The SH3 domain of Itk/Emt binds to proline-rich sequences in the cytoplasmic domain of the T cell costimulatory receptor CD28.
Engagement of the transmembrane receptor CD28 potentiates T cell survival, proliferation, and activation. The biochemical basis by which CD28 controls these outcomes is unclear, although early events following cross-linking of the receptor are characterized by tyrosine phosphorylation of CD28 and other cellular substrates. We demonstrate that following CD28 ligation, a CD28-associated tyrosine kinase activity is increased in parallel to activation of the T cell-specific tyrosine kinase Itk (Itk/Emt), while Lck and Fyn kinase activities are not increased. We show that Itk forms an inducible complex with CD28, mediated by the SH3 domain of Itk and the diproline motifs of CD28. Site-directed mutagenesis of the N-terminal diproline motif of CD28 abrogates the association of CD28 with the SH3 domain of Itk, while mutations within the C-terminal diproline motif have little effect. Peptides corresponding to the N-terminal diproline motif were more efficient at abrogating the interaction between CD28 and the SH3 domain of Itk, than peptides corresponding to the C-terminal diproline motif. In addition, peptides corresponding to the N-terminal diproline motif of CD28 activated the tyrosine kinase activity of Itk to levels similar to those observed following Ab-mediated cross-linking of CD28. Together, our data show that the SH3 domain of Itk binds to a proline-rich motif within the cytoplasmic tail of CD28, and define a mechanism by which CD28 couples to and activates a downstream tyrosine kinase.
Marengère
L E
LE
Ontario Cancer Institute, Department of Medical Biophysics, University of Toronto, Canada.
Okkenhaug
K
K
Clavreul
A
A
Couez
D
D
Gibson
S
S
Mills
G B
GB
Mak
T W
TW
Rottapel
R
R
D000595:Amino Acid Sequence; D000818:Animals; D018106:CD28 Antigens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry / Q000201:enzymology*; D004151:Dipeptides / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008213:Lymphocyte Activation; D020930:MAP Kinase Kinase Kinases; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D055232:Proline-Rich Protein Domains; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013945:Thymoma; D014407:Tumor Cells, Cultured; D018909:src Homology Domains / Q000235:genetics / Q000276:immunology*; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11
D018106:CD28 Antigens; D004151:Dipeptides; D020930:MAP Kinase Kinase Kinases; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D010455:Peptides; D011392:Proline; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; C080813:emt protein-tyrosine kinase