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. 2000 Sep;20(18):6945-57.
doi: 10.1128/MCB.20.18.6945-6957.2000.

Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation

X Shan  1 , M J Czar, S C Bunnell, P Liu, Y Liu, P L Schwartzberg, R L Wange
Affiliations

Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation

X Shan et al. Mol Cell Biol. 2000 Sep.

Abstract

Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P(2) and PI-3,4,5-P(3) in the plasma membrane. Here we describe a defect in expression of the D3 phosphoinositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregulated PH domain interactions with the plasma membrane. Inhibition of D3 phosphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitutive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to the cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell receptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosphorylation of phospholipase C-gamma1, and activation of Erk compared to those in PTEN-replete cells. These data support the idea that PH domain-mediated association with the plasma membrane is required for Itk activation, provide evidence for a negative regulatory role of PTEN in TCR stimulation, and suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.

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Figures

FIG. 1
FIG. 1
Constitutive localization of Itk to the membrane is correlated with absence of PTEN expression. (A) The cytosolic (c) and membrane (m) fractions prepared from normal human CD4+ T cells and JTAg T cells were immunoblotted with a monoclonal antibody to Itk (2F12). The membrane was stripped and reblotted with rabbit antisera to the cytosolic protein ZAP-70 (1213) and the transmembrane protein LAT (3023). (B) Whole-cell lysates (total protein, approximately 20 μg for JTAg and Jurkat E6 cells, and 10 μg for CD4+ T cells, A431 cells, and Jurkat [Upstate Biotechnology Inc. {UBI}] and Jurkat [Transduction Laboratories {TL}] cells) were immunoblotted with an antibody cocktail of four anti-PTEN antibodies (see Materials and Methods). The two A431 lysates (from UBI and TL) were included as positive controls.
FIG. 2
FIG. 2
Transcriptional expression and truncated translation of PTEN in JTAg T cells. (A) Northern blot analysis. Two major PTEN RNA species (5.5 and 2.5 kb) are indicated by open arrows. Relative levels of total RNA loading are shown as ethidium bromide staining of 28S rRNA (lower panel). (B) DNA sequencing analysis. The PTEN exon 7 sequence is shown in boldface uppercase letters. Mutation 1 (M1), containing a 2-bp deletion (in parentheses) and a 9-bp insertion, is shown in lowercase blue letters. Mutation 2 (M2), the 39-bp insertion mutation, is shown in lowercase red letters. The stop codons introduced by these mutations are underlined in blue (M1) or red (M2). Numbers refer to the codon number.
FIG. 3
FIG. 3
Effects of wortmannin on Akt phosphorylation and membrane distribution and kinase activity of Itk. JTAg T cells were treated with wortmannin (100 nM) for the indicated times prior to stimulation by cross-linking CD3 (OKT3 ascites). (A) Whole-cell lysates were immunoblotted with an anti-phospho-Akt antibody which recognizes the Ser-473-phosphorylated, active form of Akt. (B) The cytosolic (c) and membrane (m) fractions were prepared from the same samples and immunoblotted with an anti-Itk monoclonal antibody (2F12). The percentage of total Itk localized to the membrane fraction was determined by densitometric analysis of the X-ray films. (C) Itk was immunoprecipitated with rabbit polyclonal anti-Itk antisera from 107 cells treated with or without wortmannin (100 nM) and OKT3 as indicated and was subjected to an in vitro kinase assay. The inset shows an Itk blot of the Itk immunoprecipitate, indicating that equal amounts of Itk were used in the kinase assay.
FIG. 4
FIG. 4
Inhibition of PI3K with wortmannin results in a shift of plasma membrane-associated Itk into the cytoplasm. JTAg cells expressing GFP-tagged Itk were treated with vehicle (0.1% dimethyl sulfoxide) or 100 nM wortmannin and examined by fluorescence confocal microscopy at 1, 2, 3, and 4 h after the beginning of treatment. To better visualize the cytoplasmic compartment, the nuclei were stained with Hoechst 33342.
FIG. 5
FIG. 5
Reexpression of wild-type PTEN reduces Akt phosphorylation and restores the predominant cytosolic localization of Itk in JTAg T cells. Flag-tagged PTEN was expressed in JTAg T cells 18 h postelectroporation. (A) Each whole-cell lysate of 2 × 105 cell equivalents was immunoblotted for the level of PTEN expression with an anti-Flag antibody (top). The same membrane was stripped and reblotted for phosphorylated Akt (center) and Akt (bottom). (B) Cytosolic (c) and membrane (m) fractions were prepared from the same samples, and the Itk distribution pattern was examined by immunoblotting. (C) Itk was immunoprecipitated with 2F12 from 2 × 107 JTAg T cells transfected with the vector or PTEN-WT and then subjected to an in vitro kinase assay. (D) The amounts of Itk used in the kinase assay and their degree of tyrosine phosphorylation were measured by Western blot analysis.
