Limits...
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.

Show MeSH

Related in: MedlinePlus

PKCθ membrane translocation is independent of PLC activity. (A) Jurkat T cells (106) were stimulated with anti-CD3 plus anti-CD28 antibodies for 5 min. Aliquots of the cells were preincubated for 1 h with U73122 (10 μM) or with PP2 (10 μM). Cytosol (C), membrane (M), and detergent-insoluble (I) fractions were prepared, identical cell equivalents were resolved by SDS-PAGE, and the expression of PKCθ and PKCα in each fraction was determined by immunoblotting with specific antibodies. (B) Activated human peripheral blood T cells (5 × 106) were deprived of anti-CD3 antibody for 36 h, and then restimulated with anti-CD3 plus anti-CD28 antibodies for 10 min. Aliquots of the cells were preincubated for one hr with U73122 (10 μM) or LY294002 (50 μM). Subcellular fractions were prepared and analyzed as in A. (C) J.γ1 (a PLCγ1-deficient Jurkat cell line) or J.γ1.WT-2 (PLCγ1-reconstituted J.γ1) cells were stimulated and analyzed as in A. These results are representative of three similar experiments. The membrane-to-cytosol (M/C) ratio of PKC expression in each group is displayed. In A, B, and C, the numbers above the autoradiograms represent the percentage of PKCθ or -α present in each fraction (C + M + I = 100% for each group of cells), as determined by NIH Image scanning densitometry. (D) PKCθ translocation to lipid rafts is present in PLCγ1-deficient Jurkat cells. J.γ1 or J.γ1.WT-2 (20 × 106) were left unstimulated (ns) or stimulated with anti-CD3 plus anti-CD28 antibodies. The cells were lysed and the detergent-insoluble fractions were separated from the soluble fractions. The distribution of PKCθ in each fraction was determined by immunoblotting with a specific antibody. (E) The same blot was stripped and blotted with a PLCγ1-specific antibody.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199257&req=5

fig1: PKCθ membrane translocation is independent of PLC activity. (A) Jurkat T cells (106) were stimulated with anti-CD3 plus anti-CD28 antibodies for 5 min. Aliquots of the cells were preincubated for 1 h with U73122 (10 μM) or with PP2 (10 μM). Cytosol (C), membrane (M), and detergent-insoluble (I) fractions were prepared, identical cell equivalents were resolved by SDS-PAGE, and the expression of PKCθ and PKCα in each fraction was determined by immunoblotting with specific antibodies. (B) Activated human peripheral blood T cells (5 × 106) were deprived of anti-CD3 antibody for 36 h, and then restimulated with anti-CD3 plus anti-CD28 antibodies for 10 min. Aliquots of the cells were preincubated for one hr with U73122 (10 μM) or LY294002 (50 μM). Subcellular fractions were prepared and analyzed as in A. (C) J.γ1 (a PLCγ1-deficient Jurkat cell line) or J.γ1.WT-2 (PLCγ1-reconstituted J.γ1) cells were stimulated and analyzed as in A. These results are representative of three similar experiments. The membrane-to-cytosol (M/C) ratio of PKC expression in each group is displayed. In A, B, and C, the numbers above the autoradiograms represent the percentage of PKCθ or -α present in each fraction (C + M + I = 100% for each group of cells), as determined by NIH Image scanning densitometry. (D) PKCθ translocation to lipid rafts is present in PLCγ1-deficient Jurkat cells. J.γ1 or J.γ1.WT-2 (20 × 106) were left unstimulated (ns) or stimulated with anti-CD3 plus anti-CD28 antibodies. The cells were lysed and the detergent-insoluble fractions were separated from the soluble fractions. The distribution of PKCθ in each fraction was determined by immunoblotting with a specific antibody. (E) The same blot was stripped and blotted with a PLCγ1-specific antibody.

Mentions: To determine whether the reported Vav/Rac-mediated recruitment of PKCθ to the T cell membrane/cytoskeleton and its activation (Villalba et al., 2000a) are strictly dependent on activation of PLCγ1, we initially examined the effects of U73122 on the anti–CD3/CD28-induced translocation of PKCθ (or, for comparison, PKCα). This compound inhibits agonist-induced activation of PLC and the subsequent hydrolysis of inositol phospholipids in different cell types (Wang et al., 1994), including in TCR-stimulated T cells (Vassilopoulos et al., 1995). Combined anti-CD3/CD28 stimulation induced translocation of both PKC enzymes to the membrane, as evidenced by the approximately twofold increase in membrane expression of immunoreactive PKC (Fig. 1 A). As expected, U73122 pretreatment abolished the membrane translocation of PKCα and, in fact, even reduced its membrane expression below the basal level in unstimulated cells (Fig. 1 A). However, surprisingly, U73122 only minimally reduced the membrane translocation of PKCθ. As an additional control for the effectiveness of U71322 pretreatment, it also blocked the increase in intracellular calcium concentration induced by an anti-CD3 antibody (unpublished data). Conversely, PP2, an inhibitor specific for Src-family kinases, prevented the membrane translocation of PKCθ, but had only a minimal effect on PKCα translocation.


