Type II phosphatidylinositol 4-kinase beta associates with TCR-CD3 zeta chain in Jurkat cells

Phosphatidylinositol lipid signaling cascades are integral part of TCR-CD3 signaling. The mechanisms by which phosphatidylinositol kinases are coupled to TCR-CD3 complex remain elusive. Here we report an association of type II PtdIns 4-kinase with TCR-CD3 zeta chain upon cross-linking. Mapping studies have revealed that the C-terminal ITAM is critical for docking of the enzyme on the zeta chain. The association is shown to be tyrosyl phosphorylation dependent as mutation of Y-151 and Y-142 on the C-terminal ITAM disrupts interaction of the two proteins. Identification of the associated type II PtdIns 4-kinase revealed that the beta isoform of the enzyme interacts with the zeta chain in vivo.     

Interleukin-7 induces the association of phosphatidylinositol 3-kinase with the α chain of the interleukin-7 receptor

The recently characterized receptor for interleukin (IL)-7 (IL-7R) includes a unique α chain as well as a common γ chain shared with the receptors for IL-2 and IL-4. Engagement of the IL-7R activates the intracellular enzyme phosphatidylinositol (PtdIns) 3-kinase but the mechanism of PtdIns 3-kinase activation and the molecular basis of its interaction with IL-7R are not known. Here we show that IL-7 causes the 85-kDa regulatory subunit of PtdIns 3-kinase (p85), and PtdIns 3-kinase activity, to associate with the IL-7R. This interaction can be ascribed to ligand-induced phosphorylation of a single Tyr residue in the receptor's unique α chain. Herbimycin A, a specific protein tyrosine kinase inhibitor, suppresses not only tyrosine phosphorylation of the IL-7R but also its association with p85. A phosphopeptide corresponding to the sequence surrounding Tyr⁴⁴⁹ in the cytoplasmic tail of the IL-7R α chain, but not its non-phosphorylated analogue or phosphopeptides coincident with the sequences surrounding other α chain Tyr residues, efficiently competes out p85 binding. Replacement of Tyr⁴⁴⁹ with Phe results in a loss of p85 binding. Finally, soluble forms of the src homology 2 domains of p85, which bind directly to phosphotyrosyl peptides, specifically inhibit the association of p85 with the IL-7R. Thus, PtdIns 3-kinase recruitment occurs through a single, phosphotyrosine dependent recognition motif surrounding Tyr⁴⁴⁹ in the IL-7R α chain. This motif corresponds to a canonical sequence for p85 binding, Tyr(P)-X-X-Met. Since the closely related IL-2R and IL-4R also activate PtdIns 3-kinase but are devoid of such canonical motifs, our results suggest that the mechanism by which IL-7R recruits and activates PtdIns 3-kinase differs fundamentally from that used by the other receptors. PtdIns 3-kinase may, therefore, play a unique and important role in the biological response to IL-7.     

Cofilin: a missing link between T cell co-stimulation and rearrangement of the actin cytoskeleton

The actin cytoskeletal network plays a regulatory role in receptor-mediated signal-transducing events. Recently, we have shown that the small actin-depolymerizing protein cofilin represents a component of a co-stimulatory signaling pathway in human T cells. Cofilin is dephosphorylated on phosphoserine residues following co-stimulation via accessory receptors such as CD2, CD4, CD8 or CD28, but not in response to TCR engagement alone. Here we demonstrate that accessory receptor triggering induces the transient association of cofilin with the actin cytoskeleton. Only the dephosphorylated form of cofilin binds to cytoskeletal actin in vivo. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 block dephosphorylation of cofilin and its association with the actin cytoskeleton. These results suggest that cofilin provides an as yet missing link between functionally crucial T cell surface receptors and rearrangements of the actin cytoskeleton.     

