Disruption of SLP-76 interaction with Gads inhibits dynamic clustering of SLP-76 and FcepsilonRI signaling in mast cells.
We developed a confocal real-time imaging approach that allows direct observation of the subcellular localization pattern of proteins involved in proximal FcepsilonRI signaling in RBL cells and primary bone marrow-derived mast cells. The adaptor protein Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is critical for FcepsilonRI-induced calcium flux, degranulation, and cytokine secretion. In this study, we imaged SLP-76 and found it in the cytosol of unstimulated cells. Upon FcepsilonRI cross-linking, SLP-76 translocates to the cell membrane, forming clusters that colocalize with the FcepsilonRI, the tyrosine kinase Syk, the adaptor LAT, and phosphotyrosine. The disruption of the SLP-76 interaction with its constitutive binding partner, Gads, through the mutation of SLP-76 or the expression of the Gads-binding region of SLP-76, inhibits the translocation and clustering of SLP-76, suggesting that the interaction of SLP-76 with Gads is critical for appropriate subcellular localization of SLP-76. We further demonstrated that the expression of the Gads-binding region of SLP-76 in bone marrow-derived mast cells inhibits FcepsilonRI-induced calcium flux, degranulation, and cytokine secretion. These studies revealed, for the first time, that SLP-76 forms signaling clusters following FcepsilonRI stimulation and demonstrated that the Gads-binding region of SLP-76 regulates clustering of SLP-76 and FcepsilonRI-induced mast cell responses.
Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Michael A
MA
Silverman
Jonathan
J
Shoag
Jennifer
J
Wu
Gary A
GA
Koretzky
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D051790:G-Box Binding Factors / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008407:Mast Cells / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D017455:Receptors, IgE / Q000235:genetics / Q000378:metabolism*; D000072377:Syk Kinase
D048868:Adaptor Proteins, Signal Transducing; D016207:Cytokines; D051790:G-Box Binding Factors; D047908:Intracellular Signaling Peptides and Proteins; C491734:Lat protein, mouse; D008565:Membrane Proteins; C498726:Mona protein, mouse; D010750:Phosphoproteins; D017455:Receptors, IgE; C092947:SLP-76 signal Transducing adaptor proteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606897:Syk protein, mouse; C000606898:Syk protein, rat; D002118:Calcium