Activated Cdc42-associated kinase 1 (ACK1) binds the sterile α motif (SAM) domain of the adaptor SLP-76 and phosphorylates proximal tyrosines

J Biol Chem. 2017 Apr 14;292(15):6281-6290. doi: 10.1074/jbc.M116.759555. Epub 2017 Feb 10.

Abstract

The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a crucial role in T cell activation by linking antigen receptor (T cell receptor, TCR) signals to downstream pathways. At its N terminus, SLP-76 has three key tyrosines (Tyr-113, Tyr-128, and Tyr-145, "3Y") as well as a sterile α motif (SAM) domain whose function is unclear. We showed previously that the SAM domain has two binding regions that mediate dimer and oligomer formation. In this study, we have identified SAM domain-carrying non-receptor tyrosine kinase, activated Cdc42-associated tyrosine kinase 1 (ACK1; also known as Tnk2, tyrosine kinase non-receptor 2) as a novel binding partner of SLP-76. Co-precipitation, laser-scanning confocal microscopy, and in situ proximity analysis confirmed the binding of ACK1 to SLP-76. Further, the interaction was induced in response to the anti-TCR ligation and abrogated by the deletion of SLP-76 SAM domain (ΔSAM) or mutation of Tyr-113, Tyr-128, and Tyr-145 to phenylalanine (3Y3F). ACK1 induced phosphorylation of the SLP-76 N-terminal tyrosines (3Y) dependent on the SAM domain. Further, ACK1 promoted calcium flux and NFAT-AP1 promoter activity and decreased the motility of murine CD4+ primary T cells on ICAM-1-coated plates, an event reversed by a small molecule inhibitor of ACK1 (AIM-100). These findings identify ACK1 as a novel SLP-76-associated protein-tyrosine kinase that modulates early activation events in T cells.

Keywords: T cell; cell signaling; cluster of differentiation 4 (CD4); immunology; non-receptor tyrosine kinase (nRTK); protein phosphorylation; sterile α motif (SAM).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / immunology
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Amino Acid Motifs
  • Amino Acid Substitution
  • Animals
  • Humans
  • Jurkat Cells
  • Lymphocyte Activation / physiology*
  • Mice
  • Mutation, Missense
  • Phosphoproteins / genetics
  • Phosphoproteins / immunology
  • Phosphoproteins / metabolism*
  • Phosphorylation / physiology
  • Protein Domains
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / immunology
  • Protein-Tyrosine Kinases / metabolism*
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / immunology
  • Receptors, Antigen, T-Cell / metabolism*
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism*
  • Tyrosine

Substances

  • Adaptor Proteins, Signal Transducing
  • Phosphoproteins
  • Receptors, Antigen, T-Cell
  • SLP-76 signal Transducing adaptor proteins
  • Tyrosine
  • Tnk2 protein, mouse
  • Protein-Tyrosine Kinases
  • TNK2 protein, human