Association of T cell antigen CD7 with type II phosphatidylinositol-4 kinase,a key component in pathways of inositol phosphate turnover

CD7 is a 40-kDa glycoprotein that is expressed on prothymocytes and persists during T cell differentiation. CD7 has been demonstrated to generate, like other costimulatory molecules, intracellular signals that modulate T cell function. However, although it binds to phosphatidylinositol 3-kinase (PI 3-kinase), the signaling events mediated by CD7 are not completely understood. In this context, phosphatidylinositol 4-kinase (PI 4-kinase) is a key enzyme involved in a variety of events, from the modeling of the actin cytoskeleton to the activation of protein kinase C. In this study, we show for the first time that PI 4-kinase of 55 kDa can associate with CD7. The enzyme activity was insensitive to wortmannin, but was inhibited by adenosine, a characteristic for type II PI 4-kinase. Together, our findings demonstrate that type II PI 4-kinases are integral components of the CD7 signaling pathway and may play a role of CD7 in co-stimulation and thymic differentiation.     

Subcellular localization and translocation of protein kinase C isoforms zeta and epsilon in human peripheral blood lymphocytes

The calcium-independent members of the protein kinase C (PKC) family may play a significant role in T cell function. We have characterized the subcellular localization and redistribution of calcium-independent kinase C activity and of two specific members of this family (zeta and epsilon) in response to activation of human peripheral blood lymphocytes with phorbol myristate acetate (PMA) or through the TCR-CD3 complex. Both PMA and OKT3, an antibody against the TCR-associated CD3 complex, induce an increase in membrane and cytoskeletal activity with a concomitant decrease in cytosolic activity. By Western blot analysis, PKC epsilon is present in resting cytosol and membrane fractions, and is detected in the membrane following activation with PMA and in both the membrane and cytoskeleton following OKT3 activation. By contrast, PKC zeta is progressively lost from the cytoskeleton following activation with anti-CD3. Immunocytochemistry reveals distinct redistribution patterns for these enzymes in response to activation through anti-CD3 and by PMA. These findings demonstrate that signaling through the CD3 complex induces significant changes in calcium- independent PKC activity and in the intracellular distribution of specific isoenzymes, and support a role for specific functions for individual isoenzymes in T cell activation. Lastly, changes in the cytoskeletal distribution of these isoenzymes suggest a potential role in the modulation of cell structure in response to activation.      

The short length of the extracellular domain of zeta is crucial for T cell antigen receptor function

The T cell antigen receptor (TCR-CD3) consists of the pMHC-binding TCRalphabeta heterodimer and the signalling dimers CD3deltaepsilon, CD3gammaepsilon and zetazeta. The very short length of the extracellular domain (EC) of the zeta chain is preserved through evolution, however a rational explanation for this observation has not been elucidated. Here, we show that TCR-CD3 assembly is clearly defective when the murine zeta EC domain is artificially enlarged. Under these conditions, the TCR-CD3 complex is super-competent in transducing activation signals upon engagement. Furthermore, the TCR-CD3 complexes containing enlarged zeta EC domains underwent ligand-induced conformation changes with higher efficiency than TCR-CD3 complexes with an unmodified zeta EC domain. Together these data suggest that a short zeta EC domain is needed to correctly assemble the TCR-CD3 complex. When this domain is enlarged, the resulting TCR-CD3 complex is distorted leading to a hyperactive phenotype and enhanced T cell activation.     

FcepsilonRI cross-linking activates a type II phosphatidylinositol 4-kinase in RBL 2H3 cells

Crosslinking of FcepsilonRI on rat basophilic leukemia (RBL 2H3) cells leads to an increase in Phosphatidylinositol 4-kinase activity. This increase in Ptdlns 4-kinase activity is strongly correlated with its tyrosyl phosphorylation state. Characterization of the enzyme activity in anti phosphotyrosine immunoprecipitates suggests it as a type II Ptdlns 4-kinase. Membrane cholesterol depletion studies showed a reduction in type II Ptdlns 4-kinase activity suggesting that lipid rafts play an important role in activation of the enzyme. The enzyme activity was inhibited by resveratrol. In situ inhibition of type II Ptdlns 4-kinase activity showed a reduction in beta-hexosaminidase release upon FcepsilonRI cross-linking. These studies suggest that a type II Ptdlns 4-kinase is an integral component of FcepsilonRI mediated signal transduction mechanisms.     

