SLP-65 phosphorylation dynamics reveals a functional basis for signal integration by receptor-proximal adaptor proteins.
Understanding intracellular signal transduction by cell surface receptors requires information about the precise order of relevant modifications on the early transducer elements. Here we introduce the B cell line DT40 and its genetically engineered variants as a model system to determine and functionally characterize post-translational protein modifications in general. This is accomplished by a customized strategy that combines mass spectrometric analyses of protein modifications with subsequent mutational studies. When applied to the B cell receptor (BCR)-proximal effector SLP-65, this approach uncovered a differential and highly dynamic engagement of numerous newly identified phospho-acceptor sites. Some of them serve as kinase substrates in resting cells and undergo rapid dephosphorylation upon BCR ligation. Stimulation-induced phosphorylation of SLP-65 can be early and transient, or early and sustained, or late. Functional elucidation of conspicuous phosphorylation at serine 170 in SLP-65 revealed a BCR-distal checkpoint for some but not all possible B cell responses. Our data show that SLP-65 phosphorylation acts upstream for signal initiation and also downstream during selective processing of the BCR signal. Such a phenomenon defines a receptor-specific signal integrator.
Oellerich
Thomas
T
Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany.
Grønborg
Mads
M
Neumann
Konstantin
K
Hsiao
He-Hsuan
HH
Urlaub
Henning
H
Wienands
Jürgen
J
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; C113939:B cell linker protein; D010455:Peptides; D011947:Receptors, Antigen, B-Cell; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D020928:Mitogen-Activated Protein Kinases