteins RIP1 and TRAF2, which are required for the engagement of the ERK mitogen-activated protein kinase pathway. However, neither RIP1 nor TRAF2 overexpression was sufficient to activate Tpl2 and ERK. We also showed that Tpl2 activation by TNF-alpha depends on a tyrosine kinase activity that is detected in TNF-alpha-stimulated cells. Based on both genetic and biochemical evidence, we concluded that in a variety of cell types, Syk is the tyrosine kinase that plays an important role in the activation of Tpl2 upstream of ERK. These data therefore dissect the TNF receptor 1 proximal events that regulate Tpl2 and ERK and highlight a role for RIP1, TRAF2, and Syk in this pathway.     

The RING finger domain of Cbl is essential for negative regulation of the Syk tyrosine kinase

The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. Here, we used a COS-7 cell reconstitution system to address this question. Analysis of a series of C-terminally truncated Cbl mutants revealed that the N-terminal half of the protein, including the TKB and RING finger domains, was sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogated the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogated the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells was reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogated this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function.     

Redundant role of the Syk protein tyrosine kinase in mouse NK cell differentiation.

Syk and ZAP-70 subserve nonredundant functions in B and T lymphopoiesis. In the absence of Syk, B cell development is blocked, while T cell development is arrested in the absence of ZAP-70. The receptors and the signaling molecules required for differentiation of NK cells are poorly characterized. Here we investigate the role of the Syk protein tyrosine kinase in NK cell differentiation. Hemopoietic chimeras were generated by reconstituting alymphoid (B-, T-, NK-) recombinase-activating gene-2 x common cytokine receptor gamma-chain double-mutant mice with Syk-/- fetal liver cells. The phenotypically mature Syk-/- NK cells that developed in this context were fully competent in natural cytotoxicity and in calibrating functional inhibitory receptors for MHC molecules. Syk-deficient NK cells demonstrated reduced levels of Ab-dependent cellular cytotoxicity. Nevertheless, Syk-/- NK cells could signal through NK1. 1 and 2B4 activating receptors and expressed ZAP-70 protein. We conclude that the Syk protein tyrosine kinase is not essential for murine NK cell development, and that compensatory signaling pathways (including those mediated through ZAP-70) may sustain most NK cell functions in the absence of Syk.     

Prolactin receptor triggering. Evidence for rapid tyrosine kinase activation.

The mechanism of action of prolactin (PRL) has remained obscure despite the unveiling of the primary structure of PRL receptors. The present study demonstrates rapid PRL receptor-mediated tyrosine phosphorylation of at least three cellular proteins, designated p120, p97, and p40, in a rat T-lymphoma (Nb2-11C) as revealed by antiphosphotyrosine immunoblotting. One of the phosphotyrosyl proteins, p120, co-purified with activated PRL receptor complexes obtained using either anti-ligand or anti-receptor antibodies. Furthermore, in vitro incubation of affinity-purified PRL receptor complexes from PRL-stimulated cells with ATP in the presence of a tyrosine phosphatase inhibitor, resulted in a 10-15-fold increase in the phosphotyrosine content of p120, as revealed by antiphosphotyrosine immunoblotting. Parallel experiments utilizing [gamma-32P]ATP confirmed a rapid and time-dependent incorporation of phosphate into p120 in the same affinity-purified PRL receptor complexes. These data provide strong evidence for the involvement of a tyrosine kinase in PRL signal transduction and suggest the presence of a tyrosine kinase within the activated PRL receptor complex.     

Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein.     