F-actin dynamics control segregation of the TCR signaling cascade to clustered lipid rafts

Following ligand binding the TCR segregates to plasma membrane microdomains, termed lipid rafts, characterized by a highly ordered lipid structure favoring partitioning of glycosyl phosphatidyl inositol-linked costimulatory receptors and acylated signaling molecules. Here we show that the inducible association of the TCR and key signaling proteins with lipid rafts is dependent on the actin cytoskeleton through a mechanism involving raft coalescence. Although lipid rafts are required for full activation of the TCR-dependent tyrosine phosphorylation cascade and sustained signaling, triggering of TCR-proximal events, including Fyn activation and a first wave of Vav phosphorylation, is independent of lipid rafts, while a second wave of raft-dependent Vav phosphorylation occurs after raft coalescence, as also supported by the finding that Vav is phosphorylated in response to lipid raft clustering by GM1 aggregation. The constitutive association found between Vav and the CD3zeta chain suggests a model whereby the TCR-associated signaling machinery initiates raft aggregation by promoting F-actin reorganization, which permits full activation of the tyrosine phosphorylation cascade, further reorganization of the actin cytoskeleton and sustained signaling, leading to cell activation.     

Transport of phosphatidylinositol 3-phosphate into the vacuole via autophagic membranes in Saccharomyces cerevisiae

Vps34, the sole PtdIns 3-kinase in yeast, is essential for autophagy. Here, we show that the lipid-kinase activity of Vps34 is required for autophagy, implying an essential role of its product PtdIns(3) P . The protein-kinase activity of Vps15, a regulatory subunit of the PtdIns 3-kinase complex, is also required for efficient autophagy. We monitored the distribution of PtdIns(3) P in living cells using a specific indicator, the 2xFYVE domain derived from mammalian Hrs. PtdIns(3) P was abundant at endosomes and on the vacuolar membrane during logarithmic growth phase. Under starvation conditions, we observed massive transport of PtdIns(3) P into the vacuole. This accumulation was dependent on the membrane dynamics of autophagy. Notably, PtdIns(3) P was highly enriched and delivered into the vacuole as a component of autophagosome membranes but not as a cargo enclosed within them, implying direct involvement of this phosphoinositide in autophagosome formation. We also found a possible enrichment of PtdIns(3) P on the inner autophagosomal membrane compared to the outer membrane. Based on these results we discuss the function of PtdIns(3) P in autophagy.     

HIV glycoprotein gp120 inhibits TCR-CD3-mediated activation of fyn and lck

HIV major glycoprotein gp120 interacts with CD4 molecules and perturbs signaling through the TCR-CD3 complex. We examined the effects of gp120 on TCR-CD3-induced phosphorylation and activation of the src-type protein tyrosine kinases (PTK), fyn and lck. gp120 caused minimal changes in lck phosphorylation or lck enzymatic activity, but preincubation of Jurkat cells with gp120 for 20 min strongly inhibited TCR-CD3-mediated phosphorylation and activation of lck and fyn, as well as phosphorylation of CD3 zeta. Inhibition of TCR-CD3 signaling in T cells preincubated with gp120 was paralleled by inhibition of T cell proliferation to the antigen tetanus toxoid. Neither surface CD4 expression nor CD4-lck association was affected by gp120. Furthermore, gp120 inhibited lck phosphorylation induced by cross-linking of TCR-CD3 and CD4 suggesting that the inhibition of lck phosphorylation could not be simply accounted for by sequestration of CD4 molecules. gp120 selectively enhanced the phosphorylation of the lck peptide containing the autoinhibitory tyrosine residue Tyr505 relative to the lck peptide containing the positive regulatory residue Tyr394, suggesting that a qualitative alteration in lck may underlie the inhibition of TCR-CD3 signaling by gp120.      

