SAP increases FynT kinase activity and is required for phosphorylation of SLAM and Ly9.
The free Src homology 2 (SH2) domain protein SAP, encoded by the X-linked lymphoproliferative disease gene SH2D1A, controls signal transduction initiated by engagement of the SLAM-related receptors in T and NK cells. Here we demonstrate that SAP is required for phosphorylation of both SLAM and Ly9 in thymocytes and peripheral T cells. Furthermore, in vitro protein interaction studies and yeast two-hybrid analyses indicated that SAP binds directly to FynT and Lck. While SAP bound to both the SH3 domain and to the kinase domain of FynT, SAP bound solely to the kinase domain of Lck. The existence of a strong interaction between SAP and the SH3 domain of FynT prompted us to study the role of SAP in modulating the activity of FynT. In vitro addition of SAP to the autoinhibited form of FynT caused a large increase in FynT catalytic activity. By contrast, the SAP mutant R78E, which is unable to bind to the FynT SH3 domain, did not increase FynT activity and also displayed a reduced adaptor function upon transfection into T cells. Our results demonstrate that SAP is an adaptor that bridges SLAM and Ly9 with Src-like protein tyrosine kinases (PTKs), and has the ability to activate FynT.
Simarro
Maria
M
Division of Immunology, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA. msimarro@rics.bwh.harvard.edu
Lanyi
Arpad
A
Howie
Duncan
D
Poy
Florence
F
Bruggeman
Joost
J
Choi
Michelle
M
Sumegi
Janos
J
Eck
Michael J
MJ
Terhorst
Cox
C
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D002460:Cell Line; D006023:Glycoproteins / Q000378:metabolism*; D007136:Immunoglobulins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D010766:Phosphorylation; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000819:agonists / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D011956:Receptors, Cell Surface; D015398:Signal Transduction; D000071179:Signaling Lymphocytic Activation Molecule Associated Protein; D000071176:Signaling Lymphocytic Activation Molecule Family; D000071177:Signaling Lymphocytic Activation Molecule Family Member 1; D013601:T-Lymphocytes / Q000201:enzymology / Q000276:immunology*; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000378:metabolism
D015703:Antigens, CD; D006023:Glycoproteins; D007136:Immunoglobulins; D047908:Intracellular Signaling Peptides and Proteins; C498972:Ly9 protein, mouse; D011518:Proto-Oncogene Proteins; D011956:Receptors, Cell Surface; C499569:Sh2d1a protein, mouse; D000071179:Signaling Lymphocytic Activation Molecule Associated Protein; D000071176:Signaling Lymphocytic Activation Molecule Family; D000071177:Signaling Lymphocytic Activation Molecule Family Member 1; D014443:Tyrosine; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn