Screening strategy for the isolation of Jurkat T-cell clones with impaired DAG-induced Ras activation. (A) FACS analysis of induced CD69 expression on Jurkat or JPRM441 cells following overnight PMA stimulation. Numbers indicate the mean fluorescent intensity. (B) FACS analysis of GFP and induced CD69 expression on Jurkat and JPRM441 cells, 40 h after transient cotransfection with GFP (10 μg) and vector or RasV12 (10 μg). Numbers indicate the percentage of live cells in each quadrant. All experiments in Fig. 1 and all subsequent figures are representative examples of at least three independent experiments unless indicated otherwise.

Isolation of a RasGRP1-deficient Jurkat T-cell clone. (A) Western blot analysis of RasGRP1 expression in Jurkat and JPRM441 whole-cell lysates (WCL) or RasGRP1 immunoprecipitations (m133 IP). The expression was quantified and set to a relative 100% expression level in wild-type Jurkat cells. (B) Northern blot analysis of RasGRP1, TCRα, TCRβ, TCRζ, and CD3ɛ gene expression in Jurkat (lanes 1) and JPRM441 (lanes 2) cell lines. RNA levels are indicated by β-actin hybridization of the same blots. (C) RT-PCR analysis of RasGRP-1, -2, -3, and -4 gene expression on 1:10 serial dilutions of Jurkat and JPRM441 cDNAs and on cDNA derived from human peripheral blood mononuclear cells, and a panel of lymphoid and nonlymphoid cell lines (cell type): lane 1, peripheral blood mononuclear cells; lane 2, peer (γδ T cell); lane 3, RAJI (B cell); lane 4, K562 (myeloid cell); lane 5, U937 (myeloid cell); lane 6, RPMI8226 (multiple myeloma cell); and lane 7, HELA (cervix carcinoma). RT-PCR for HPRT gene expression demonstrates equal RNA levels.

Restored induction of CD69 in stimulated JPRM441 cells reconstituted with wild-type RasGRP1 cDNA. (A) Strategy to mimic TCR signaling in JPRM441 through cross-linking of the introduced chimeric CD25ζζ protein. A FACS analysis for CD25 demonstrates equal levels of expression of CD25ζζ in the stable clones Jurkat-CD25ζζ and JPRM441-CD25ζζ. (B) Western blot analysis of RasGRP1 expression in stable cell lines expressing wild-type RasGRP1 cDNA or ΔDAGRasGRP1 cDNA in JPRM441-CD25ζζ, yielding the indicated cell lines. Western blot analyses of PKCθ, Erk-1 and -2, and P38 expression demonstrate equal levels of these four proteins in all the lines. WCL, whole-cell lysates. (C) The indicated cell lines were left unstimulated or were stimulated overnight with PMA or soluble anti-CD25 and goat anti-mouse antibodies and analyzed by FACS for induced CD69 expression. Numbers in the histograms indicate mean fluorescent intensities.

Defective PMA-Ras-Erk signaling due to RasGRP1 deficiency. (A) Resting and PMA-stimulated Jurkat and JPRM441 cells were analyzed for the extent of Ras activation at the indicated time points by a GST-Raf pull-down assay. Bands were quantified, and the percentage of Ras activation was obtained through normalization to total Ras expression levels. (B) Western blot analysis of PMA-induced activation of Erk-1 and -2 in Jurkat and JPRM441 cells. The mean levels of phosphorylation of Erk-1 (P-Erk1) and -2 and the standard deviations from three independent experiments were calculated using normalization for α-tubulin expression and are indicated in the box. These mean levels of activation were also plotted as relative percentages for the various time points. (C) Western blot analysis of PMA- and ionomycin-induced (ion.) phosphorylation of P38 or PMA-induced phosphorylation of JNK-1 and -2 in Jurkat and JPRM441 cells. The same blot was subjected to α-tubulin Western blot analysis as a loading control. (D) Luciferase assay for the transcriptional activity of AP-1 in resting or PMA-stimulated Jurkat and JPRM441 cells. The bar graph represents the mean luciferase values and standard deviations from three independent assays.

