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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-induced PKCθ translocation does not depend on PLCγ1 activity. (A) Jurkat-TAg cells were cotransfected with the indicated combinations of empty vector (Vector) or Vav (5 μg each), and/or dominant negative PLCγ1 (PLCz; 15 μg) together with a PKCθ regulatory domain-GFP expression plasmid (5 μg). After 2 d, cells were fixed and GFP localization was analyzed by confocal imaging. A portion of the cells in each group was stimulated for the final 10 min of culture with anti-CD3 (1 μg/ml). PKCθ-GFP (green) and F-actin (red) localization was analyzed by confocal microscopy. The right column panels in the resting or stimulated groups represent a threefold enlargement of a single cell marked with an arrow in the middle column panels. The bars in the lower right micrograph correspond to 20 μ. (B) PLCz blocks Vav- or anti-CD3–induced NFAT activation. Jurkat-TAg cells (10 × 106) were transfected with the indicated combinations of Vav (5 μg) and/or PLCz (15 μg) in the presence of NFAT-Luc (5 μg) and β-Gal (1.5 μg). Cells were left unstimulated or stimulated for the final 6 h of culture with anti-CD3 or with PMA (100 ng/ml) plus ionomycin (1 μg/ml). Luciferase activity was determined after 48 h of culture, and normalized to the activity of a cotransfected β-galactosidase plasmid. Data represent percentage of the response induced by PMA plus ionomycin, and are average ± standard deviation of two experiments performed in duplicate.
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fig3: Vav-induced PKCθ translocation does not depend on PLCγ1 activity. (A) Jurkat-TAg cells were cotransfected with the indicated combinations of empty vector (Vector) or Vav (5 μg each), and/or dominant negative PLCγ1 (PLCz; 15 μg) together with a PKCθ regulatory domain-GFP expression plasmid (5 μg). After 2 d, cells were fixed and GFP localization was analyzed by confocal imaging. A portion of the cells in each group was stimulated for the final 10 min of culture with anti-CD3 (1 μg/ml). PKCθ-GFP (green) and F-actin (red) localization was analyzed by confocal microscopy. The right column panels in the resting or stimulated groups represent a threefold enlargement of a single cell marked with an arrow in the middle column panels. The bars in the lower right micrograph correspond to 20 μ. (B) PLCz blocks Vav- or anti-CD3–induced NFAT activation. Jurkat-TAg cells (10 × 106) were transfected with the indicated combinations of Vav (5 μg) and/or PLCz (15 μg) in the presence of NFAT-Luc (5 μg) and β-Gal (1.5 μg). Cells were left unstimulated or stimulated for the final 6 h of culture with anti-CD3 or with PMA (100 ng/ml) plus ionomycin (1 μg/ml). Luciferase activity was determined after 48 h of culture, and normalized to the activity of a cotransfected β-galactosidase plasmid. Data represent percentage of the response induced by PMA plus ionomycin, and are average ± standard deviation of two experiments performed in duplicate.

