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Translocation of PKC[theta] in T cells is mediated by a nonconventional, PI3-K- and Vav-dependent pathway, but does not absolutely require phospholipase C.

Villalba M, Bi K, Hu J, Altman Y, Bushway P, Reits E, Neefjes J, Baier G, Abraham RT, Altman A - J. Cell Biol. (2002)

Bottom Line: Using three independent approaches, i.e., a selective PLC inhibitor, a PLCgamma1-deficient T cell line, or a dominant negative PLCgamma1 mutant, we demonstrate that CD3/CD28-induced membrane recruitment and COOH-terminal phosphorylation of PKCtheta are largely independent of PLC.Membrane or lipid raft recruitment of PKCtheta (but not PKCalpha) was absent in T cells treated with phosphatidylinositol 3-kinase (PI3-K) inhibitors or in Vav-deficient T cells, and was enhanced by constitutively active PI3-K. 3-phosphoinositide-dependent kinase-1 (PDK1) also upregulated the membrane translocation of PKCtheta;, but did not associate with it.These results provide evidence that a nonconventional PI3-K- and Vav-dependent pathway mediates the selective membrane recruitment and, possibly, activation of PKCtheta in T cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA.

ABSTRACT
PKCtheta plays an essential role in activation of mature T cells via stimulation of AP-1 and NF-kappaB, and is known to selectively translocate to the immunological synapse in antigen-stimulated T cells. Recently, we reported that a Vav/Rac pathway which depends on actin cytoskeleton reorganization mediates selective recruitment of PKCtheta to the membrane or cytoskeleton and its catalytic activation by anti-CD3/CD28 costimulation. Because this pathway acted selectively on PKCtheta, we addressed here the question of whether the translocation and activation of PKCtheta in T cells is regulated by a unique pathway distinct from the conventional mechanism for PKC activation, i.e., PLC-mediated production of DAG. Using three independent approaches, i.e., a selective PLC inhibitor, a PLCgamma1-deficient T cell line, or a dominant negative PLCgamma1 mutant, we demonstrate that CD3/CD28-induced membrane recruitment and COOH-terminal phosphorylation of PKCtheta are largely independent of PLC. In contrast, the same inhibitory strategies blocked the membrane translocation of PKCalpha. Membrane or lipid raft recruitment of PKCtheta (but not PKCalpha) was absent in T cells treated with phosphatidylinositol 3-kinase (PI3-K) inhibitors or in Vav-deficient T cells, and was enhanced by constitutively active PI3-K. 3-phosphoinositide-dependent kinase-1 (PDK1) also upregulated the membrane translocation of PKCtheta;, but did not associate with it. These results provide evidence that a nonconventional PI3-K- and Vav-dependent pathway mediates the selective membrane recruitment and, possibly, activation of PKCtheta in T cells.

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Vav is required for PKCθ translocation to the cap-like membrane structures. (A) Lymph node–derived T cells from Vav−/− or wild type mice were activated with anti-CD3 for 5 d and rested for two additional days. The cells were then plated over poly-l-lysine–coated glass slides and left unstimulated (resting) or stimulated for 10 min with anti-CD3 plus anti-CD28 antibodies. The cells were fixed and analyzed for PKCθ (red) or F-actin (green) localization. The overlay images are also shown. Arrowheads indicate T cells with capped PKCθ and F-actin. One cell labeled with an arrow in each left panel is magnified threefold in the right panels. The bars in the lower right micrograph correspond to 10 μ. (B) Quantitation of the results displayed in A. These results represent the average of two experiments, in which at least 100 cells were evaluated for PKCθ or F-actin membrane clustering.
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fig4: Vav is required for PKCθ translocation to the cap-like membrane structures. (A) Lymph node–derived T cells from Vav−/− or wild type mice were activated with anti-CD3 for 5 d and rested for two additional days. The cells were then plated over poly-l-lysine–coated glass slides and left unstimulated (resting) or stimulated for 10 min with anti-CD3 plus anti-CD28 antibodies. The cells were fixed and analyzed for PKCθ (red) or F-actin (green) localization. The overlay images are also shown. Arrowheads indicate T cells with capped PKCθ and F-actin. One cell labeled with an arrow in each left panel is magnified threefold in the right panels. The bars in the lower right micrograph correspond to 10 μ. (B) Quantitation of the results displayed in A. These results represent the average of two experiments, in which at least 100 cells were evaluated for PKCθ or F-actin membrane clustering.

Mentions: Next, we decided to study the components of the unique pathway involved in the membrane translocation of PKCθ. First, we examined the role of Vav by comparing T cells from wild-type versus Vav-deficient T cells (Fig. 4). F-actin localization was determined in parallel. In order to expand the T cell population from the vav−/− mice, their lymph node cells were activated with an anti-CD3 mAb in the presence of IL-2, and then rested prior to restimulation. In T cells derived from vav+/+ mice, combined CD3/CD28 engagement induced actin polymerization, with a tendency of F-actin to polarize in a cap-like structure in a fraction of the cells. In agreement with previous results (Fischer et al., 1998; Holsinger et al., 1998), this outcome was clearly reduced in stimulated T cells derived from vav−/− mice (Fig. 4 A).