PMA-MAPK signaling in RasGRP1-reconstituted cells. Results of an analysis of the PMA-induced phosphorylation of Erk-1 and -2 or JNK-1 and -2 and the PMA- and ionomycin-induced (ion.) phosphorylation of P38 in Jurkat-CD25ζζ, JPRM441-CD25ζζ, and wtRasGRP1- or ΔDAGRasGRP1-reconstituted JPRM441-CD25ζζ cells are shown. The mean levels of phosphorylation of Erk-1 and -2 and the standard deviations from three independent experiments were calculated using normalization for α-tubulin expression and are indicated in the box.

TCR-induced tyrosine-phosphorylated proteins and calcium mobilization. (A) Induced tyrosine phosphorylation patterns upon TCRβ or CD25ζζ cross-linking in the indicated cell lines by 4G10 Western blot analysis are shown. Positions of known tyrosine-phosphorylated proteins are indicated. Equal protein loadings are indicated by α-tubulin expression on the same blot. (B) Measurement of intracellular Ca²⁺ levels using the indicator Fluo-3-AM in Jurkat, Jurkat-CD25ζζ, and JPRM441-CD25ζζ cells. Arrows indicate the administration of C305 cross-linking TCRβ or anti-CD25 cross-linking CD25ζζ.

Impaired TCR-Erk signal transduction in RasGRP1-deficient cells. (A) Western blot analysis of activation of Erk-1 and -2 by cross-linking CD25ζζ on Jurkat-CD25ζζ and JPRM441-CD25ζζ cells. The mean levels of phosphorylation of Erk-1 (P-Erk1) and -2 and the standard deviations from three independent experiments were calculated and are plotted as in Fig. 4B. (B) Analysis of anti-CD25-induced phosphorylation of Erk-1 and -2 in Jurkat-CD25ζζ, JPRM441-CD25ζζ, and wtRasGRP1- or ΔDAGRasGRP1-reconstituted JPRM441-CD25ζζ cells. The mean levels of phosphorylation of Erk-1 and -2 and the standard deviations from three independent experiments were calculated usingnormalization for α-tubulin expression and are indicated in the box. (C) Western blot analysis of SOS1 and phospho-p36 (LAT) coimmunoprecipitating (IP) with Grb2 in the indicated cell lines following CD25ζζ cross-linking. An analysis of whole-cell lysates (WCL) indicates equal levels of expression or phosphorylation of the three proteins in the two cell lines.

Both RasGRP1 and novel PKC family members are required for PMA-Ras-Erk signaling. (A) Jurkat and JPRM441 cells were treated with DMSO control or with the PKC inhibitor Rottlerin for 1 h. Resting and PMA-stimulated Jurkat and JPRM441 cells were analyzed for the extent of Ras activation. Bands were quantified, and the percentages of Ras activation were obtained through normalization to total Ras expression levels. (B) Following Rottlerin or control treatment, the phosphorylation of Erk-1 (P-Erk1) and -2 was determined by Western blot analysis of PMA-induced or resting Jurkat and JPRM441 cells. Activation of the two kinases was quantified using normalization for α-tubulin expression on the same blot. Numbers are the means and standard deviations from three independent experiments. (C) Purified human CD4 T cells were pretreated with Rottlerin or with DMSO control and analyzed for the amount of PMA-induced phosphorylation of Erk-1 and -2 as described for panel B.

