Regulation of T-cell adhesion and megakaryopoiesis by immune adaptor ADAP

• Main Author: Xiong, Y.
• Other Authors: Liu, H.
• Published: University of Liverpool 2017
• Subjects: 570
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.733929

The immune adaptor ADAP possesses versatile roles in a variety of immune cells, including T cells, dendritic cells, macrophages, and platelets, etc. The most extensivelystudied role of ADAP is that it couples TCR activation to integrin activation and T-cell adhesion. However, the regulation of this adaptor during integrin activation and T-cell adhesion remains unclear. Meantime, the functions of ADAP linked to other immune cells are largely unknown. Work in this thesis have identified Ubc9, the sole SUMO E2 conjugase, as an essential regulator of ADAP in T-cell adhesion. We show that ADAP interacted directly with Ubc9 in vitro and in vivo, and the association was further strengthened in response to anti-CD3 stimulation. The Ubc9 binding domain on ADAP was mapped to a nuclear localisation sequence (aa 674-700) within ADAP. Knockdown of Ubc9 by shRNA or expression of the Ubc9-binding-deficient ADAP mutant significantly decreased TCRinduced integrin adhesion to ICAM-1 and fibronectin, as well as LFA-1 clustering, while having little effect on the TCR proximal signalling responses and TCR-induced IL-2 transcription. Furthermore, downregulation of Ubc9 impaired TCR-mediated Rac1 activation and attenuated the membrane targeting of Rap1 but not RIAM. Taken together, our data demonstrate for the first time that ADAP forms a functional interplay with Ubc9 and Ubc9 plays a selective role in integrin-mediated T-cell adhesion via modulation of Rap1 membrane recruitment and Rac1 activation. Another important finding of this thesis is the identification of a negative regulatory role for ADAP in the megakaryopoiesis. Here we show that in the bone marrow and spleen of ADAP-/- mice, a significant increase in the number of megakaryocytes were observed, and the ADAP-deficient megakaryocytes exhibited potentiated capacity in differentiation and development compared to the WT megakaryocytes. Mechanistically, ADAP directly interacted with STAT1, an indispensable modulator in megakaryopoiesis. Analysis on the activation of STAT1 showed that depletion of ADAP resulted in potentiated STAT1 phosphorylation and transcriptional activity, as well as upregulations of STAT1-regulatory genes. Collectively, these results suggest a novel role of ADAP in megakaryocytes, where ADAP attenuates megakaryopoiesis by direct interaction with STAT1 and negatively modulates the STAT1 activities. In summary, the work in this thesis have illustrated the diverse roles of ADAP in TCR-mediated integrin activation and megakaryopoiesis, and altogether contributed to our current knowledge of the many facets of ADAP in immunity.