Roles of the proline-rich domain in SLP-76 subcellular localization and T cell function.
The adaptor protein Src homology (SH)2 domain-containing and leukocyte-specific phosphoprotein of 76 kDa (SLP-76) is critical for signal transduction in multiple hematopoietic lineages. It links proximal and distal T cell receptor signaling events through its function as a molecular scaffold in the assembly of multimolecular signaling complexes. Here we studied the functional roles of sub-domains within the SLP-76 proline-rich region, specifically the Gads binding domain and the recently defined P1 domain. To gain a further understanding of the functions mediated by this region, we used three complementary approaches as follows: reconstitution of SLP-76-deficient cells with functional domain deletion mutants, blocking molecular associations through the expression of a dominant negative protein fragment, and directed localization of SLP-76 to assess the role of the domains in SLP-76 recruitment. We find the Gads binding domain and the P1 domain are both necessary for optimal SLP-76 function, and in the absence of these two regions, SLP-76 is functionally inert. Furthermore, we provide direct evidence that SLP-76 localization and, in turn, function are dependent upon association with Gads.
Singer
Andrew L
AL
Signal Transduction Program, Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Bunnell
Stephen C
SC
Obstfeld
Amrom E
AE
Jordan
Martha S
MS
Wu
Jennifer N
JN
Myung
Peggy S
PS
Samelson
Lawrence E
LE
Koretzky
Gary A
GA
D048868:Adaptor Proteins, Signal Transducing; D000409:Alanine / Q000737:chemistry; D015703:Antigens, CD / Q000096:biosynthesis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis; D001120:Arginine / Q000737:chemistry; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D019070:Cell Lineage; D005434:Flow Cytometry; D017353:Gene Deletion; D005799:Genes, Dominant; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D037181:Lectins, C-Type; D008156:Luciferases / Q000378:metabolism; D008213:Lymphocyte Activation; D008239:Lysine / Q000737:chemistry; D021962:Membrane Microdomains / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011392:Proline / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D018909:src Homology Domains; D000099192:CD69 Antigens
D048868:Adaptor Proteins, Signal Transducing; D000409:Alanine; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D001120:Arginine; D002118:Calcium; D037181:Lectins, C-Type; D008156:Luciferases; D008239:Lysine; D010750:Phosphoproteins; D011392:Proline; D000099192:CD69 Antigens; C092947:SLP-76 signal Transducing adaptor proteins