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

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.

LFA-1 activates focal adhesion kinases FAK1/PYK2 to generate LAT-GRB2-SKAP1 complexes that terminate T-cell conjugate formation

“…Enhanced TCR ligation at the nascent IS would activate ZAP-70 to induce classic activating LAT- PLCγ1/SLP-76 complexes. ITK, RLK and ACK (Cdc42-associated kinase 1) phosphorylate SLP-76 (refs 71 , 72 , 73 ), whereas PLCγ1 and GADs-SLP-76 mobilize calcium and activate the ERK pathway 9 11 . However, with the maturation of the IS and subsequent clustering, the cross-linking of LFA-1 (either alone or in conjunction with the TCR) would become increasing dominant with increased numbers of LAT-GRB-2 SKAP1 complexes at the expense of activating LAT-GADs-SLP-76- complexes.…”

Small Molecules Targeting Activated Cdc42-Associated Kinase 1 (ACK1/TNK2) for the Treatment of Cancers

“…ACK1 combines with Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) and phosphorylates it at Tyr113, Tyr128, and Tyr145, which allows pSLP-76 to further recruit many other signaling molecules to form multimeric complexes and guarantees the differentiation of the thymus and the function of mature T cells. − In addition, Thaker et al found that ACK1 could affect the movement of T cells. Exogenous ACK1 can decrease the random mobility of T cells on ICAM-1-coated plates.…”

Research Progress of the Functional Role of ACK1 in Breast Cancer

“…The biological functions of some domains have been reported. For instance, the SAM domain was involved with the membrane localization, dimerization, and activation of ACK1 [18, 19]. The CRIB domain mediates the interaction between ACK1 and Cdc42 [3, 20], and the PPXY motif mediates the interaction between ACK1 and WW domain [21].…”