Regulation and function of SKAP-55 non-canonical motif binding to the SH3c domain of adhesion and degranulation-promoting adaptor protein.
The immune cell adaptor adhesion and degranulation promoting adaptor protein (ADAP) and its binding to T-cell adaptor Src kinase-associated protein of 55 kDa (SKAP-55) play a key role in the modulation of T-cell adhesion. While primary binding occurs via SKAP-55 SH3 domain binding to a proline-rich region in ADAP, a second interaction occurs between the ADAP C-terminal SH3 domain (ADAP-SH3c) and a non-canonical RKXXY294XXY297 motif in SKAP-55. Increasing numbers of non-canonical SH3 domain binding motifs have been identified in a number of biological systems. The presence of tyrosine residues in the SKAP-55 RKXXY294XXY297 motif suggested that phosphorylation might influence this unusual SH3 domain interaction. Here, we show that the Src kinase p59fyn can induce the in vivo phosphorylation of the motif, and this event blocks ADAP-SH3c domain binding to the peptide motif. The importance of tyrosine phosphorylation was confirmed by plasmon resonance interaction analysis showing that phosphorylation of Tyr294 residue plays a central role in mediating dissociation, whereas phosphorylation of the second Tyr297 had no effect. Although loss of this secondary interaction did not result in the disruption of the complex, the Y294F mutation blocked T-cell receptor-induced up-regulation of lymphocyte function-associated antigen-1-mediated adhesion to intercellular adhesion molecule-1 and interleukin-2 promoter activity. Our findings identify a RKXXY294 motif in SKAP-55 that mediates unique ADAP SH3c domain binding and is needed for LFA-1-mediated adhesion and cytokine production.
Duke-Cohan
Jonathan S
JS
Department of Medical Oncology, Harvard Medical School, Boston, Massachusetts 02115. Electronic address: jonathan_duke-cohan@dfci.harvard.edu.
Kang
Hyun
H
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Departments of Medicine and Pathology, Harvard Medical School, Boston, Massachusetts 02115.
Liu
Hebin
H
Molecular Immunology Section, Department of Immunology, Division of Investigative Sciences, Division of Medicine, Imperial College, Hammersmith Campus, Du Cane Road, London W12 ONN, United Kingdom.
Rudd
Christopher E
CE
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Departments of Medicine and Pathology, Harvard Medical School, Boston, Massachusetts 02115; Molecular Immunology Section, Department of Immunology, Division of Investigative Sciences, Division of Medicine, Imperial College, Hammersmith Campus, Du Cane Road, London W12 ONN, United Kingdom. Electronic address: c.rudd@imperial.ac.uk.
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002448:Cell Adhesion; D002522:Chlorocebus aethiops; D016207:Cytokines / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D018909:src Homology Domains
D016207:Cytokines; D016169:Lymphocyte Function-Associated Antigen-1; D010750:Phosphoproteins; C108336:SKAP1 protein, human