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 membrane translocation is independent of PLC activity. (A) Jurkat T cells (106) were stimulated with anti-CD3 plus anti-CD28 antibodies for 5 min. Aliquots of the cells were preincubated for 1 h with U73122 (10 μM) or with PP2 (10 μM). Cytosol (C), membrane (M), and detergent-insoluble (I) fractions were prepared, identical cell equivalents were resolved by SDS-PAGE, and the expression of PKCθ and PKCα in each fraction was determined by immunoblotting with specific antibodies. (B) Activated human peripheral blood T cells (5 × 106) were deprived of anti-CD3 antibody for 36 h, and then restimulated with anti-CD3 plus anti-CD28 antibodies for 10 min. Aliquots of the cells were preincubated for one hr with U73122 (10 μM) or LY294002 (50 μM). Subcellular fractions were prepared and analyzed as in A. (C) J.γ1 (a PLCγ1-deficient Jurkat cell line) or J.γ1.WT-2 (PLCγ1-reconstituted J.γ1) cells were stimulated and analyzed as in A. These results are representative of three similar experiments. The membrane-to-cytosol (M/C) ratio of PKC expression in each group is displayed. In A, B, and C, the numbers above the autoradiograms represent the percentage of PKCθ or -α present in each fraction (C + M + I = 100% for each group of cells), as determined by NIH Image scanning densitometry. (D) PKCθ translocation to lipid rafts is present in PLCγ1-deficient Jurkat cells. J.γ1 or J.γ1.WT-2 (20 × 106) were left unstimulated (ns) or stimulated with anti-CD3 plus anti-CD28 antibodies. The cells were lysed and the detergent-insoluble fractions were separated from the soluble fractions. The distribution of PKCθ in each fraction was determined by immunoblotting with a specific antibody. (E) The same blot was stripped and blotted with a PLCγ1-specific antibody.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: PKCθ membrane translocation is independent of PLC activity. (A) Jurkat T cells (106) were stimulated with anti-CD3 plus anti-CD28 antibodies for 5 min. Aliquots of the cells were preincubated for 1 h with U73122 (10 μM) or with PP2 (10 μM). Cytosol (C), membrane (M), and detergent-insoluble (I) fractions were prepared, identical cell equivalents were resolved by SDS-PAGE, and the expression of PKCθ and PKCα in each fraction was determined by immunoblotting with specific antibodies. (B) Activated human peripheral blood T cells (5 × 106) were deprived of anti-CD3 antibody for 36 h, and then restimulated with anti-CD3 plus anti-CD28 antibodies for 10 min. Aliquots of the cells were preincubated for one hr with U73122 (10 μM) or LY294002 (50 μM). Subcellular fractions were prepared and analyzed as in A. (C) J.γ1 (a PLCγ1-deficient Jurkat cell line) or J.γ1.WT-2 (PLCγ1-reconstituted J.γ1) cells were stimulated and analyzed as in A. These results are representative of three similar experiments. The membrane-to-cytosol (M/C) ratio of PKC expression in each group is displayed. In A, B, and C, the numbers above the autoradiograms represent the percentage of PKCθ or -α present in each fraction (C + M + I = 100% for each group of cells), as determined by NIH Image scanning densitometry. (D) PKCθ translocation to lipid rafts is present in PLCγ1-deficient Jurkat cells. J.γ1 or J.γ1.WT-2 (20 × 106) were left unstimulated (ns) or stimulated with anti-CD3 plus anti-CD28 antibodies. The cells were lysed and the detergent-insoluble fractions were separated from the soluble fractions. The distribution of PKCθ in each fraction was determined by immunoblotting with a specific antibody. (E) The same blot was stripped and blotted with a PLCγ1-specific antibody.
Mentions: To determine whether the reported Vav/Rac-mediated recruitment of PKCθ to the T cell membrane/cytoskeleton and its activation (Villalba et al., 2000a) are strictly dependent on activation of PLCγ1, we initially examined the effects of U73122 on the anti–CD3/CD28-induced translocation of PKCθ (or, for comparison, PKCα). This compound inhibits agonist-induced activation of PLC and the subsequent hydrolysis of inositol phospholipids in different cell types (Wang et al., 1994), including in TCR-stimulated T cells (Vassilopoulos et al., 1995). Combined anti-CD3/CD28 stimulation induced translocation of both PKC enzymes to the membrane, as evidenced by the approximately twofold increase in membrane expression of immunoreactive PKC (Fig. 1 A). As expected, U73122 pretreatment abolished the membrane translocation of PKCα and, in fact, even reduced its membrane expression below the basal level in unstimulated cells (Fig. 1 A). However, surprisingly, U73122 only minimally reduced the membrane translocation of PKCθ. As an additional control for the effectiveness of U71322 pretreatment, it also blocked the increase in intracellular calcium concentration induced by an anti-CD3 antibody (unpublished data). Conversely, PP2, an inhibitor specific for Src-family kinases, prevented the membrane translocation of PKCθ, but had only a minimal effect on PKCα translocation.

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