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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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Inhibition of PLC does not block PKCθ COOH-terminal phosphorylation. Jurkat T cells (2 × 106) were left unstimulated or stimulated with anti-CD3 plus anti-CD28 antibodies (1 μg/ml each) for the indicated times. Cell aliquots were preincubated for 1 h with U73122 or U73343 (10 μM). Cytosol and insoluble fractions were prepared, resolved by SDS-PAGE, and blotted with anti-phospho-PKCθ (p-PKCθ) or anti-PKCθ antibodies. The insoluble fraction represents the combined membrane and cytoskeleton fractions, which was not further fractionated in order to minimize dephosphorylation of p-PKCθ.
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fig2: Inhibition of PLC does not block PKCθ COOH-terminal phosphorylation. Jurkat T cells (2 × 106) were left unstimulated or stimulated with anti-CD3 plus anti-CD28 antibodies (1 μg/ml each) for the indicated times. Cell aliquots were preincubated for 1 h with U73122 or U73343 (10 μM). Cytosol and insoluble fractions were prepared, resolved by SDS-PAGE, and blotted with anti-phospho-PKCθ (p-PKCθ) or anti-PKCθ antibodies. The insoluble fraction represents the combined membrane and cytoskeleton fractions, which was not further fractionated in order to minimize dephosphorylation of p-PKCθ.

Mentions: Activation of PKC enzymes is associated with their auto- or heterophosphorylation, events that regulate the enzymatic activity (Newton, 1997; Parekh et al., 2000). Although the regulation of PKCθ localization and/or activity by phosphorylation has not been analyzed in detail, a recent study indicated that an antibody specific for phosphorylated Thr-538 in the activation loop of PKCθ reacted specifically with the active, membrane-localized fraction of PKCθ (Bauer et al., 2001). We used another antibody specific for Ser-695 in the COOH-terminal tail of PKCθ, which is a potential autophosphorylation (Keranen et al., 1995) or heterophosphorylation (Ziegler et al., 1999; Parekh et al., 2000) site based on its homology with other PKC enzymes in order to assess the role of PLCγ1 in PKC phosphorylation. This site has very recently been implicated as a positive regulatory site in PKCθ (Liu et al., 2002). As expected, this antibody did not recognize PKCθ in unstimulated T cells, even though PKCθ was readily detected by a PKCθ-specific antibody (Fig. 2, two top panels). Anti-CD3 plus anti-CD28 stimulation induced the expected translocation of PKCθ to the insoluble fraction, which represents the pooled membranes and cytoskeleton. Unlike the PKCθ-specific antibody, the phospho-PKCθ–specific antibody only recognized PKCθ from activated cells, which was exclusively associated with the insoluble fraction. Importantly, pretreatment of the cells with a selective PLC inhibitor (U73122, two middle panels) or its nonfunctional analog (U73343, two bottom panels) had no significant effect on the induction and membrane translocation of phospho-PKCθ (Fig. 2).


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)

Inhibition of PLC does not block PKCθ COOH-terminal phosphorylation. Jurkat T cells (2 × 106) were left unstimulated or stimulated with anti-CD3 plus anti-CD28 antibodies (1 μg/ml each) for the indicated times. Cell aliquots were preincubated for 1 h with U73122 or U73343 (10 μM). Cytosol and insoluble fractions were prepared, resolved by SDS-PAGE, and blotted with anti-phospho-PKCθ (p-PKCθ) or anti-PKCθ antibodies. The insoluble fraction represents the combined membrane and cytoskeleton fractions, which was not further fractionated in order to minimize dephosphorylation of p-PKCθ.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199257&req=5

fig2: Inhibition of PLC does not block PKCθ COOH-terminal phosphorylation. Jurkat T cells (2 × 106) were left unstimulated or stimulated with anti-CD3 plus anti-CD28 antibodies (1 μg/ml each) for the indicated times. Cell aliquots were preincubated for 1 h with U73122 or U73343 (10 μM). Cytosol and insoluble fractions were prepared, resolved by SDS-PAGE, and blotted with anti-phospho-PKCθ (p-PKCθ) or anti-PKCθ antibodies. The insoluble fraction represents the combined membrane and cytoskeleton fractions, which was not further fractionated in order to minimize dephosphorylation of p-PKCθ.
Mentions: Activation of PKC enzymes is associated with their auto- or heterophosphorylation, events that regulate the enzymatic activity (Newton, 1997; Parekh et al., 2000). Although the regulation of PKCθ localization and/or activity by phosphorylation has not been analyzed in detail, a recent study indicated that an antibody specific for phosphorylated Thr-538 in the activation loop of PKCθ reacted specifically with the active, membrane-localized fraction of PKCθ (Bauer et al., 2001). We used another antibody specific for Ser-695 in the COOH-terminal tail of PKCθ, which is a potential autophosphorylation (Keranen et al., 1995) or heterophosphorylation (Ziegler et al., 1999; Parekh et al., 2000) site based on its homology with other PKC enzymes in order to assess the role of PLCγ1 in PKC phosphorylation. This site has very recently been implicated as a positive regulatory site in PKCθ (Liu et al., 2002). As expected, this antibody did not recognize PKCθ in unstimulated T cells, even though PKCθ was readily detected by a PKCθ-specific antibody (Fig. 2, two top panels). Anti-CD3 plus anti-CD28 stimulation induced the expected translocation of PKCθ to the insoluble fraction, which represents the pooled membranes and cytoskeleton. Unlike the PKCθ-specific antibody, the phospho-PKCθ–specific antibody only recognized PKCθ from activated cells, which was exclusively associated with the insoluble fraction. Importantly, pretreatment of the cells with a selective PLC inhibitor (U73122, two middle panels) or its nonfunctional analog (U73343, two bottom panels) had no significant effect on the induction and membrane translocation of phospho-PKCθ (Fig. 2).

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