FIG. 6
FIG. 6
PTEN expression in JTAg cells prevents plasma membrane association of Itk. GFP-tagged Itk was coexpressed in JTAg cells with vector (pSRα-Flag-Srf I), PTEN-WT, or the inactive mutant, PTEN-C/S, and examined by fluorescence confocal microscopy. To better visualize the cytoplasmic compartment, the nuclei were stained with Hoechst 33342.
FIG. 7
FIG. 7
Targeting of Itk to the membrane and sensitivity of Itk to CD3 stimulation requires an intact PH domain. Six micrograms of each pSRα-Itk-myc construct or vector was electroporated into 1.2 × 107 JTAg T cells to get about two- to threefold overexpression of the recombinant Itk over endogenous Itk. (A) The levels of Itk expression were monitored by Western blot analysis of the whole-cell lysates with an anti-myc antibody (9E10) (top) or an anti-Itk antibody (2F12) (bottom). (B) The cytosol (c) and membrane (m) fractions from pSR-Itk(WT) and pSR-Itk(R29C) transfectants were electrophoresed on a 6% Tris-glycine gel to resolve the endogenous and transfected Itk's and then immunoblotted with an anti-Itk antibody (2F12). (C) The recombinant Itk was immunoprecipitated with an anti-myc antibody (9E10) from 2 × 107 JTAg T cells transfected with either pSR-Itk(WT) or pSR-Itk(R29C) and was analyzed in an in vitro kinase assay.
FIG. 8
FIG. 8
Effect of PTEN expression on signaling pathways downstream of the TCR. JTAg T cells were transiently transfected with PTEN as described for Fig. 5 and stimulated with OKT3 for the indicated times (prime, minutes; double prime, seconds). (A) The expression level of PTEN and degree of Akt Ser-473 phosphorylation were determined by immunoblotting of whole-cell lysates. (B) The extent of protein tyrosine phosphorylation initiated upon CD3 cross-linking (OKT3) was determined by antiphosphotyrosine (4G10) Western blotting of whole-cell lysates. (C) PLC-γ1 was immunoprecipitated and blotted for phosphotyrosine (4G10) (top). The membrane was stripped and reblotted for PLC-γ1 (second panel). Erk activation was assessed either by immunoblotting of the whole-cell lysates with anti-active Erk (P-Erk) (third panel) or by detection of the electrophoretic shift of phosphorylated Erk-2 on a 10% Tris-glycine gel (bottom panel). (D) A total of 1.2 × 107 JTAg T cells were cotransfected with 10 μg of pNF-AT-Luc and 5 μg of the pβ-Gal control plasmid. Cells also received 30 μg of either the pSRα-PTEN-Flag or the pSRα-Flag (empty vector) plasmid. Lysates were prepared 16 h after transfection and were analyzed for luciferase and β-galactosidase activities. Relative light units (RLU) of the luciferase activity normalized for β-galactosidase activity are shown.
FIG. 9
FIG. 9
Multiple TCR-stimulated signaling pathways are sensitive to the level of D3-phosphorylated phosphoinositides. Opposing activities of PI3K (heavy green arrow) and PTEN (red arrow) are shown regulating the pool of D3-phosphorylated phosphoinositides. The potential for D3-phosphorylated phosphoinositides to regulate signaling molecules activated in response to TCR activation is depicted by light green arrows. Of the signaling proteins indicated, Itk, Tec, PLC-γ1, Akt, and Vav all contain PH domains that preferentially bind to PI-3,4,5-P3. Ras does not contain a PH domain, but it is subject to regulation by SOS and Ras-GAP, which both contain PI-3,4,5-P3-binding PH domains. Likewise, the various isozymes of PKC do not possess PH domains but are all phosphorylated upon their activation loop by PDK1, which has a PH domain that binds PI-3,4,5-P3 with high affinity.
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References

    1. Alessi D R, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings B A. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 1996;15:6541–6551. - PMC - PubMed
    1. Andjelkovic M, Maira S M, Cron P, Parker P J, Hemmings B A. Domain swapping used to investigate the mechanism of protein kinase B regulation by 3-phosphoinositide-dependent protein kinase 1 and Ser473 kinase. Mol Cell Biol. 1999;19:5061–5072. - PMC - PubMed
    1. August A, Sadra A, Dupont B, Hanafusa H. Src-induced activation of inducible T cell kinase (ITK) requires phosphatidylinositol 3-kinase activity and the pleckstrin homology domain of inducible T cell kinase. Proc Natl Acad Sci USA. 1997;94:11227–11232. - PMC - PubMed
    1. Beals C R, Sheridan C M, Turck C W, Gardner P, Crabtree G R. Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3. Science. 1997;275:1930–1934. - PubMed
    1. Besson A, Robbins S M, Yong V W. PTEN/MMAC1/TEP1 in signal transduction and tumorigenesis. Eur J Biochem. 1999;263:605–611. - PubMed

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