Activated Cdc42-associated kinase 1 (ACK1) binds the SAM domain of adaptor SLP-76 and phosphorylates proximal tyrosines [Immunology]

The adaptor protein Src homology 2 domain-containing leukocyte phospho-protein of 76 kDa (SLP-76) plays a crucial role in T-cell activation by linking antigen receptor (TCR) signals to downstream pathways. At its N-terminus, SLP-76 has three key N-terminus tyrosines (Y113, Y128 and Y145, or 3Y) as well as a sterile-α motif (SAM) domain, whose function is unclear. We previously showed 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 Y113, Y128 and Y145 to phenylalanine (3Y3F). ACK1 induced phosphorylation of 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.

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