HePTP in lipid rafts. (a) Anti-HePTP (top panel), anti-PKC θ (second panel), anti-LAT (third panel), and anti-CD45 (bottom panel) immunoblots of detergent extracts of resting Jurkat T cells fractionated on a sucrose gradient. Lipid rafts are in fractions 2 to 4, mostly in fraction 3. (b) Similar experiment with Jurkat T cells stimulated with anti-TCR mAbs. M_rs (in thousands) are shown to the left of the gels.

PKC θ is required for HePTP translocation to the immune synapse. (a) Confocal microscopy of CD8⁺ antigen-specific, TCR-transgenic OT-I T cells in contact with APCs for 20 min and stained for HePTP (green) and PKC θ (red). (b) Immunofluorescence microscopy of Jurkat T cells in contact with Raji B cells without (top row) or with (all other rows) staphylococcal enterotoxin E and stained for HePTP (green), B cells (red), and DNA (blue). The centers of the contacts between the T cell and the APC are indicated with arrows; B cells are labeled “B” in contrast panels. (c) Quantitation of formed immune synapses in numerous fields of view. (d) Immunofluorescence microscopy of CD4⁺ antigen-specific, TCR-transgenic OT-II T cells in contact with APCs in the absence (left panels) or presence (right panels) of OVA peptide and stained for HePTP (green) and DNA (blue). The upper panels show control OT-II T cells; the lower panels show T cells from OT-II × pkcθ^−/− mice, as indicated. B cells are labeled “B” in contrast panels. wt, wild type; DAPI, 4′,6′-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide.

Phosphorylation of HePTP by different PKC isoenzymes. (a) Autoradiogram of GST-HePTP incubated with the indicated PKC isoenzymes in the presence of [γ-³²P]ATP for 30 min. M_rs (in thousands) are shown to the left of the gel. (b) Tryptic peptide maps (peptides labeled 1, 2, and 3) of the phosphorylated HePTP proteins from panel a. Exposure times were as follows: 5 days for PKC δ, PKC ɛ, and PKC η; 17 h for PKC θ; and 20 h for PKC ζ and PKC μ. (c) Phosphoamino acid analysis of peptides 1 and 3 from the PKC θ map, showing that both contain only phosphoserine.

Role of HePTP phosphorylation at S225. (a) Anti-HA immunoblots of detergent extracts of Jurkat T cells transfected with HePTP (top panel), HePTP-S225D (middle panel), or HePTP-S225A (bottom panel) fractionated on a sucrose gradient. Lipid rafts are in fractions 2 to 4, mostly in fraction 3. n.s., nonstimulated. (b) Luciferase assay (relative light units [RLU]) of Jurkat T cells transfected with an NFAT/AP-1 reporter construct and empty vector (HePTP, HePTP-S225D, or HePTP-S225A, as indicated) and either left unstimulated or treated with OKT3 anti-CD3ɛ mAb for 7 h. The luciferase activity was normalized using a cotransfected Renilla luciferase. The data represent the means, and the error bars standard deviations, from triplicate determinations; this experiment is representative of three independent experiments. wt, wild type. (c) Anti-HA immunoblot to show the expressed HePTP in the same cell samples. (d) Elimination of HePTP by RNA interference augments PKC θ-induced Erk activation. Jurkat T cells were transfected with a nonspecific RNA oligonucleotide (lanes 1 to 3) or with an HePTP-specific siRNA (lanes 4 to 6) and cotransfected with expression plasmids for the constitutively active PKC θ-AE (lanes 2 and 5) or myristylated PKC θ (lanes 3 and 6). The four panels represent blots with different antibodies, as indicated. M_rs (in thousands) are shown to the left of the gels.

HePTP and Erk activation in the immune synapse. (a) Immunoblots of lipid raft fractions from resting (lane 1) or activated (lane 2) Jurkat T cells with antibodies against total Erk (upper left panel), phospho-Erk (lower left panel), HePTP (top middle panel), Lck (second middle panel), phospho-Y505 Lck (bottom middle panel), or antiphosphotyrosine (right panel). M_rs (in thousands) are shown to the left of the gels. (b) Confocal microscopy of CD8⁺ antigen-specific, TCR-transgenic OT-I T cells in contact with APCs for 0 min (subpanels a) or 20 min (subpanels b to d) and stained for HePTP (green), phospho-Erk (pErk; red), and DNA (blue). Subpanels c are enlargements of the indicated subpanels b to show phospho-Erk location in the immune synapse (IS) and nucleus (N). DAPI, 4′,6′-diamidino-2-phenylindole. (c) Three-dimensional reconstruction of a representative OT-I T cell stained for HePTP (green) and phospho-Erk (red) in contact with an APC. The reconstructed cell is shown in the z plane, i.e., the direction from which the APC would see the T cell (left panel); 45° between the z and y planes (middle panel); and in the y plane (right panel). The center of the immune synapse and the location of staining in the nucleus are indicated.

Phosphorylation of HePTP by PKC at S225. (a) Autoradiogram of in vitro kinase assay containing HePTP alone (lane 1), PKC-M alone (lane 2), GST plus PKC-M (lane 3), GST-HePTP plus PKC (lane 4), or the indicated S/T-to-A mutants of GST-HePTP plus PKC-M (lanes 5 to 9) in the presence of [γ-³²P]ATP. M_rs (in thousands) are shown to the left of the gel. (b) Tryptic peptide maps (peptides labeled 1, 2, and 3) of the phosphorylated HePTP proteins from panel a. Exposure time for all maps was 21 h. Arrows indicate missing peptides. wt, wild type.

PKC θ phosphorylates HePTP at S225 in primary T cells. (a) Upper panel, anti-(K/R)-X-pS-Φ-K immunoblot of GST-HePTP (lanes 1 and 3) and GST-HePTP-S225A (lanes 2 and 4) incubated with kinase buffer alone (lanes 1 and 2) or with PKC (lanes 3 and 4). Lower panel, anti-HePTP blot of the same filter. (b) Upper panel, anti-(K/R)-X-pS-Φ-K immunoblot of anti-HePTP immunoprecipitates from primary human T cells left untreated (lane 1) or stimulated with anti-CD3 and anti-CD28 Dynabeads (lane 2) or 50 nM phorbol ester (lane 3) for 10 min before lysis. Lower panel, anti-HePTP immunoblot of the same immunoprecipitates. (c) Upper panel, anti-(K/R)-X-pS-Φ-K immunoblot of anti-HePTP immunoprecipitates from primary human T cells left untreated (lane 1), stimulated with anti-CD3 (lane 2), or treated with 30 μM rottlerin plus anti-CD3/CD28 beads (lane 3) or with 60 μM rottlerin plus anti-CD3/CD28 beads (lane 4). Lower panel, anti-HePTP immunoblot of the same immunoprecipitates. M_rs (in thousands) are shown to the left of the gels.