Volume 9, Issue 5, Pages 595-605 (November 1998) Adaptor Function for the Syk Kinases–Interacting Protein 3BP2 in IL-2 Gene Activation  Marcel Deckert, Sophie Tartare-Deckert, Jerry Hernandez, Robert Rottapel, Amnon Altman  Immunity  Volume 9, Issue 5, Pages 595-605 (November 1998) DOI: 10.1016/S1074-7613(00)80657-3

Figure 1 Structural Features and Expression of 3BP2 (A) Alignment of the predicted amino acid sequences of human (h) or mouse (m) 3BP2 and the yeast two-hybrid clone SKIP-2. The PH, proline-rich (Pro-rich), and SH2 domains are represented by black, hatched, and gray boxes, respectively. (B) Expression of 3BP2 mRNA. A 32P-labeled SKIP-2 cDNA probe was used to perform a Northern blot analysis on RNA samples from the indicated human tissues (Clontech). A low molecular weight tyrosine phosphatase (LMPTP) cDNA probe was used as a control (bottom panel). (C) RT-PCR analysis of 3BP2 transcript expression in human tissues. (D) Expression of 3BP2 protein in murine T cell hybridomas and human cell lines. Cell lysates were subjected to SDS-PAGE and immunoblotting with a rabbit anti-mouse 3BP2 antibody. (E) Expression of 3BP2 protein in murine lymphoid tissues. Immunity 1998 9, 595-605DOI: (10.1016/S1074-7613(00)80657-3)

Figure 2 Cellular Localization of 3BP2 (A) Ectopic expression of 3BP2. COS-1 cells were transiently transfected with an expression plasmid encoding an NH2-terminal HA epitope-tagged 3BP2, and the expressed protein was detected by staining with the anti-HA mAb 12CA5 (right panel). Arrows show accumulations of 3BP2 at the cell membrane. Phalloidin counterstaining was used to visualize the actin cytoskeleton (left panel). (B) Expression and localization of 3BP2 in Jurkat T cells. The cells were transfected with HA-tagged 3BP2 cDNA. After 24 hr, the cells were treated with 2 μg/ml OKT3 for 5 or 15 min, or left unstimulated (no), fixed, and stained with an anti-HA mAb. Arrows show characteristic accumulations of 3BP2 in large patches at the cell membrane, and arrowheads indicate cytoplasmic accumulation of 3BP2. (C) 3BP2 localization in fractionated T cells. HA-3BP2-transfected unstimulated or OKT3-stimulated Jurkat-TAg cells were fractionated into cytoplasmic (C), membrane (M), and detergent-insoluble (I) fractions as described in Experimental Procedures, and the fractions were subjected to SDS-PAGE and immunoblotting with an anti-HA (top panel), -Zap-70 (middle panel), or -Vav (bottom panel) mAbs. Immunity 1998 9, 595-605DOI: (10.1016/S1074-7613(00)80657-3)

Figure 3 Interaction of 3BP2 with Syk and Fyn (A) Yeast two-hybrid interaction. Syk, Zap-70, or Fyn cDNAs, or the indicated Syk mutants, were fused to the DNA-binding domain of Lex9, while the indicated 3BP2 cDNAs were fused to the Gal4 activation domain. Yeast were cotransformed with the indicated plasmid combinations, and interactions were assayed using a β-galactosidase filter assay. The Table summarizes three independent experiments: (−) no interaction; (+++) strong interaction. Similar results were obtained by assessing yeast growth in histidine-deficient medium. A Lex9-lamin expression vector was used as a negative control. (B) Mammalian two-hybrid interaction. 3BP2 or Vav were fused to the activation domain of VP16, whereas Syk or Zap-70 were fused to the DNA-binding domain of Gal4. These constructs and a luciferase reporter plasmid were transfected into 293T cells, and interactions were detected by measuring luciferase activity using a mammalian two-hybrid system. (C) Interaction of 3BP2 with Zap-70 in intact T cells. Jurkat cells were either left unstimulated or stimulated with 0.1 mM pervanadate (PV) for 5 min and lysed. Lysates were immunoprecipitated (IP) using a polyclonal rabbit anti-3BP2 antiserum, or normal rabbit serum (NRS) as a negative control, followed by a goat anti-rabbit antibody conjugated to agarose beads. The immunoprecipitates, as well as whole-cell lysates (WCL; 1 × 106 cell equivalents) were blotted with anti-phophotyrosine or anti-Zap-70 mAbs, followed by chemiluminescent detection. Immunity 1998 9, 595-605DOI: (10.1016/S1074-7613(00)80657-3)

Figure 4 Transcriptional Activation of NFAT by 3BP2 (A) Dose-dependent activation of NFAT by 3BP2. Jurkat-TAg cells were transfected with the indicated quantities of empty pMT3 expression vector, or of the same vector expressing HA-tagged 3BP2, in the presence of an NFAT reporter construct. NFAT reporter activity was determined in the absence or presence of an anti-CD3 mAb (OKT3; 2 μg/ml) stimulation. The bottom panel shows the expression levels of 3BP2 detected by immunoblotting with an anti-HA mAb. (B) Schematic structures of the 3BP2 constructs used in this study. (C) Jurkat-TAg cells were cotransfected with 5 μg of empty pMT3 expression vector, or of the indicated 3BP2 cDNA, and an NFAT reporter construct. NFAT reporter activity was determined in unstimulated cells. The bottom panel represents an anti-HA immunoblot showing the expression of the transfected 3BP2 proteins. Immunity 1998 9, 595-605DOI: (10.1016/S1074-7613(00)80657-3)