Translocation of PKC[theta] in T cells is mediated by a nonconventional, PI3-K- and Vav-dependent pathway, but does not absolutely require phospholipase C.

Villalba M, Bi K, Hu J, Altman Y, Bushway P, Reits E, Neefjes J, Baier G, Abraham RT, Altman A - J. Cell Biol. (2002)

Vav is required for PKCθ translocation to the cap-like membrane structures. (A) Lymph node–derived T cells from Vav−/− or wild type mice were activated with anti-CD3 for 5 d and rested for two additional days. The cells were then plated over poly-l-lysine–coated glass slides and left unstimulated (resting) or stimulated for 10 min with anti-CD3 plus anti-CD28 antibodies. The cells were fixed and analyzed for PKCθ (red) or F-actin (green) localization. The overlay images are also shown. Arrowheads indicate T cells with capped PKCθ and F-actin. One cell labeled with an arrow in each left panel is magnified threefold in the right panels. The bars in the lower right micrograph correspond to 10 μ. (B) Quantitation of the results displayed in A. These results represent the average of two experiments, in which at least 100 cells were evaluated for PKCθ or F-actin membrane clustering.
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fig4: Vav is required for PKCθ translocation to the cap-like membrane structures. (A) Lymph node–derived T cells from Vav−/− or wild type mice were activated with anti-CD3 for 5 d and rested for two additional days. The cells were then plated over poly-l-lysine–coated glass slides and left unstimulated (resting) or stimulated for 10 min with anti-CD3 plus anti-CD28 antibodies. The cells were fixed and analyzed for PKCθ (red) or F-actin (green) localization. The overlay images are also shown. Arrowheads indicate T cells with capped PKCθ and F-actin. One cell labeled with an arrow in each left panel is magnified threefold in the right panels. The bars in the lower right micrograph correspond to 10 μ. (B) Quantitation of the results displayed in A. These results represent the average of two experiments, in which at least 100 cells were evaluated for PKCθ or F-actin membrane clustering.
Mentions: Next, we decided to study the components of the unique pathway involved in the membrane translocation of PKCθ. First, we examined the role of Vav by comparing T cells from wild-type versus Vav-deficient T cells (Fig. 4). F-actin localization was determined in parallel. In order to expand the T cell population from the vav−/− mice, their lymph node cells were activated with an anti-CD3 mAb in the presence of IL-2, and then rested prior to restimulation. In T cells derived from vav+/+ mice, combined CD3/CD28 engagement induced actin polymerization, with a tendency of F-actin to polarize in a cap-like structure in a fraction of the cells. In agreement with previous results (Fischer et al., 1998; Holsinger et al., 1998), this outcome was clearly reduced in stimulated T cells derived from vav−/− mice (Fig. 4 A).

Bottom Line: Using three independent approaches, i.e., a selective PLC inhibitor, a PLCgamma1-deficient T cell line, or a dominant negative PLCgamma1 mutant, we demonstrate that CD3/CD28-induced membrane recruitment and COOH-terminal phosphorylation of PKCtheta are largely independent of PLC.Membrane or lipid raft recruitment of PKCtheta (but not PKCalpha) was absent in T cells treated with phosphatidylinositol 3-kinase (PI3-K) inhibitors or in Vav-deficient T cells, and was enhanced by constitutively active PI3-K. 3-phosphoinositide-dependent kinase-1 (PDK1) also upregulated the membrane translocation of PKCtheta;, but did not associate with it.These results provide evidence that a nonconventional PI3-K- and Vav-dependent pathway mediates the selective membrane recruitment and, possibly, activation of PKCtheta in T cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA.

ABSTRACT
PKCtheta plays an essential role in activation of mature T cells via stimulation of AP-1 and NF-kappaB, and is known to selectively translocate to the immunological synapse in antigen-stimulated T cells. Recently, we reported that a Vav/Rac pathway which depends on actin cytoskeleton reorganization mediates selective recruitment of PKCtheta to the membrane or cytoskeleton and its catalytic activation by anti-CD3/CD28 costimulation. Because this pathway acted selectively on PKCtheta, we addressed here the question of whether the translocation and activation of PKCtheta in T cells is regulated by a unique pathway distinct from the conventional mechanism for PKC activation, i.e., PLC-mediated production of DAG. Using three independent approaches, i.e., a selective PLC inhibitor, a PLCgamma1-deficient T cell line, or a dominant negative PLCgamma1 mutant, we demonstrate that CD3/CD28-induced membrane recruitment and COOH-terminal phosphorylation of PKCtheta are largely independent of PLC. In contrast, the same inhibitory strategies blocked the membrane translocation of PKCalpha. Membrane or lipid raft recruitment of PKCtheta (but not PKCalpha) was absent in T cells treated with phosphatidylinositol 3-kinase (PI3-K) inhibitors or in Vav-deficient T cells, and was enhanced by constitutively active PI3-K. 3-phosphoinositide-dependent kinase-1 (PDK1) also upregulated the membrane translocation of PKCtheta;, but did not associate with it. These results provide evidence that a nonconventional PI3-K- and Vav-dependent pathway mediates the selective membrane recruitment and, possibly, activation of PKCtheta in T cells.

Show MeSH
Related in: MedlinePlus