Approaches to define antigen receptor-induced serine kinase signal transduction pathways.
In the present report we describe the properties of a novel phospho-specific antiserum that has opened a route to the characterization of antigen receptor-activated serine kinase pathways in lymphocytes. The basis for the present work was that Ser-21 in glycogen synthase kinase 3alpha is robustly phosphorylated following antigen receptor triggering. We predicted accordingly that antigen receptors would also stimulate phosphorylation of other proteins with a similar sequence. To test this idea we raised an antibody against the phospho-peptide RARTSpSFAEP, where pS is a phospho-serine corresponding to the glycogen synthase kinase 3alpha Ser-21 sequence. The resulting antiserum was called phospho antibody for proteomics-1 (PAP-1). The present study describes the properties of PAP-1 and shows that it can reveal quite striking differences in the phospho-proteome of different cell types and is able to pinpoint new targets in important signal transduction pathways. PAP-1 was used to map protein phosphorylations regulated by the antigen receptor in T cells. One of these PAP-1-reactive proteins was purified and revealed to be a previously unrecognized target for antigen receptor signal transduction, namely an "orphan" adapter SLY (Src homology 3 (SH3) domain-containing protein expressed in lymphocytes). The use of sera detecting specific phosphorylation sites is thus proved as a powerful method for the discovery of novel downstream components of antigen receptor signals in T cells.
Astoul
Emmanuelle
E
Lymphocyte Activation Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
Laurence
Arian D
AD
Totty
Nick
N
Beer
Sandra
S
Alexander
Denis R
DR
Cantrell
Doreen A
DA
D020816:Amino Acid Motifs; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D002845:Chromatography; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D038362:Glycogen Synthase Kinase 3 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D000073718:Pancreatitis-Associated Proteins; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010857:Pigment Epithelium of Eye / Q000166:cytology; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013601:T-Lymphocytes / Q000378:metabolism; D018909:src Homology Domains
D000073718:Pancreatitis-Associated Proteins; D010455:Peptides; D011518:Proto-Oncogene Proteins; C000612141:REG3A protein, human; D011948:Receptors, Antigen, T-Cell; D012694:Serine; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; C448021:glycogen synthase kinase 3 alpha