Novel PKC kinases signal to a RasGRP1-like molecule in RasGRP1-null cells. (A) Immunoblotting for Myc expression in Myc-tagged RasGRP1-transfected 293T cells in combination with the indicated RNAi constructs. (B) Immunoblotting for Express expression in Express-tagged PKCθ(KR)-transfected 293T cells in combination with the indicated siRNA construct. (C) Western blot analysis for RasGRP1, and Grb2 of MACS-purified Jurkat and JPRM441 cells transfected with the indicated constructs. Numbers indicate the relative percentages of RasGRP1 expression. (D) The indicated purified, transfected pools were preincubated with DMSO control or Rottlerin, stimulated as indicated, and analyzed for Erk phosphorylation by Western blot analysis. Numbers indicate the relative levels of phosphorylated Erk-1 (P-Erk1) and -2, obtained by normalizing for protein loading through Grb2. Panel D is representative of two independent experiments.

Novel PKC kinases phosphorylate and signal through RasGRP1. (A) Coimmunoprecipitations of Myc-tagged RasGRP-1, -2, -3, and -4 with Express-tagged, kinase-dead PKCθ or PKCα. The indicated constructs were cotransfected into 293T cells. One-fifteenth of the NP-40 lysates was used to verify expression in whole-cell lysates (WCL). The remainder was used for Myc immunoprecipitation (IP). Samples were subjected to Western blotting for Express-tag (PKCθ or PKCα). The same blot was stripped and reprobed for Myc expression (RasGRP's). (B) PKCθ in vitro kinase assay. The indicated constructs were expressed in 293T cells. Following Myc immunoprecipitations, three-fourths of the material was offered as a substrate in an in vitro kinase reaction using purified, active PKCθ. One-fifteenth (whole-cell lysate) and one-fourth (Myc IP) of the material were subjected to Western blotting for Myc expression (RasGRP's). The in vitro kinase assay was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred, and exposed to film. Relative phosphorylation was determined by determining the autoradiograph/Myc expression ratio from the Myc immunoprecipitations. (C) FACS analysis of GFP and induced CD69 expression. The active alleles of RasV12 (10 μg), PKCθ(AE) (20 μg), and PKCα(AE) (20 μg) or the control vector (10 μg) were cotransfected with GFP (10 μg) in the indicated cell lines, and cells were analyzed 40 h later for the amount of CD69 expression. (D) Western blot analysis for phosphorylated RasGRP1. Jurkat T cells were pretreated with DMSO control or Rottlerin and stimulated as indicated. The immunoprecipitated RasGRP1 was analyzed for phosphorylation on threonine 184 using a phospho-specific antibody (51). Backblotting for RasGRP1 identified phospho-RasGRP1 as the top band on the P-T184 blot. The lower band most likely reflects phosphorylated RasGRP3 that can also be immunoprecipitated with the M133 antibody. Analysis of whole-cell lysates demonstrates the proper inducible phosphorylation of Erk kinases, and expression of RasGRP1 and Grb2 indicates equal protein loadings in all samples. Panel D is representative of two independent experiments.

Model of TCR-induced Ras-Raf-Erk activation in T lymphocytes. T lymphocytes express two RasGEF molecules, SOS and RasGRP1, and hypothetically can achieve Ras activation through two different pathways. One pathway, (1), utilizes SOS, involving the formation of a phospho-LAT-Grb2-SOS complex to bring SOS in proximity with Ras-GDP. The second pathway, (2), requires generation of DAG by PLCγ. DAG recruits RasGRP1 and PKCθ to the membrane through their C1 domains. Possibly, DAG can also recruit molecules Y (e.g., PKCη) and X (RasGRP3). Novel PKC kinases phosphorylate RasGRP1 on threonine 184 (or RasGRP3 on threonine 133) and other residues, likely leading to increased GEF activity of RasGRP1. RasGRP1 subsequently converts Ras-GDP to active Ras-GTP. Disruption of the PKCθ-RasGRP1 pathway results in impaired TCR-induced Ras and Erk activation, while formation of the phospho-LAT-Grb2-SOS complex is unaffected. RasGRP1-induced Ras activation is therefore unique and cannot be compensated for by SOS. MHC, major histocompatibility complex.