Increases in tyrosine phosphorylation are detectable before phospholipase C activation after T cell receptor stimulation.
Antiphosphotyrosine immunoblots were used to characterize tyrosine phosphorylated proteins after stimulation of the human TCR. Increased tyrosine phosphorylation was evident on at least 12 substrates within 2 min after ligation of the TCR with mAb. Analysis of the time course for increased tyrosine phosphorylation revealed distinct patterns. Increased phosphorylation of 135-kDa and 100-kDa substrates was evident within 5 s, whereas increased phosphorylation of the TCR-zeta-chain required several minutes after treatment with anti-CD3 mAb. This rapid cellular tyrosine phosphorylation occurred independent of the cell cycle, as it occurred after stimulation of resting T cells, T cell blasts, and the Jurkat T cell leukemia line. When the TCR complex was cross-linked together with the CD4 receptor by heteroconjugate anti-CD3/CD4 mAb, an increased magnitude of tyrosine phosphorylation occurred, although no new substrates could be detected. The increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates was specific in that anti-HLA class I, anti-CD6, anti-CD7, and anti-CD28 antibodies did not cause increased tyrosine phosphorylation. Anti-CD4 stimulation of resting T cells did not cause increased tyrosine phosphorylation of pp100 and pp135, suggesting that the CD4-associated kinase, lck, does not account for the tyrosine phosphorylation observed after TCR stimulation. Similarly, pharmacologic treatment of cells with phorbol ester and calcium ionophore did not cause increased tyrosine phosphorylation of these substrates, indicating that activation of protein kinase C or phospholipase C does not account for these early increases in tyrosine phosphorylation. The time of onset of pp100 phosphorylation, and the magnitude of phosphorylation correlated with the magnitude of calcium mobilization when cells were stimulated with different forms of TCR stimulation. When cells were labeled with [3H]myoinositol and analyzed after stimulation by anti-CD3 mAb, increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates preceded the activation of phospholipase C, as measured by the appearance of inositol 1,4,5-trisphosphate. This occurred in both T cell blasts and in the Jurkat T cell line. Thus, these findings show that increased tyrosine phosphorylation is the earliest yet detected signal observed after ligation of the TCR complex, and furthermore suggest that tyrosine phosphorylation might link the TCR to the phosphatidylinositolbisphosphate hydrolysis signaling pathway.
Naval Medical Research Institute, Bethesda, MD 20814.
C H
CH
June
M C
MC
Fletcher
J A
JA
Ledbetter
L E
LE
Samelson
D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000502:physiology; D017252:CD3 Complex; D015704:CD4 Antigens / Q000502:physiology; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism*; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011940:Receptor Aggregation; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D015398:Signal Transduction; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D015704:CD4 Antigens; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases