Effects of I domain deletion on the function of the beta2 integrin lymphocyte function-associated antigen-1.
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A subset of integrin alpha subunits contain an I domain, which is important for ligand binding. We have deleted the I domain from the beta2 integrin lymphocyte function-asssociated antigen-1 (LFA-1) and expressed the resulting non-I domain-containing integrin (DeltaI-LFA-1) in an LFA-1-deficient T cell line. DeltaI-LFA-1 showed no recognition of LFA-1 ligands, confirming the essential role of the I domain in ligand binding. Except for I domain monoclonal antibodies (mAbs), DeltaI-LFA-1 was recognized by a panel of anti-LFA-1 mAbs similarly to wild-type LFA-1. However, DeltaI-LFA-1 had enhanced expression of seven mAb epitopes that are associated with beta2 integrin activation, suggesting that it exhibited an "active" conformation. In keeping with this characteristic, DeltaI-LFA-1 induced constitutive activation of alpha4beta1 and alpha5beta1, suggesting intracellular signaling to these integrins. This "cross-talk" was not due to an effect on beta1 integrin affinity. However, the enhanced activity was susceptible to inhibition by cytochalasin D, indicating a role for the cytoskeleton, and also correlated with clustering of beta1 integrins. Thus, removal of the I domain from LFA-1 created an integrin with the hallmarks of a constitutively active receptor mediating signals into the cell. These findings suggest a key role for the I domain in controlling integrin activity. (+info)
Differential activation of MAP kinase family members triggered by CD99 engagement.
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The molecular basis for the modulatory properties of CD99 is not well understood. Treatment of human Jurkat T lymphocytes with anti-CD99 antibody led to activation of three mitogen-activated protein kinase (MAPK) members, ERK, JNK, and p38 MAPK, along with homotypic aggregation. While phosphorylation of ERK and JNK was inhibited by the pretreatment of a PKC inhibitor, bisindolylmaleimide I, activation of p38 MAPK was upregulated by the same pretreatment. The signaling pathways to MAPKs by CD99 engagement were independent of PI-3 kinase, distinguishing from those by CD3 engagement. Among MAPKs, ERK pathway was essential for homotypic aggregation together with intracytoplasmic Ca(2+). (+info)
Fyn-binding protein (Fyb)/SLP-76-associated protein (SLAP), Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the Arp2/3 complex link T cell receptor (TCR) signaling to the actin cytoskeleton.
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T cell receptor (TCR)-driven activation of helper T cells induces a rapid polarization of their cytoskeleton towards bound antigen presenting cells (APCs). We have identified the Fyn- and SLP-76-associated protein Fyb/SLAP as a new ligand for Ena/ vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Upon TCR engagement, Fyb/SLAP localizes at the interface between T cells and anti-CD3-coated beads, where Evl, a member of the Ena/VASP family, Wiskott-Aldrich syndrome protein (WASP) and the Arp2/3 complex are also found. In addition, Fyb/SLAP is restricted to lamellipodia of spreading platelets. In activated T cells, Fyb/SLAP associates with Ena/VASP family proteins and is present within biochemical complexes containing WASP, Nck, and SLP-76. Inhibition of binding between Fyb/SLAP and Ena/VASP proteins or WASP and the Arp2/3 complex impairs TCR-dependent actin rearrangement, suggesting that these interactions play a key role in linking T cell signaling to remodeling of the actin cytoskeleton. (+info)
Essential role for the C-terminal noncatalytic region of SHIP in FcgammaRIIB1-mediated inhibitory signaling.
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The inositol phosphatase SHIP binds to the FcgammaRIIB1 receptor and plays a critical role in FcgammaRIIB1-mediated inhibition of B-cell proliferation and immunoglobulin synthesis. The molecular details of SHIP function are not fully understood. While point mutations of the signature motifs in the inositol phosphatase domain abolish SHIP's ability to inhibit calcium flux in B cells, little is known about the function of the evolutionarily conserved, putative noncatalytic regions of SHIP in vivo. In this study, through a systematic mutagenesis approach, we identified the inositol phosphatase domain of SHIP between amino acids 400 and 866. Through reconstitution of a SHIP-deficient B-cell line with wild-type and mutant forms of SHIP, we demonstrate that the catalytic domain alone is not sufficient to mediate FcgammaRIIB1/SHIP-dependent inhibition of B-cell receptor signaling. Expression of a truncation mutant of SHIP that has intact phosphatase activity but lacks the last 190 amino acids showed that the noncatalytic region in the C terminus is essential for inhibitory signaling. Mutation of two tyrosines within this C-terminal region, previously identified as important in binding to Shc, showed a reduced inhibition of calcium flux. However, studies with an Shc-deficient B-cell line indicated that Shc-SHIP complex formation is not required and that other proteins that bind these tyrosines may be important in FcgammaRIIB1/SHIP-mediated calcium inhibition. Interestingly, membrane targeting of SHIP lacking the C terminus is able to restore this inhibition, suggesting a role for the C terminus in localization or stabilization of SHIP interaction at the membrane. Taken together, these data suggest that the noncatalytic carboxyl-terminal 190 amino acids of SHIP play a critical role in SHIP function in B cells and may play a similar role in several other receptor systems where SHIP functions as a negative regulator. (+info)
The tumor necrosis factor-related apoptosis-inducing ligand receptors TRAIL-R1 and TRAIL-R2 have distinct cross-linking requirements for initiation of apoptosis and are non-redundant in JNK activation.
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Overexpression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induces apoptosis and activation of NF-kappaB in cultured cells. In this study, we have demonstrated differential signaling capacities by both receptors using either epitope-tagged soluble TRAIL (sTRAIL) or sTRAIL that was cross-linked with a monoclonal antibody. Interestingly, sTRAIL was sufficient for induction of apoptosis only in cell lines that were killed by agonistic TRAIL-R1- and TRAIL-R2-specific IgG preparations. Moreover, in these cell lines interleukin-6 secretion and NF-kappaB activation were induced by cross-linked or non-cross-linked anti-TRAIL, as well as by both receptor-specific IgGs. However, cross-linking of sTRAIL was required for induction of apoptosis in cell lines that only responded to the agonistic anti-TRAIL-R2-IgG. Interestingly, activation of c-Jun N-terminal kinase (JNK) was only observed in response to either cross-linked sTRAIL or anti-TRAIL-R2-IgG even in cell lines where both receptors were capable of signaling apoptosis and NF-kappaB activation. Taken together, our data suggest that TRAIL-R1 responds to either cross-linked or non-cross-linked sTRAIL which signals NF-kappaB activation and apoptosis, whereas TRAIL-R2 signals NF-kappaB activation, apoptosis, and JNK activation only in response to cross-linked TRAIL. (+info)
Protein kinase D. A selective target for antigen receptors and a downstream target for protein kinase C in lymphocytes.
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Protein kinase Cs (PKCs) are activated by antigen receptors in lymphocytes, but little is known about proximal targets for PKCs in antigen receptor-mediated responses. In this report, we define a role for diacylglycerol-regulated PKC isoforms in controlling the activity of the serine/threonine kinase protein kinase D (PKD; also known as PKC mu) in T cells, B cells, and mast cells. Antigen receptor activation of PKD is a rapid and sustained response that can be seen in T cells activated via the T cell antigen receptor, B cells activated via the B cell antigen receptor, and in mast cells triggered via the high-affinity receptor for IgE (FcepsilonR1). Herein, we show that antigen receptor activation of PKD requires the activity of classical/novel PKCs. Moreover, PKC activity is sufficient to bypass the requirement for antigen receptor signals in the induction of PKD activity. These biochemical and genetic studies establish a role for antigen receptor-regulated PKC enzymes in the control of PKD activity. Regulation of PKD activity through upstream PKCs reveals a signaling network that exists between different members of the PKC superfamily of kinases that can operate to amplify and disseminate antigen receptor signals generated at the plasma membrane. (+info)
Agrin-induced acetylcholine receptor clustering is mediated by the small guanosine triphosphatases Rac and Cdc42.
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During neuromuscular junction formation, agrin secreted from motor neurons causes muscle cell surface acetylcholine receptors (AChRs) to cluster at synaptic sites by mechanisms that are insufficiently understood. The Rho family of small guanosine triphosphatases (GTPases), including Rac and Cdc42, can mediate focal reorganization of the cell periphery in response to extracellular signals. Here, we investigated the role of Rac and Cdc42 in coupling agrin signaling to AChR clustering. We found that agrin causes marked muscle-specific activation of Rac and Cdc42 in differentiated myotubes, as detected by biochemical measurements. Moreover, this activation is crucial for AChR clustering, since the expression of dominant interfering mutants of either Rac or Cdc42 in myotubes blocks agrin-induced AChR clustering. In contrast, constitutively active Rac and Cdc42 mutants cause AChR to aggregate in the absence of agrin. By indicating that agrin-dependent activation of Rac and Cdc42 constitutes a critical step in the signaling pathway leading to AChR clustering, these findings suggest a novel role for these Rho-GTPases: the coupling of neuronal signaling to a key step in neuromuscular synaptogenesis. (+info)
p135 src homology 2 domain-containing inositol 5'-phosphatase (SHIPbeta ) isoform can substitute for p145 SHIP in fcgamma RIIB1-mediated inhibitory signaling in B cells.
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The inositol 5'-phosphatase, SHIP (also referred to as SHIP-1 or SHIPalpha), is expressed in all cells of the hematopoietic lineage. Depending on the cell type being investigated and the state of differentiation, SHIP isoforms of several different molecular masses (170, 160, 145, 135, 125, and 110 kDa) have been seen in immunoblots. However, the function of the individual isoforms and the effect of expressing multiple isoforms simultaneously are not understood. Some of these SHIP isoforms have recently been characterized at the level of primary sequence. In this report, we investigated the function of the recently characterized 135-kDa SHIP isoform (SHIPbeta), which appears to possess the catalytic domain but lacks some of the protein-protein interaction motifs at the C terminus. By reconstituting SHIP-deficient DT40 B cells with either SHIPbeta or the better-characterized p145 SHIPalpha, we addressed the function of SHIPbeta in the complete absence of SHIPalpha. We observed that SHIPbeta had enzymatic activity comparable with SHIPalpha and that SHIPbeta was able to reconstitute F(c)gammaRIIB1-mediated inhibition of B cell receptor-induced signaling events such as calcium flux and Akt and mitogen-activated protein kinase activation. SHIPbeta was readily phosphorylated in response to B cell receptor cross-linking with the inhibitory receptor F(c)gammaRIIB1 and SHIPbeta also interacted with the adapter protein Shc. During these studies we also observed that the SHIPalpha or SHIPbeta interaction with Grb2 is not required for F(c)gammaRIIB1-mediated inhibition of calcium flux. These data suggest that SHIPbeta, which is normally expressed in B cells along with SHIPalpha, functions comparably with SHIPalpha and that these two isoforms are not likely to be antagonistic in their function in vivo. (+info)