Functional interaction of protein kinase Calpha with the tyrosine kinases Syk and Src in human platelets

There is a high degree of cross-talk between tyrosine phosphorylation and the serine/threonine phosphorylation signaling pathways. Here we show a physical and functional interaction between the classical protein kinase C isoform (cPKC), PKCalpha, and two major nonreceptor tyrosine kinases in platelets, Syk and Src. In the presence of the cPKC-selective inhibitor Go6976, platelet 5-hydroxytryptamine release was abolished in response to co-activation of glycoproteins VI and Ib-IX-V by the snake venom alboaggregin A, whereas platelet aggregation was substantially inhibited. Of the two platelet cPKCs, PKCalpha but not PKCbeta was activated, occurring in an Syk- and phospholipase C-dependent manner. Syk and PKCalpha associate in a stimulation-dependent manner, requiring Syk but not PKC activity. PKCalpha and Syk also co-translocate from the cytosol to the plasma membrane upon platelet activation, in a manner dependent upon the activities of both kinases. Although PKCalpha is phosphorylated on tyrosine downstream of Syk, we provide evidence against phosphorylation of Syk by PKCalpha, consistent with a lack of effect of PKCalpha inhibition on Syk activity. PKCalpha also associates with Src; although in contrast to interaction with Syk, PKCalpha activity is required for the association of these kinases but not the stimulation-induced translocation of Src to the cell membrane. Finally, the activity of Src is negatively regulated by PKC, as shown by potentiation of Src activity in the presence of the PKC inhibitors GF109203X or Go6976. Therefore, there is a complex interplay between PKCalpha, Syk, and Src involving physical interaction, phosphorylation, translocation within the cell, and functional activity regulation.     

The non-receptor tyrosine kinase Syk is a target of Cbl-mediated ubiquitylation upon B-cell receptor stimulation.

The negative regulator Cbl functions as a ubiquitin ligase towards activated receptor tyrosine kinases and facilitates their transport to lysosomes. Whether Cbl ubiquitin ligase activity mediates its negative regulatory effects on cytoplasmic tyrosine kinases of the Syk/ZAP-70 family has not been addressed, nor is it known whether these kinases are regulated via ubiquitylation during lymphocyte B-cell receptor engagement. Here we show that B-cell receptor stimulation in Ramos cells induces the ubiquitylation of Syk tyrosine kinase which is inhibited by a dominant-negative mutant of Cbl. Intact tyrosine kinase-binding and RING finger domains of Cbl were found to be essential for Syk ubiquitylation in 293T cells and for in vitro Syk ubiquitylation. These same domains were also essential for Cbl-mediated negative regulation of Syk as measured using an NFAT-luciferase reporter in a lymphoid cell. Association with Cbl did not alter the kinase activity of Syk. Altogether, our results support an essential role for Cbl ubiquitin ligase activity in the negative regulation of Syk, and establish that ubiquitylation provides a mechanism of Cbl-mediated negative regulation of cytoplasmic targets.     

Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon Fc epsilon RI cross-linking.

Tyrosine phosphorylation of several cellular proteins is one of the earliest signaling events induced by cross-linking of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells or basophils. Tyrosine kinases activated during this process include the Src family kinases, Lyn, c-Yes, and c-Src, and members of another subfamily, Syk and PTK72 (identical or highly related to Syk). Recently, some of us described two novel tyrosine kinases, Emb and Emt, whose expression was limited to subsets of hematopoietic cells, including mast cells. Emb turned out to be identical to Btk, a gene product defective in human X-linked agammaglobulinemia and in X-linked immunodeficient (xid) mice. Here we report that Fc epsilon RI cross-linking induced rapid phosphorylation on tyrosine, serine, and threonine residues and activation of Btk in mouse bone marrow-derived mast cells. A small fraction of Btk translocated from the cytosol to the membrane compartment following receptor cross-linking. Tyrosine phosphorylation of Btk was not induced by either a Ca2+ ionophore (A23187), phorbol 12-myristate 13-acetate, or a combination of the two reagents. Co-immunoprecipitation between Btk and receptor subunit beta or gamma was not detected. The data collectively suggest that Btk is not associated with Fc epsilon but that its activation takes place prior to protein kinase C activation and plays a novel role in the Fc epsilon RI signaling pathway.     

syk protein tyrosine kinase regulates Fc receptor gamma-chain-mediated transport to lysosomes.

