Ubc9 Binds to ADAP and Is Required for Rap1 Membrane Recruitment, Rac1 Activation, and Integrin-Mediated T Cell Adhesion

• Published in: The Journal of immunology (1950) Vol. 199; no. 12; pp. 4142 - 4154
• Main Authors: Xiong, Yiwei, Ye, Chengjin, Yang, Naiqi, Li, Madanqi, Liu, Hebin
• Format: Journal Article
• Language: English
• Published: United States American Association of Immunologists 15.12.2017
• Subjects: Adaptor Proteins, Signal Transducing - metabolism; Adhesion; Amino Acid Sequence; Animals; CD3 antigen; Cell activation; Cell Adhesion; Cell adhesion & migration; Cell Membrane - metabolism; Cercopithecus aethiops; COS Cells; Degranulation; Enzyme Activation; Fibronectin; Gene Knockdown Techniques; Guanosine triphosphatases; Humans; Integrins - physiology; Intercellular adhesion molecule 1; Interleukin 2; Jurkat Cells; LFA-1 antigen; Localization; Lymphocyte Function-Associated Antigen-1 - physiology; Lymphocytes; Lymphocytes T; Mice; Models, Immunological; Monomeric GTP-Binding Proteins - metabolism; Phosphoproteins - genetics; Phosphoproteins - metabolism; Phosphorylation; Protein Binding; Protein Processing, Post-Translational; rac1 GTP-Binding Protein - metabolism; Rac1 protein; rap1 GTP-Binding Proteins - metabolism; Rap1 protein; Receptors, Antigen, T-Cell - immunology; Recombinant Fusion Proteins - metabolism; Recruitment; Ribonucleic acid; RNA; RNA, Small Interfering - pharmacology; Signal Transduction - physiology; T cell receptors; T-Lymphocytes - cytology; Transcription; Ubiquitin; Ubiquitin-Conjugating Enzymes - antagonists & inhibitors; Ubiquitin-Conjugating Enzymes - genetics; Ubiquitin-Conjugating Enzymes - physiology
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.1700572

Although the immune adaptor adhesion and degranulation-promoting adaptor protein (ADAP) acts as a key mediator of integrin inside-out signaling leading to T cell adhesion, the regulation of this adaptor during integrin activation and clustering remains unclear. We now identify Ubc9, the sole small ubiquitin-related modifier E2 conjugase, as an essential regulator of ADAP where it is required for TCR-induced membrane recruitment of the small GTPase Rap1 and its effector protein RapL and for activation of the small GTPase Rac1 in T cell adhesion. We show that Ubc9 interacted directly with ADAP in vitro and in vivo, and the association was increased in response to anti-CD3 stimulation. The Ubc9-binding domain on ADAP was mapped to a nuclear localization sequence (aa 674-700) within ADAP. Knockdown of Ubc9 by short hairpin RNA or expression of the Ubc9-binding-deficient ADAP mutant significantly decreased TCR-induced integrin adhesion to ICAM-1 and fibronectin, as well as LFA-1 clustering, although it had little effect on the TCR proximal signaling responses and TCR-induced IL-2 transcription. Furthermore, downregulation of Ubc9 impaired TCR-mediated Rac1 activation and attenuated the membrane targeting of Rap1 and RapL, but not Rap1-interacting adaptor molecule. Taken together, our data demonstrate for the first time, to our knowledge, that Ubc9 acts as a functional binding partner of ADAP and plays a selective role in integrin-mediated T cell adhesion via modulation of Rap1-RapL membrane recruitment and Rac1 activation.