Figure 5 Role of the 3BP2 SH2 Domain in Transcriptional Activation and Its Binding to Signaling Intermediates (A) Activation of NFAT, AP-1, and the IL-2 promoter by 3BP2. Jurkat-TAg cells were transfected with the empty pMT3 expression vector, or with the same vector expressing HA-tagged 3BP2 or an SH2-truncated 3BP2 mutant (HA-3BP2 1–486), in the presence of NFAT, AP-1, or IL-2 promoter reporter constructs. NFAT or AP-1 reporter activity was determined in the absence or presence of OKT3 (2 μg/ml) stimulation. To measure IL-2 promoter activity, the cells were stimulated with OKT3 plus 10 ng/ml of PMA. (B) Inhibition of TCR-induced NFAT activation by the isolated SH2 domain of 3BP2. NFAT activity in cells transfected with an empty pMT3 vector or a pMT3 vector encoding the HA-tagged form of the 3BP2 SH2 domain (residues 454–559) was assayed in the absence or presence of OKT3 or ionomycin (1 μg/ml) stimulation. The inset at the top represents an anti-HA immunoblot showing the expression of the transfected 3BP2 SH2 domain. (C) In vitro interaction of 3BP2 with Zap-70 and tyrosine-phosphorylated signaling molecules. Jurkat cells lysates from unstimulated cells or from cells stimulated for 3 min with an anti-TCR mAb (C305; 2 μg/ml) or 0.1 mM pervanadate were incubated with 5 μg GST-3BP2 SH2 fusion protein (+), or GST alone (−), followed by glutathione-sepharose beads. Bound proteins were subjected to SDS-PAGE analysis and immunoblotting with the indicated antibodies, followed by chemiluminescent detection. For reference, whole- cell lysate (WCL) aliquots were immunoblotted with the same antibodies (right panels). Positions of the relevant proteins are indicated by the arrows. The open arrowhead on the left indicates nonspecific cross-reactivity of the anti-LAT antibody, which possesses anti-GST reactivity, against the GST-3BP2 SH2 fusion protein. Immunity 1998 9, 595-605DOI: (10.1016/S1074-7613(00)80657-3)

Figure 6 Cooperation between Syk-Family Kinases and 3BP2 in NFAT Activation (A) Defective 3BP2 signaling in Zap-70-deficient cells. Wild-type and Zap-70-deficient P116 Jurkat cells were cotransfected with empty vector or HA-3BP2 and assayed for NFAT activity, either in the absence or presence of OKT3 stimulation. Data were calculated as percentage of the maximal response obtained in the presence of PMA and ionomycin. The inset shows the expression of endogenous Zap-70 and transfected HA-3BP2 in both cell clones. (B) Cooperation between Syk and 3BP2 in NFAT activation. Jurkat-TAg cells were cotransfected with 1 μg DNA of the indicated combinations of HA-3BP2 and/or Syk expression vectors and tested for NFAT activity after 24 hr. An anti-HA immunoblot showing the expression of the epitope-tagged 3BP2 and Syk proteins is shown in the inset on top. Immunity 1998 9, 595-605DOI: (10.1016/S1074-7613(00)80657-3)

Figure 7 Requirements for 3BP2-Induced Gene Transcription (A) Direct NFAT activation by 3BP2. Jurkat-TAg cells were transfected with an empty or an HA-tagged 3BP2-expressing pMT3 vector in the presence of an NFAT reporter derived from the TNFα promoter plus an HA-tagged NFAT-1c expression vector. The cells were left unstimulated or stimulated for the final 6 hr of culture with OKT3 or ionomycin (1 μg/ml each). CAT activity in cell extracts was determined by colorimetric analysis. The results represent the average of two independent experiments ± SEM. Expression of the transfected NFAT-1c or 3BP2 proteins was monitored by immunoblotting aliquots of cell lysates with an anti-HA mAb (inset). (B) Inhibition of 3BP2-induced NFAT and IL-2 promoter activation by CsA. Jurkat-TAg cells were transfected with an empty or an HA-tagged 3BP2-expressing pMT3 vector in the presence of NFAT, AP-1, or IL-2 promoter reporter constructs and cultured in the absence (−) or presence (+) of CsA (100 ng/ml). NFAT or AP-1 reporter activity in unstimulated cells was determined as in Figure 4. IL-2 promoter activity was assessed in OKT3 (2 μg/ml) plus PMA (50 ng/ml)-stimulated cells. The inset displays the expression of the 3BP2 protein revealed by anti-HA immunoblotting. (C) Requirement of functional Ras for 3BP2-dependent NFAT activation. Jurkat-TAg cells were cotransfected with an NFAT reporter construct and either empty or 3BP2-expressing pMT3 plasmid, in the absence or the presence of a plasmid encoding the dominant-negative mutant of Ras (RasN17) as indicated. Reporter activity was determined in unstimulated cells or in cells stimulated with OKT3 or ionomycin. Immunity 1998 9, 595-605DOI: (10.1016/S1074-7613(00)80657-3)