B- and T-cell receptors, as well as most Fc receptors (FcR), are part of a large family of membrane proteins named immunoreceptors and are expressed on all cells of the immune system. Immunoreceptors' biological functions rely on two of their fundamental attributes: signal transduction and internalization. The signals required for these two functions are present in the chains associated with immunoreceptors, within conserved amino acid motifs called immunoreceptor tyrosine-based activation motifs (ITAMs). We have examined the role of the protein tyrosine kinase (PTK) syk, a critical effector of immunoreceptor-mediated cell signalling through ITAMs, in FcR-associated gamma-chain internalization and lysosomal targeting. A point mutation in the immunoreceptor-associated gamma-chain ITAM affecting syk activation, as well as overexpression of a syk dominant negative mutant, inhibited signal transduction without affecting receptor coated-pit localization or internalization. In contrast, blocking of gamma-chain-mediated syk activation impaired FcR transport from endosomes to lysosomes and selectively inhibited the presentation of certain T-cell epitopes. Therefore, activation of the PTK syk is dispensable for receptor internalization, but necessary for cell signalling and for gamma-chain-mediated FcR delivery to lysosomes.     

Nucleocytoplasmic trafficking of the Syk protein tyrosine kinase

The protein tyrosine kinase Syk couples the B-cell receptor (BCR) for antigen to multiple intracellular signaling pathways and also modulates cellular responses to inducers of oxidative stress in a receptor-independent fashion. In B cells, Syk is found in both the nuclear and cytoplasmic compartments but contains no recognizable nuclear localization or export signals. Through the analysis of a series of deletion mutants, we identified the presence of an unconventional shuttling sequence near the junction of the catalytic domain and the linker B region that accounts for Syk's subcellular localization. This localization is altered following prolonged engagement of the BCR, which causes Syk to be excluded from the nucleus. Nuclear exclusion requires the receptor-mediated activation of protein kinase C and new protein synthesis. Both of these processes also potentiate the activation of caspase 3 in cells in response to oxidative stress in a manner that is dependent on the localization of Syk outside of the nucleus. In contrast, restriction of Syk to the nucleus greatly diminishes the stress-induced activation of caspase 3.     

Insulin receptor serine kinase activation by casein kinase 2 and a membrane tyrosine kinase

The insulin receptor (IR) tyrosine kinase can apparently directly phosphorylate and activate one or more serine kinases. The identities of such serine kinases and their modes of activation are still unclear. We have described a serine kinase (here designated insulin receptor serine (IRS) kinase) from rat liver membranes that co-purifies with IR on wheat germ agglutinin-agarose. The kinase was activated after phosphorylation of the membrane glycoproteins by casein kinase-1, casein kinase-2, or casein kinase-3 (Biochem Biophys Res Commun 171:75–83, 1990). In this study, IRS kinase was further characterized. The presence of vanadate or phosphotyrosine in reaction mixtures was required for activation to be observed. Phosphoserine and phosphothreonine are only about 25% as effective as phosphotyrosine, whereas sodium fluoride and molybdate were ineffective in supporting activation. Vanadate and phosphotyrosine support IRS kinase activation by apparently inhibiting phosphotyrosine protein phosphatases present among the membrane glycoproteins. IR -subunit, myelin basic protein, and microtubule-associated protein-2 are good substrates for IRS kinase. The kinase prefers Mn²⁺ (K_a=1.3 mM) as a metal cofactor. Mg²⁺ (K_a=3.3 mM) is only 30% as effective as Mn²⁺. The kinase activity is stimulated by basic polypeptides, with greater than 30-fold activation achieved with polylysine and protamine. Our results suggest that both serine/threonine and tyrosine phosphorylation are required for activation of IRS kinase. Serine phosphorylation is catalyzed by one of the casein kinases, whereas tyrosine phosphorylation is catalyzed by a membrane tyrosine kinase, possibly IR tyrosine kinase. (Mol Cell Biochem121: 167–174, 1993)     