Mentions: Based on recent findings that a functional interaction between the Vav/Rac pathway and PKCθ is required for T cell activation (Dienz et al., 2000; Hehner et al., 2000; Villalba et al., 2000a; Moller et al., 2001), we considered the Vav/Rac pathway as a candidate for a selective PLCγ1-independent mechanism that recruits PKCθ to the membrane. Therefore, we next used a dominant negative PLCγ1 mutant (PLCz), which was previously found to inhibit PLCγ1-dependent functions in various cells (Chen et al., 1996), to investigate whether the Vav-induced PKCθ translocation depends on PLCγ1. Cells were cotransfected with the regulatory domain of PKCθ fused to the NH2 terminus of green fluorescent protein (GFP) (Villalba et al., 2000a) plus combinations of empty vector, PLCz, and/or wild-type Vav. The intracellular localization of GFP (PKCθ) and polymerized actin (F-actin) were analyzed by confocal microscopy (Fig. 3 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-induced PKCθ translocation does not depend on PLCγ1 activity. (A) Jurkat-TAg cells were cotransfected with the indicated combinations of empty vector (Vector) or Vav (5 μg each), and/or dominant negative PLCγ1 (PLCz; 15 μg) together with a PKCθ regulatory domain-GFP expression plasmid (5 μg). After 2 d, cells were fixed and GFP localization was analyzed by confocal imaging. A portion of the cells in each group was stimulated for the final 10 min of culture with anti-CD3 (1 μg/ml). PKCθ-GFP (green) and F-actin (red) localization was analyzed by confocal microscopy. The right column panels in the resting or stimulated groups represent a threefold enlargement of a single cell marked with an arrow in the middle column panels. The bars in the lower right micrograph correspond to 20 μ. (B) PLCz blocks Vav- or anti-CD3–induced NFAT activation. Jurkat-TAg cells (10 × 106) were transfected with the indicated combinations of Vav (5 μg) and/or PLCz (15 μg) in the presence of NFAT-Luc (5 μg) and β-Gal (1.5 μg). Cells were left unstimulated or stimulated for the final 6 h of culture with anti-CD3 or with PMA (100 ng/ml) plus ionomycin (1 μg/ml). Luciferase activity was determined after 48 h of culture, and normalized to the activity of a cotransfected β-galactosidase plasmid. Data represent percentage of the response induced by PMA plus ionomycin, and are average ± standard deviation of two experiments performed in duplicate.
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fig3: Vav-induced PKCθ translocation does not depend on PLCγ1 activity. (A) Jurkat-TAg cells were cotransfected with the indicated combinations of empty vector (Vector) or Vav (5 μg each), and/or dominant negative PLCγ1 (PLCz; 15 μg) together with a PKCθ regulatory domain-GFP expression plasmid (5 μg). After 2 d, cells were fixed and GFP localization was analyzed by confocal imaging. A portion of the cells in each group was stimulated for the final 10 min of culture with anti-CD3 (1 μg/ml). PKCθ-GFP (green) and F-actin (red) localization was analyzed by confocal microscopy. The right column panels in the resting or stimulated groups represent a threefold enlargement of a single cell marked with an arrow in the middle column panels. The bars in the lower right micrograph correspond to 20 μ. (B) PLCz blocks Vav- or anti-CD3–induced NFAT activation. Jurkat-TAg cells (10 × 106) were transfected with the indicated combinations of Vav (5 μg) and/or PLCz (15 μg) in the presence of NFAT-Luc (5 μg) and β-Gal (1.5 μg). Cells were left unstimulated or stimulated for the final 6 h of culture with anti-CD3 or with PMA (100 ng/ml) plus ionomycin (1 μg/ml). Luciferase activity was determined after 48 h of culture, and normalized to the activity of a cotransfected β-galactosidase plasmid. Data represent percentage of the response induced by PMA plus ionomycin, and are average ± standard deviation of two experiments performed in duplicate.
Mentions: Based on recent findings that a functional interaction between the Vav/Rac pathway and PKCθ is required for T cell activation (Dienz et al., 2000; Hehner et al., 2000; Villalba et al., 2000a; Moller et al., 2001), we considered the Vav/Rac pathway as a candidate for a selective PLCγ1-independent mechanism that recruits PKCθ to the membrane. Therefore, we next used a dominant negative PLCγ1 mutant (PLCz), which was previously found to inhibit PLCγ1-dependent functions in various cells (Chen et al., 1996), to investigate whether the Vav-induced PKCθ translocation depends on PLCγ1. Cells were cotransfected with the regulatory domain of PKCθ fused to the NH2 terminus of green fluorescent protein (GFP) (Villalba et al., 2000a) plus combinations of empty vector, PLCz, and/or wild-type Vav. The intracellular localization of GFP (PKCθ) and polymerized actin (F-actin) were analyzed by confocal microscopy (Fig. 3 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