hSiah2 is a new Vav binding protein which inhibits Vav-mediated signaling pathways - PubMed (original) (raw)

hSiah2 is a new Vav binding protein which inhibits Vav-mediated signaling pathways

A Germani et al. Mol Cell Biol. 1999 May.

Abstract

The hematopoietic proto-oncogene vav has been characterized as a Rac1-GDP/GTP exchanger protein which regulates cytoskeletal reorganization as well as signaling pathways leading to the activation of stress-activated protein kinases (SAPK/JNKs). Furthermore, vav overexpression enhances basal and T-cell receptor (TCR)-mediated stimulation of the nuclear factor of activated T cells (NFAT). We report here the interaction between Vav and hSiah2, a mammalian homolog of Drosophila Seven in absentia (Sina) that has been implicated in R7 photoreceptor cell formation during Drosophila eye development via the proteasome degradation pathway. Vav and hSiah2 interact in vitro and in vivo and colocalize in the cytoplasm of hematopoietic cells. The Src homology domain of Vav and the C-terminal region of hSiah2 are required for this interaction. We provide evidence for a negative regulation by hSiah2 of Vav-induced basal and TCR-mediated NFAT-dependent transcription. Overexpression of hSiah2 also inhibits the onco-Vav-induced JNK activation. Although the Vav-interacting domain is located in the C-terminal portion of hSiah2, the N-terminal region of hSiah2 is necessary for the inhibitory role that seems to be independent of the proteasome degradation.

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Figures

FIG. 1

FIG. 1

Vav interacts with hSiah2 in the yeast two-hybrid system. (A) Schematic representation of hSiah2 and the clones obtained from the two-hybrid screening. (B) Protein interaction in the two-hybrid system. The L40 reporter strain was cotransformed with 1 μg of the indicated pLex- and pGAD-derived plasmids, and interactions were detected as β-galactosidase activity.

FIG. 2

FIG. 2

In vitro binding between hSiah2 and Vav. (A) Schematic representation of GST-hSiah2 fusion proteins. (B) Binding of Vav to GST-hSiah2 fusion proteins. Total-cell lysates from 107 Jurkat T cells were incubated at 4°C for 2 h with 1 μg of the fusion proteins or GST alone. (C) (Top) Lysates from unstimulated (0 min) and CD3-plus-CD28-stimulated (1 min and 30 min) Jurkat T cells (107) were incubated with 1 μg of GST-v240. The resulting complexes were resolved by SDS-PAGE, and the Western blot (Wb) was developed with anti-Vav MAb. The left-hand lane contains total lysate from 2 × 105 cells. (Bottom) Western blot analysis with antiphosphotyrosine antibody (anti-PTyr) of the total-cell extracts used in the top panel.

FIG. 3

FIG. 3

hSiah2 interacts with Vav in mammalian cells. COS-7 cells (A) or T-Ag Jurkat cells (B), transiently transfected with 4 and 20 μg, respectively, of either pKES-Vav (Vav), myc-tagged pCAN-v240 (myc-v240), or pCAN-v460 (myc-v460) alone or with a combination of the vectors (Vav+myc-v240; Vav+myc-v460), were lysed in NP-40 buffer and immunoprecipitated with anti-myc MAb and protein G-Sepharose beads. Immunoprecipitates (IP) were detected with anti-Vav MAb (top). Expression of transfected hSiah2 (clones v240 and v460) was verified by reprobing the nitrocellulose membrane with anti-myc MAb (bottom). Wb, Western blot.

FIG. 4

FIG. 4

Immunolocalization of Vav and hSiah2 by confocal immunofluorescence microscopy. RBL cells were labeled with preimmune Siah antiserum (A), Siah antiserum depleted of the immunizing peptide (B), anti-Vav MAb (C and D), and anti-hSiah2 rabbit polyclonal antibody (E and F) as described in Materials and Methods. Colocalization of red fluorescence from Vav and green fluorescence from hSiah2 produced a yellow signal, indicating an overlap in the distribution of the two proteins (G and H). In panels D, F, and H, cells were stimulated (Stim.) by FcɛRI cross-linking. Panels A and B were obtained with a much higher transmission rate in order for the signal to be detectable.

FIG. 5

FIG. 5

Vav-mediated NFAT activation is inhibited by hSiah2. (A) T-Ag Jurkat cells (107) were transfected with NFAT and pSVβ-galactosidase reporter plasmids (5 and 1 μg, respectively) and 20 μg of either an empty vector (vector), pEF-Vav (Vav), pCAN-v240 (v240), pCAN-v460 (v460), or a combination of the vectors as indicated. A total of 106 cells were either left unstimulated or stimulated after 24 h with anti-CD3 plus anti-CD28 for 8 h. Luciferase activity was measured and corrected for β-galactosidase activity, and the results were expressed as average fold induction relative to unstimulated cells transfected with the empty vector. The data are representative of four independent experiments. The basal activity and the maximum NFAT responses were approximately 600 and 2 × 105 AU, respectively. (B) T-Ag Jurkat cell lysates from panel A were analyzed by immunoblotting for expression of Vav and hSiah2 (v240 and v460). Wb, Western blot.

FIG. 6

FIG. 6

Overexpression of hSiah2 inhibits Vav-induced NFAT activity in a dose-dependent manner. T-Ag Jurkat cells (107) were transfected with NFAT and pSVβ-galactosidase reporter plasmids (5 and 1 μg, respectively), 10 μg of pEF-Vav (Vav), and increasing concentrations of pBK-CMV hSiah2 or pBK-CMV hSiah1 as indicated. Luciferase activity was determined and normalized to β-galactosidase activity to correct for transfection efficiency.

FIG. 7

FIG. 7

Inhibitory effect of hSiah2 on onco-Vav-mediated JNK activation. COS-7 (A) or T-Ag Jurkat (C) cells were transfected with 1 μg (A) or 5 μg (C) of pcDNA3-HA-JNK together with 3 μg (A) or 15 μg (C) of expression vectors containing cDNA for the indicated plasmids. The total amount of transfected DNA was kept constant by using empty pcDNA3 vector. COS-7 cells treated with 1 μg of anisomycin per ml for 20 min were used as a control. The kinase reaction was performed in anti-HA immunoprecipitates from the corresponding cellular lysates with purified GST–c-Jun as a substrate (A and C, top panels). The levels of HA-JNK protein were confirmed by Western blot (Wb) analysis with anti-HA antibody (bottom panels). Values in the histogram represent the means and the standard errors of four independent experiments. COS-7 (B) and T-Ag Jurkat (D) cell lysates from panels A and C were analyzed by immunoblotting for expression of Vav and myc-epitope-tagged hSiah2 (v240 and v460). The additional bands in these blots could be due to degradative events caused by onco-Vav overexpression.

FIG. 8

FIG. 8

hSiah2 does not decrease the half-life of Vav. COS-7 cells were cotransfected with 4 μg of myc-tagged pEF-Vav (myc-Vav) and pCAN-v240 (myc-v240) or pCAN-v460 (myc-v460). At 48 h after transfection, the cells were pulse-labeled for 1 h with [35S]methionine, chased with cold methionine for the indicated times, and then lysed as described in Materials and Methods. Vav and hSiah2 proteins were immunoprecipitated (IP) with anti-myc antibody and analyzed by SDS-PAGE and autoradiography.

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