p56Lck and p59Fyn regulate CD28 binding to phosphatidylinositol 3-kinase, growth factor receptor-bound protein GRB-2, and T cell-specific protein-tyrosine kinase ITK: implications for T-cell costimulation.
T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, zeta-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.
Raab
M
M
Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Cai
Y C
YC
Bunnell
S C
SC
Heyeck
S D
SD
Berg
L J
LJ
Rudd
C E
CE
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D018106:CD28 Antigens / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D066246:ErbB Receptors / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D018106:CD28 Antigens; D018076:DNA, Complementary; D051380:GRB2 Adaptor Protein; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051076:Proto-Oncogene Proteins c-fyn; C080813:emt protein-tyrosine kinase; D019061:src-Family Kinases