T cell activation-induced CrkII binding to the Zap70 protein tyrosine kinase is mediated by Lck-dependent phosphorylation of Zap70 tyrosine 315.
The Zap70 protein tyrosine kinase controls TCR-linked signal transduction pathways and is critical for T cell development and responsiveness. Following engagement of TCR, the Zap70 undergoes phosphorylation on multiple tyrosine residues that are implicated in the regulation of its catalytic activity and interaction with signaling effector molecules downstream of the TCR. We have shown previously that the CT10 regulator of kinase II (CrkII) adapter protein interacts with tyrosine-phosphorylated Zap70 in TCR-engaged T cells, and now extend these studies to show that Tyr315 in the Zap70 interdomain B region is the site of interaction with CrkII. A point mutation of Tyr315 (Y315F) eliminated the CrkII-Zap70 interaction capacity. Phosphorylation of Tyr315 and Zap70 association with CrkII were both dependent upon the Lck protein tyrosine kinase. Previous studies demonstrated the Tyr315 is the Vav-Src homology 2 (SH2) binding site, and that replacement of Tyr315 by Phe impaired the function of Zap70 in TCR signaling. However, fluorescence polarization-based binding studies revealed that the CrkII-SH2 and the Vav-SH2 bind a phosphorylated Tyr315-Zap70-derived peptide with affinities of a similar order of magnitude (Kd of 2.5 and 1.02 microM, respectively). The results suggest therefore that the biological functions attributed to the association of Zap70 with Vav following T cell activation may equally reflect the association of Zap70 with CrkII, and further support a regulatory role for CrkII in the TCR-linked signal transduction pathway.
Gelkop
Sigal
S
Department of Microbiology and Immunology, Faculty of Health Sciences, and the Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva, Israel.
Gish
Gerrald D
GD
Babichev
Yael
Y
Pawson
Tony
T
Isakov
Noah
N
D001665:Binding Sites; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000502:physiology*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D050737:Proto-Oncogene Proteins c-crk / Q000378:metabolism*; D051567:Proto-Oncogene Proteins c-vav / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000378:metabolism*
D050737:Proto-Oncogene Proteins c-crk; D051567:Proto-Oncogene Proteins c-vav; D014443:Tyrosine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human