PMC:1887720 / 19025-22646 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1887720","sourcedb":"PMC","sourceid":"1887720","source_url":"https://www.ncbi.nlm.nih.gov/pmc/1887720","text":"TCR-induced Proliferation and Actin Polymerization Proceed Normally in WAS−/−ΔGBD Mice.\nAlthough activated cdc42 can bind WASp and trigger WASp VCA domain–mediated Arp2/3 activity (2, 3), it is unclear whether this interaction is required and/or solely responsible for induction of WASp effector function after TCR stimulation. To address this issue, the relevance of the GBD–cdc42 interaction to WASp effects on T cell activation was studied using mice in which a CD2 promoter/enhancer-driven WASp cDNA lacking the GBD (WASpΔGBD; Fig. 1 A) was expressed on the C57BL/6 WAS−/− background. After confirmation that the WASpΔGBD species does not bind activated cdc42 (Fig. 1 B) and demonstration that the WASpΔGBD transgene is expressed in T lineage cells (Fig. 1 C), WAS−/−ΔGBD mice were characterized with respect to T cell phenotype and function. In contrast to WAS−/− mice, in which thymocyte and peripheral T cell numbers are markedly reduced and thymocyte maturation impaired (18), the WAS−/−ΔGBD mice showed no defects in either T cell numbers or development (not depicted). Analysis of TCR-induced proliferation also revealed proliferative responses to anti-CD3 antibody or anti-CD3 and anti-CD28 antibodies to be essentially comparable in WAS−/−ΔGBD and control thymocytes and peripheral T cells, and markedly reduced in WAS−/− cells (Fig. 1 D). Similarly, immunofluorescence analysis of FITC phalloidin–stained WAS−/−ΔGBD thymocytes revealed CD3/CD28-induced actin polymerization, which is abrogated in WAS−/− cells, to be essentially normal in WAS−/−ΔGBD cells (Fig. 1 E). Together, these observations indicate the coupling of TCR engagement to proliferation and actin polymerization to be unaffected by absence of the WASp GBD and thus imply that WASp effects on TCR-induced transcriptional activation and cytoskeletal arrangement can be evoked by mechanisms other than cdc42–GBD interaction.\nFigure 1. The GBD of WASp is not essential for WASp activity. (A) Scheme that shows the domain organization of wild-type and a GBD-deleted (WASpΔGBD) WASp cDNA used to derive WAS−/−ΔGBD mice. Positions of all tyrosine residues within WASp are indicated. (B) Lysates obtained from WASp or WASpΔGBD-expressing Cos-7 cells were incubated with GTPγS-loaded GST-cdc42 fusion protein bound to glutathione sepharose beads and the complexes were then resolved by SDS-PAGE and sequential immunoblotting analysis with anti-WASp and anti-cdc42 antibodies (top two panels). Immunoblotting analysis demonstrating WASp and WASpΔGBD expression in Cos-7 transfectants (Tfn) is shown in the bottom panel. (C) Immunoblotting analysis showing the WASp species detected in thymocytes and lymphocytes from wild-type (WT), WAS−/−, and WAS−/−ΔGBD mice using an anti-WASp antibody. (D) Lymphocytes and thymocytes isolated from 4–8-wk-old wild-type (WT), WAS−/−, and WAS−/−ΔGBD mice were cultured for 48 h in medium alone or with either 1 μg/ml anti-CD3 antibody or 0.2 μg/ml anti-CD3 plus anti-CD28 antibody. Antigen receptor–evoked proliferative responses were determined after an 18-h pulse with [3H]thymidine. Values represent the means (± SEM) of four independent experiments. (E) Thymocytes from wild-type (WT), WAS−/−, and WAS−/−ΔGBD mice were isolated and stimulated with anti-CD3 plus anti-CD28 antibodies for 30 min on ice followed by cross-linking with a secondary antibody. Cells were fixed with 5% paraformaldehyde and F-actin content was quantified by flow cytometric analysis of FITC phalloidin–stained resting (bold lines) and stimulated (dashed lines) cells. The results are representative of three independent experiments.\n\nP","divisions":[{"label":"Title","span":{"begin":0,"end":87}},{"label":"Figure caption","span":{"begin":1902,"end":3620}}],"tracks":[{"project":"2_test","denotations":[{"id":"14707117-9889097-59645195","span":{"begin":181,"end":182},"obj":"9889097"},{"id":"14707117-10219243-59645196","span":{"begin":184,"end":185},"obj":"10219243"},{"id":"14707117-10544204-59645197","span":{"begin":980,"end":982},"obj":"10544204"}],"attributes":[{"subj":"14707117-9889097-59645195","pred":"source","obj":"2_test"},{"subj":"14707117-10219243-59645196","pred":"source","obj":"2_test"},{"subj":"14707117-10544204-59645197","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#eca993","default":true}]}]}}