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

J Immunol. 2017 Dec 15;199(12):4142-4154. doi: 10.4049/jimmunol.1700572. Epub 2017 Nov 10.


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.

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism*
  • Amino Acid Sequence
  • Animals
  • Apoptosis Regulatory Proteins
  • COS Cells
  • Cell Adhesion
  • Cell Membrane / metabolism*
  • Chlorocebus aethiops
  • Enzyme Activation
  • Gene Knockdown Techniques
  • Humans
  • Integrins / physiology
  • Jurkat Cells
  • Lymphocyte Function-Associated Antigen-1 / physiology
  • Mice
  • Models, Immunological
  • Monomeric GTP-Binding Proteins / metabolism*
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein Binding
  • Protein Processing, Post-Translational
  • RNA, Small Interfering / pharmacology
  • Receptors, Antigen, T-Cell / immunology
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction / physiology
  • T-Lymphocytes / cytology*
  • Ubiquitin-Conjugating Enzymes / antagonists & inhibitors
  • Ubiquitin-Conjugating Enzymes / genetics
  • Ubiquitin-Conjugating Enzymes / physiology*
  • rac1 GTP-Binding Protein / metabolism*
  • rap1 GTP-Binding Proteins / metabolism*


  • Adaptor Proteins, Signal Transducing
  • Apoptosis Regulatory Proteins
  • FYB1 protein, human
  • Integrins
  • Lymphocyte Function-Associated Antigen-1
  • Phosphoproteins
  • RAC1 protein, human
  • RASSF5 protein, human
  • RNA, Small Interfering
  • Receptors, Antigen, T-Cell
  • Recombinant Fusion Proteins
  • SKAP1 protein, human
  • Ubiquitin-Conjugating Enzymes
  • Monomeric GTP-Binding Proteins
  • rac1 GTP-Binding Protein
  • rap1 GTP-Binding Proteins
  • ubiquitin-conjugating enzyme UBC9