Mapping out regions on the surface of the aspartate receptor that are essential for kinase activation

The aspartate receptor of bacterial chemotaxis is representative of a large family of taxis receptors widespread in prokaryotes. The homodimeric receptor associates with cytoplasmic components to form a receptor-kinase signaling complex. Within this complex the receptor is known to directly contact the histidine kinase CheA, the coupling protein CheW, and other receptor dimers. However, the locations and extents of the contact regions on the receptor surface remain ambiguous. The present study applies the protein-interactions-by-cysteine-modification (PICM) method to map out surfaces on the aspartate receptor that are essential for kinase stimulation in the assembled receptor-kinase complex. The approach utilizes 52 engineered cysteine positions scattered over the surface of the receptor periplasmic and cytoplasmic domains. When the bulky, anionic probe 5-fluorescein-maleimide is coupled to these positions, large effects on receptor-mediated kinase stimulation are observed at eight cytoplasmic locations. By contrast, no large effects are observed for probe attachment at exposed positions in the periplasmic domain. The results indicate that essential receptor surface regions are located near the hairpin turn at the distal end of the cytoplasmic domain and in the cytoplasmic adaptation site region. These surface regions include the docking sites for CheA, CheW, and other receptor dimers, as well as surfaces that transmit information from the receptor adaptation sites to the kinase. Smaller effects observed in the cytoplasmic linker or HAMP region suggest this region may also play a role in kinase regulation. A comparison of the activity perturbations caused by a dianionic, bulky probe (5-fluorescein-maleimide), a zwitterionic, bulky probe (5-tetramethyl-rhodamine-maleimide), and a nonionic, smaller probe (N-ethyl-maleimide) reveals the roles of probe size and charge in generating the observed effects on kinase activity. Overall, the results indicate that interactions between the periplasmic domains of different receptor dimers are not required for kinase activation in the signaling complex. By contrast, the observed spatial distribution of protein contact surfaces on the cytoplasmic domain is consistent with both (i) distinct docking sites for cytoplasmic proteins and (ii) interactions between the cytoplasmic domains of different dimers to form a trimer-of-dimers.     

A critical role for Syk protein tyrosine kinase in Fc receptor-mediated antigen presentation and induction of dendritic cell maturation

Dendritic cells (DCs) are the only APCs capable of initiating adaptive immune responses. The initiation of immune responses requires that DCs 1) internalize and present Ags; and 2) undergo a differentiation process, called "maturation", which transforms DCs into efficient APCs. DC maturation may be initiated by the engagement of different surface receptors, including certain cytokine receptors (such as TNFR), Toll-like receptors, CD40, and FcRs. The early activation events that link receptor engagement and DC maturation are not well characterized. We found that FcR engagement by immune complexes induced the phosphorylation of Syk, a protein tyrosine kinase acting immediately downstream of FcRs. Syk was dispensable for DC differentiation in vitro and in vivo, but was strictly required for immune complexes internalization and subsequent Ag presentation to T lymphocytes. Importantly, Syk was also required for the induction of DC maturation and IL-12 production after FcR engagement, but not after engagement of other surface receptors, such as TNFR or Toll-like receptors. Therefore, protein tyrosine phosphorylation by Syk represents a novel pathway for the induction of DC maturation.     

The tyrosine kinase activity of the epidermal-growth-factor receptor is necessary for phospholipase A2 activation.