The extracellular part of zeta is buried in the T cell antigen receptor complex

The zeta chain is a key component of the T cell antigen receptor (TCR-CD3) complex, required for the expression of the receptor on the cell surface. It contains an extremely small extracellular (EC) part of nine amino acids. Interestingly, the length, but not the sequence, of the zeta EC has been highly conserved through evolution. Here, we examined the effect of increasing the length of human zeta EC on TCR-CD3 assembly and surface expression. Appending a 30 kDa polypeptide to the N-terminus of zeta completely abolished assembly and transport of the TCR-CD3 to the cell surface. Addition of only 17 amino acids, including the HA-tag (HAzeta), strongly reduced the efficiency of TCR-CD3 assembly and led to reduced expression on the surface, suggesting that the short zeta EC region is located within the receptor complex. In Blue Native gels (BN-PAGE) these receptors had a normal size, indicating that they have a stoichiometry of alphabetagammaepsilondeltaepsilonzetazeta. In resting TCR-CD3s the HA-tag, and thus the zeta EC region, was not accessible for anti-HA antibody binding, demonstrating that it was indeed buried in a cavity within the receptor complex. However, prolonged stimulation with antigen permitted the access of the anti-HA antibody, thus suggesting that stimulation led to architectural changes in the TCR-CD3.     

Molecular mechanisms in the TCR (TCR alpha beta-CD3 delta epsilon, gamma epsilon) interaction with zeta 2 homodimers: clues from a 'phenotypic revertant' clone.

The association between the TCRalphabeta-CD3gammaepsilondeltaepsilon hexamers and zeta2 homodimers in the endoplasmic reticulum (ER) constitutes a key step in TCR assembly and export to the T cell surface. Incompletely assembled TCR-CD3 complexes are degraded in the ER or the lysosomes. A previously described Jurkat variant (J79) has a mutation at position 195 on the TCR Calpha domain causing a phenylalanine to valine exchange. This results in a lack of association between TCRalphabeta-CD3gammaepsilondeltaepsilon hexamers and zeta2 homodimers. Two main hypotheses could explain this phenomenon in J79 cells: TCR-CD3 hexamers may be incapable of interacting with zeta2 due to a structural change in the TCR Calpha region; alternatively, TCR-CD3 hexamers may be incapable of interacting with zeta2 due to factors unrelated to either molecular complex. In order to assess these two possibilities, the TCR-CD3 membrane-negative J79 cells were treated with ethylmethylsulfonate and clones positive for TCR membrane expression were isolated. The characterization of the J79r58 phenotypic revertant cell line is the subject of this study. The main question was to assess the reason for the TCR re-expression. The TCR on J79r58 cells appears qualitatively and functionally equivalent to wild-type TCR complexes. Nucleotide sequence analysis confirmed the presence of the original mutation in the TCR Calpha region but failed to detect compensatory mutations in alpha, beta, gamma, delta, epsilon or zeta chains. Thus, mutated J79-TCR-CD3 complexes can interact with zeta2 homodimers. Possible mechanisms for the unsuccessful TCR-CD3 interaction with zeta2 homodimers are presented and discussed.     

Asymmetrical protein kinase A activity establishes neutrophil cytoskeletal polarity and enables chemotaxis

Neutrophil chemotaxis requires precise spatial organization of the actin cytoskeleton and integrin activation to polarize the cell and enable migration. Protein kinase A (PKA) activity regulates integrin activation and actin cytoskeletal organization, suggesting that PKA is a key element in the mechanism regulating neutrophil chemotaxis. Our hypothesis is that asymmetrical PKA activity is critical for establishing neutrophil adhesive and cytoskeletal polarity required for migration during chemotaxis. To test this hypothesis, we first determined that global treatment with the PKA inhibitor KT5720 decreased formylated Met-Leu-Phe (fMLF)-induced migration. The ability of PKA inhibitors to reduce migration correlated with increased overall beta2 integrin cell-surface expression, affinity activation, and cellular adhesion. We next determined whether asymmetrical PKA activity was sufficient to induce migration. Exposure to gradient of the PKA inhibitors KT5720 or H-89 or a stearated, cell-permeant peptide (St-Ht31), which inhibits PKA binding to anchorage proteins, stimulated neutrophil migration in a chemotaxis chamber. Global treatment with KT5720 abolished the ability of fMLF to polarize the neutrophil actin cytoskeleton. In contrast to global treatment with KT5720, a point source of KT5720 was sufficient to polarize the actin cytoskeleton. The ability of KT5720 and St-Ht31 to stimulate migration was abolished by pretreatment with the phosphatidylinositol-3 kinase (PI-3K) inhibitors wortmannin and LY294002. These data suggest that asymmetrical PKA activity is necessary and sufficient for actin cytoskeletal polarization and migration during neutrophil chemotaxis. In addition, our data suggest PI-3K is an effector of PKA during chemotaxis.     

Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils.

Leukocyte chemoattractants are known to stimulate signaling pathways that involve Rho family GTPases. Direct evidence for the regulation of the leukocyte cytoskeleton by Rho GTPases and their effector targets is limited. The p21-activated kinases (PAKs) are specific targets of activated GTP-bound Rac and Cdc42, and have been proposed as regulators of chemoattractant-driven actin cytoskeletal changes in fibroblasts. PAK1 colocalizes with F-actin to cortical actin structures in stimulated fibroblasts, and activated PAK1 mutants induce membrane ruffling and polarized cytoskeletal rearrangements. We investigated whether PAK1 was associated with remodeling of the actin cytoskeleton in activated human neutrophils. We monitored the redistribution of PAK1 and F-actin into the actin cytoskeleton after stimulation of human neutrophils with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) or the particulate stimulus, opsonized zymosan (OZ). PAK1 exhibited a similar distribution as F-actin in fMLP-stimulated leukocytes, localizing in membrane ruffles and to lamellipodia at the leading edge of polarized cells. Addition of OZ induced phagocytic uptake of this particulate stimulus, and PAK1 re-localized to the F-actin-rich pseudopodia and phagocytic cups associated with this process. Once the OZ was internalized, there was little PAK1 localized around the ingested particles, suggesting that PAK1 may be regulating the cytoskeletal extensions and events required for engulfment of bacteria, but not the subsequent steps of internalization. Localization of PAK1 and F-actin in cytoskeletal structures was abolished by the actin polymerization inhibitor cytochalasin D and the phosphatidylinositol 3-kinase inhibitor wortmannin. Our data suggest that PAK1 may regulate a subset of cytoskeletal dynamics initiated by chemoattractant and phagocytic stimuli in human neutrophils.     

Involvement of phosphatidylinositol 5-phosphate in insulin-stimulated glucose uptake in the L6 myotube model of skeletal muscle

The phosphoinositide phospholipid PtdIns5P has previously been implicated in insulin-stimulated translocation of the glucose transporter GLUT4 into the plasma membrane of adipocytes, but its potential role in glucose transport in muscle has not been explored. The involvement of PtdIns5P in insulin-stimulated glucose uptake was therefore investigated in myotubes of the skeletal muscle cell line L6. Stimulation with insulin produced a transient increase in PtdIns5P, which was abolished by the over-expression of the highly active PtdIns5P 4-kinase PIP4Kα. PIP4Kα over-expression also abolished both the enhanced glucose uptake and the robust peak of PtdIns(3,4,5)P ₃ production stimulated by insulin in myotubes. Delivery of exogenous PtdIns5P into unstimulated myotubes increased Akt phosphorylation, promoted GLUT4 relocalisation from internal membrane to plasma membrane fractions and its association with plasma membrane lawns and also stimulated glucose uptake in a tyrosine kinase and phosphoinositide 3-kinase (PI 3-kinase)-dependent fashion. Our results are consistent with a role for insulin-stimulated PtdIns5P production in regulating glucose transport by promoting PI 3-kinase signalling.     

