The Autoimmunity Risk Variant LYP-W620 Cooperates with CSK in the Regulation of TCR Signaling
Figure 5
LYP is phosphorylated in tyrosine.
A, Lysates of PBLs stimulated with Abs against CD3 and CD28 molecules were subjected to IP with anti-LYPAb and immunoblotted with anti-phospho-Y Ab 4G10 to detect LYP Y-phosphorylation. After stripping, the membrane was probed with anti-LYP Ab to verify equal loading of LYP. Zap70 phosphorylation with anti-Phospho-Y319 was tested to check activation of the cells. The blot showing LYP Tyr phosphorylation (P-Y) was measured by densitometry and the data were expressed as arbitrary units under the blot. B, Lysates of Jurkat cells transfected with myc-LYPR-DA and the indicated kinases were subjected to IP to detect Lyp Y-phosphorylation as in A. Expression of the kinases transfected was detected by Western blot with anti-HA antibody, where HA was present, or with specific antibodies for the untagged kinases. C, LYP Y-phoshophorylation was studied in different Jurkat derived cell lines deficient in LCK (JCaM1.6) and Zap70 (P116) for comparison with Jurkat parental cells. Phosphorylation of endogenous LYP after IP was detected as aforementioned. D, In vitro phosphorylation of myc-LYP-RDA by recombinant LCK, LYP was immunoprecipitated from HEK293 transfected cells and active recombinant LCK was added to the beads along with ATP and the kinase buffer. The reaction was incubated at 30°C for 30 min. and LYP phosphorylation was detected as before. E, HEK293 cells were transfected with several myc-LYPR-DA Tyr to Phe mutants along with LCK. Phosphorylation of LYP was detected by IB with 4G10 Ab after IP of LYP. F, Lysates of Jurkat cells transfected with myc-LYPR-DA or myc-LYPW-DA along with LCK were subjected to IP and phosphorylation was detected as before. G, Activation of a luciferase reporter gene driven by the IL-2 minimal promoter in Jurkat cells cotransfected with different LYP plasmids, as indicated. The insert shows the expression of LYP proteins by IB.