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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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PKCθ translocation is indirectly mediated by PDK1. (A) Jurkat E6.1 cells (106) were stimulated with anti-CD3 plus anti-CD28 antibodies or PMA for 8 min. Subcellular fractions were prepared and analyzed for endogenous PKCθ and PDK1 expression by immunoblotting with specific antibodies. (B) Jurkat-TAg cells were transiently cotransfected with an Xpress-tagged PKCθ expression vector plus c-Myc–tagged PDK1 or PDK1 plus VavΔPH. 40 h later, the cells were left unstimulated, or stimulated with anti-CD3 antibody. A fraction of the stimulated cells was pretreated for LY294002. Subcellular fractionation and immunoblotting were performed as in Fig. 5 and transfected PKCθ was detected by anti-Xpress immunoblotting. (C) Jurkat-TAg cells were transfected as in (B), and the cells were left unstimulated or stimulated with anti-CD3 or PMA. Transfected PKCθ and PDK1 expression in different fractions was determined by immunoblotting with the corresponding tag-specific antibodies.
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fig6: PKCθ translocation is indirectly mediated by PDK1. (A) Jurkat E6.1 cells (106) were stimulated with anti-CD3 plus anti-CD28 antibodies or PMA for 8 min. Subcellular fractions were prepared and analyzed for endogenous PKCθ and PDK1 expression by immunoblotting with specific antibodies. (B) Jurkat-TAg cells were transiently cotransfected with an Xpress-tagged PKCθ expression vector plus c-Myc–tagged PDK1 or PDK1 plus VavΔPH. 40 h later, the cells were left unstimulated, or stimulated with anti-CD3 antibody. A fraction of the stimulated cells was pretreated for LY294002. Subcellular fractionation and immunoblotting were performed as in Fig. 5 and transfected PKCθ was detected by anti-Xpress immunoblotting. (C) Jurkat-TAg cells were transfected as in (B), and the cells were left unstimulated or stimulated with anti-CD3 or PMA. Transfected PKCθ and PDK1 expression in different fractions was determined by immunoblotting with the corresponding tag-specific antibodies.

Mentions: When Jurkat T cells were stimulated with a combination of anti-CD3 plus -CD28 antibodies (or with PMA), endogenous PKCθ was clearly translocated to the membrane fraction; however, under the same conditions we could not detect similar translocation of endogenous PDK1 (Fig. 6 A), indicating that in T cells, PDK1 intracellular localization is not regulated by TCR/CD28 stimulation. To assess more directly whether PDK1 can influence the translocation of PKCθ, we cotransfected Jurkat-TAg cells with PKCθ plus PDK1 expression vectors. PDK1 coexpression enhanced the membrane and cytoskeleton translocation of PKCθ, and this effect was only partially sensitive to a PI3-K inhibitor (Fig. 6, B and C). Interestingly, this enhanced PDK1-induced translocation of PKCθ was largely reversed by coexpression of the dominant negative (ΔPH) Vav mutant. Even under these overexpression conditions, no PDK1 was detected in the membrane and cytoskeletal fractions of the stimulated cells (Fig. 6 C). In other, functional assays we found that coexpression of PDK1 with PKCθ did not enhanced the PKCθ-induced activation of NF-κB and AP-1 reporter genes (unpublished data). These results suggest that, although PDK1 may be involved in the maturation (perhaps via activation loop phosphorylation; Bauer et al., 2001) of PKCθ in a similar manner to other PKC enzymes (Chou et al., 1998; Dutil et al., 1998; Le Good et al., 1998; Dutil and Newton, 2000; Toker and Newton, 2000), it does not directly translocate PKCθ to the membrane by associating with it in T cells.


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)

PKCθ translocation is indirectly mediated by PDK1. (A) Jurkat E6.1 cells (106) were stimulated with anti-CD3 plus anti-CD28 antibodies or PMA for 8 min. Subcellular fractions were prepared and analyzed for endogenous PKCθ and PDK1 expression by immunoblotting with specific antibodies. (B) Jurkat-TAg cells were transiently cotransfected with an Xpress-tagged PKCθ expression vector plus c-Myc–tagged PDK1 or PDK1 plus VavΔPH. 40 h later, the cells were left unstimulated, or stimulated with anti-CD3 antibody. A fraction of the stimulated cells was pretreated for LY294002. Subcellular fractionation and immunoblotting were performed as in Fig. 5 and transfected PKCθ was detected by anti-Xpress immunoblotting. (C) Jurkat-TAg cells were transfected as in (B), and the cells were left unstimulated or stimulated with anti-CD3 or PMA. Transfected PKCθ and PDK1 expression in different fractions was determined by immunoblotting with the corresponding tag-specific antibodies.
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Related In: Results  -  Collection

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fig6: PKCθ translocation is indirectly mediated by PDK1. (A) Jurkat E6.1 cells (106) were stimulated with anti-CD3 plus anti-CD28 antibodies or PMA for 8 min. Subcellular fractions were prepared and analyzed for endogenous PKCθ and PDK1 expression by immunoblotting with specific antibodies. (B) Jurkat-TAg cells were transiently cotransfected with an Xpress-tagged PKCθ expression vector plus c-Myc–tagged PDK1 or PDK1 plus VavΔPH. 40 h later, the cells were left unstimulated, or stimulated with anti-CD3 antibody. A fraction of the stimulated cells was pretreated for LY294002. Subcellular fractionation and immunoblotting were performed as in Fig. 5 and transfected PKCθ was detected by anti-Xpress immunoblotting. (C) Jurkat-TAg cells were transfected as in (B), and the cells were left unstimulated or stimulated with anti-CD3 or PMA. Transfected PKCθ and PDK1 expression in different fractions was determined by immunoblotting with the corresponding tag-specific antibodies.
Mentions: When Jurkat T cells were stimulated with a combination of anti-CD3 plus -CD28 antibodies (or with PMA), endogenous PKCθ was clearly translocated to the membrane fraction; however, under the same conditions we could not detect similar translocation of endogenous PDK1 (Fig. 6 A), indicating that in T cells, PDK1 intracellular localization is not regulated by TCR/CD28 stimulation. To assess more directly whether PDK1 can influence the translocation of PKCθ, we cotransfected Jurkat-TAg cells with PKCθ plus PDK1 expression vectors. PDK1 coexpression enhanced the membrane and cytoskeleton translocation of PKCθ, and this effect was only partially sensitive to a PI3-K inhibitor (Fig. 6, B and C). Interestingly, this enhanced PDK1-induced translocation of PKCθ was largely reversed by coexpression of the dominant negative (ΔPH) Vav mutant. Even under these overexpression conditions, no PDK1 was detected in the membrane and cytoskeletal fractions of the stimulated cells (Fig. 6 C). In other, functional assays we found that coexpression of PDK1 with PKCθ did not enhanced the PKCθ-induced activation of NF-κB and AP-1 reporter genes (unpublished data). These results suggest that, although PDK1 may be involved in the maturation (perhaps via activation loop phosphorylation; Bauer et al., 2001) of PKCθ in a similar manner to other PKC enzymes (Chou et al., 1998; Dutil et al., 1998; Le Good et al., 1998; Dutil and Newton, 2000; Toker and Newton, 2000), it does not directly translocate PKCθ to the membrane by associating with it in T cells.

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