Effects of wortmannin and latrunculin A on slow endocytosis at the frog neuromuscular junction

Phosphoinositides are key regulators of synaptic vesicle cycling and endocytic traffic; the actin cytoskeleton also seems to be involved in modulating these processes. We investigated the effects of perturbing phosphoinositide signalling and actin dynamics on vesicle cycling in frog motor nerve terminals, using fluorescence and electron microscopy, and electrophysiology. Antibody staining for β-actin revealed that actin surrounds but does not overlap with synaptic vesicle clusters. Latrunculin A, which disrupts actin filaments by binding actin monomers, and wortmannin, an inhibitor of phosphatidyl inositol-3-kinase (PI3-kinase), each disrupted the pattern of presynaptic actin staining, but not vesicle clusters in resting terminals. Latrunculin A, but not wortmannin, also reduced vesicle mobilization and exocytosis. Both drugs inhibited the stimulation-induced uptake of the styryl dye FM1-43 and blocked vesicle reformation from internalized membrane objects after tetanic stimulation. These results are consistent with a role of PI3-kinase and the actin cytoskeleton in the slow pathway of vesicle endocytosis, used primarily by reserve pool vesicles.     

Focal adhesion kinase is involved in type III group B streptococcal invasion of human brain microvascular endothelial cells

Group B streptococcus (GBS), the leading cause of neonatal meningitis, has been shown to invade human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. GBS invasion of HBMEC has been shown to require the host cell actin cytoskeleton rearrangements. The present study examined the mechanisms underlying actin cytoskeleton rearrangements that are involved in type III GBS invasion of HBMEC. We showed that type III GBS invasion was inhibited by genistein, a general tyrosine kinase inhibitor (mean 54% invasion decrease at 100 microM), and LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor (mean 70% invasion decrease at 50 microM), but not by PP2, an inhibitor of the Src family tyrosine kinases. We subsequently showed that the focal adhesion kinase (FAK) was the one of the host proteins tyrosine phosphorylated by type III GBS. Over-expression of a dominant negative form of the FAK C-terminal domain significantly decreased type III GBS invasion of HBMEC (mean 51% invasion decrease). In addition, we showed that FAK phosphorylation correlated with its association of paxillin, an adapter protein of actin filament, and PI3-kinase subunit p85. This is the first demonstration that FAK phosphorylation and its association with paxillin and PI3 kinase play a key role in type III GBS invasion of HBMEC.     

Inhibition of Cytoskeletal Assembly by Cytochalasin B Prevents Signaling Through Tyrosine Phosphorylation and Secretion Triggered by Collagen but Not by Thrombin

Activation of platelets leads to cytoskeletal assembly that is responsible for platelet motility and internal contraction. We have evaluated the involvement of the cytoskeleton in platelet activation by two strong agonists, collagen and thrombin. Activation was assessed by measuring changes in cytoskeletal assembly, externalization of activation-dependent markers and expression of procoagulant activity, and tyrosine phosphorylation of proteins, in both the absence and the presence of cytochalasin B. Activation of platelets with collagen and thrombin induced morphological changes and increased the expression of CD62P, CD63, glycoprotein IV, and binding of annexin V to platelets. Moreover, both activating agents induced actin polymerization, increased the association of other contractile proteins, and promoted tyrosine phosphorylation of multiple proteins, some of which were associated with the cytoskeleton. The presence of cytochalasin B blocked the previous events when collagen was used as the activating agent, although binding of annexin V still occurred. In contrast, platelet response to thrombin was not completely prevented by the presence of cytochalasin B. Thus, activation by collagen requires a functional cytoskeleton to trigger signaling through tyrosine phosphorylation and secretion. This is not the case for thrombin, which is capable of activating signaling mechanisms in the presence of strong inhibitors of cytoskeletal assembly. Moreover, the expression of a procoagulant surface in platelets still occurs even when platelet motility has been inhibited.     

Immune pathology associated with altered actin cytoskeleton regulation

The actin cytoskeleton plays a crucial role in a variety of important cellular processes required for normal immune function, including locomotion, intercellular interactions, endocytosis, cytokinesis, signal transduction, and maintenance of cell morphology. Recent studies have uncovered not only many of the components and mechanisms that regulate the cortical actin cytoskeleton but have also revealed significant immunopathological consequences associated with genetic alteration of actin cytoskeletal regulatory genes. These advances have provided new insights into the role of cortical actin cytoskeletal regulation in a number of immune cell functions and have identified cytoskeletal regulatory proteins critical for normal immune system activity and susceptibility to autoimmunity.