Cytosolic glutathione transferases (GSTs) were purified from the rat spleen by S-hexyl-GSH-Sepharose chromatography, and two major forms were identified as GSTs 2-2 and 7-7 (GST P). Besides these forms an acidic form (pI 5.8) was purified by chromatofocusing at pH 7-4 and it accounted for about 1% of the total GST activity bound to S-hexyl-GSH-Sepharose. Two-dimensional gel electrophoresis revealed that it is a homodimer (subunit Mr 26,000 with pI 5.8). Immunoblot analysis demonstrated that it was immunologically related to GSTs 2-2 and 1-1, and its N-terminal amino acid was apparently blocked, similarly to other forms of the class Alpha. This form had a low activity towards cumene hydroperoxide or 4-hydroxynon-2-enal, indicating that this form differed from GSTs 10-10 and 8-8 as well as from GSTs 1-1 and 2-2. These results suggest that it is a new form of GST belonging to the class Alpha.     

Protein kinase B is regulated in platelets by the collagen receptor glycoprotein VI

Phosphoinositide 3-kinase (PI3K) is a critical component of the signaling pathways that control the activation of platelets. Here we have examined the regulation of protein kinase B (PKB), a downstream effector of PI3K, by the platelet collagen receptor glycoprotein (GP) VI and thrombin receptors. Stimulation of platelets with collagen or convulxin (a selective GPVI agonist) resulted in PI3K-dependent, and aggregation independent, Ser(473) and Thr(308) phosphorylation of PKBalpha, which results in PKB activation. This was accompanied by translocation of PKB to cell membranes. The phosphoinositide-dependent kinase PDK1 is known to phosphorylate PKBalpha on Thr(308), although the identity of the kinase responsible for Ser(473) phosphorylation is less clear. One candidate that has been implicated as being responsible for Ser(473) phosphorylation, either directly or indirectly, is the integrin-linked kinase (ILK). In this study we have examined the interactions of PKB, PDK1, and ILK in resting and stimulated platelets. We demonstrate that in platelets PKB is physically associated with PDK1 and ILK. Furthermore, the association of PDK1 and ILK increases upon platelet stimulation. It would therefore appear that formation of a tertiary complex between PDK1, ILK, and PKB may be necessary for phosphorylation of PKB. These observations indicate that PKB participates in cell signaling downstream of the platelet collagen receptor GPVI. The role of PKB in collagen- and thrombin-stimulated platelets remains to be determined.     

Signaling through mutants of the IgA receptor CD89 and consequences for Fc receptor gamma-chain interaction

The prototypic receptor for IgA (FcalphaRI, CD89) is expressed on myeloid cells and can trigger phagocytosis, tumor cell lysis, and release of inflammatory mediators. The functions of FcalphaRI and activating receptors for IgG (FcgammaRI and FcgammaRIII) are dependent on the FcR gamma-chain dimer. This study increases our understanding of the molecular basis of the FcalphaRI-FcR gamma-chain transmembrane interaction, which is distinct from that of other activatory FcRs. FcalphaRI is unique in its interaction with the common FcR gamma-chain, because it is based on a positively charged residue at position 209, which associates with a negatively charged amino acid of FcR gamma-chain. We explored the importance of the position of this positive charge within human FcalphaRI for FcR gamma-chain association and FcalphaRI functioning with the use of site-directed mutagenesis. In an FcalphaRI R209L/A213H mutant, which represents a vertical relocation of the positive charge, proximal and distal FcR gamma-chain-dependent functions, such as calcium flux, MAPK phosphorylation, and IL-2 release, were similar to those of wild-type FcalphaRI. A lateral transfer of the positive charge, however, completely abrogated FcR gamma-chain-dependent functions in an FcalphaRI R209L/M210R mutant. By coimmunoprecipitation, we have demonstrated the loss of a physical interaction between FcR gamma-chain and FcalphaRI M210R mutant, thus explaining the loss of FcR gamma-chain-dependent functions. In conclusion, not only the presence of a basic residue in the transmembrane region of FcalphaRI, but also the orientation of FcalphaRI toward the FcR gamma-chain dimer is essential for FcR gamma-chain association. This suggests the involvement of additional amino acids in the FcalphaRI-FcR gamma-chain interaction.