Figure 1.
LYP binds to CSK in an inducible manner.
A, Total lysates (TL) of HEK293 cells transiently transfected with LYP tagged with the myc epitope and HA-CSK, including the empty vector pEF as control, and treated or untreated with pervanadate (PV) for 5 min were subjected to immunoprecipitation (IP) and immunoblot (IB) with the indicated antibodies. B, Lysates from Jurkat cells transfected with plasmids that express myc-LYPR-DA or myc-LYPW-DA treated with PV for 5 minutes were subjected to IP with anti-myc Ab and then blotted with anti-HA (upper panel) to detect CSK. Similar expression of the proteins in the assay was detected by IB in the TL. C, Lysates of Jurkat cells untreated (resting cells), treated with PV or stimulated with anti-CD3 and anti-CD28 Abs for 5 min were subjected to IP with anti-CSK Ab, anti-LYP Ab, or an irrelevant Ab used as control, and inmunoblotted against endogenous LYP and CSK. D, T lymphocytes obtained from peripheral blood of healthy donors were incubated for the indicated times at 37°C with medium alone or in the presence of anti-CD3 and anti-CD28 Ab. Lysates from these cells were immunoprecipitated with anti-CSK or an irrelevant Ab (IgG) to show specificity, and the presence of LYP and CSK in the precipitates was detected with specific Abs by IB. LYP blot was measured by densitometry and the values obtained, shown under the blot, are expressed in arbitrary units.
Figure 2.
P1 and P2 LYP motifs bind to CSK.
A, Lysates of HEK293 cells transfected with LYP and a deletion mutant of LYP tagged with the myc epitope that lacks the CTH PRM, tagged with the myc epitope, along with HA-CSK plasmids were immunoprecipitated and immunoblotted with the indicated Abs. Expression of these proteins was verified by IB in TL. B, Lysates from 3×108 Jurkat cells were divided equally into five tubes and were subjected to pull-down assays with the indicated PRMs fused to GST. The presence of CSK in the precipitates was detected by IB with CSK Ab and GST-peptides were detected using a GST Ab. TL shows the presence of CSK in the lysate. C, Lysates of HEK293 cells transfected with different LYP mutants in the P1 and P2 motifs, tagged with the myc epitope, along with HA-CSK were subjected to IP and IB as indicated. D, Interaction of HA-CSK with a myc-LYP mutant in the C-terminus of the P1 motif, IV, (Ile626Ala,Val627 Ala) was verified by IB after LYP IP in transiently transfected HEK293 cells. E, Lysates of HEK293 cells transfected with HA-CSK and different mutants of LYP in the P1, the P2, or in both motifs, tagged with the myc epitope, were immunoprecipitated and immunoblotted with the indicated Abs. F, Arg to Phe LYP mutants in P1 and P2 PRM tagged with myc were expressed in HEK293 cells and interaction with HA-CSK was detected by immunoblot after LYP immunoprecipitation. CSK blots in panels B, C, D, E and F were scanned and the values obtained were expressed as arbitrary units under the blot.
Figure 3.
CSK SH3 and SH2 domains are involved in the association with LYP.
A, Schematic representation of the NMR structure of the Pro rich motif P1 of Pep (orange) bound to the SH3 domain of CSK (blue) (PDB code 1JEG). P1 resid ues are numbered according to the LYP sequence (613IPPPLPVRTPESFIVVEE630). Arg620 is involved in intermolecular polar contacts with Asp27 and Gln26 of CSK; hydrogen bonds are shown by dashed lines. In addition, Arg620 could establish an intramolecular hydrogen bond with Ser624. B, Jurkat cells were electroporated with HA-CSK wild type and several mutants of CSK SH3 domain along myc-LYPR as indicated. Interaction was detected by IB after LYP IP. C, Several HA-CSK mutants in the SH3 and SH2 domains were tested for interaction with myc-LYP-RDA. Jurkat cells were left untreated or treated with PV and LYP was immunoprecipitated from cell lysates. CSK interaction was detected by IB with specific anti-HA antibody. D, Activation of a luciferase reporter gene driven by the IL-2 minimal promoter in Jurkat cells cotransfected with different CSK plasmids, as indicated. The insert shows the IB of the CSK proteins expressed.
Figure 4.
LYP/CSK interaction is not required to regulate TCR signaling.
A, Activation of a luciferase reporter gene driven by the IL-2 minimal promoter in Jurkat cells co-transfected with different myc-LYP plasmids, as indicated. The insert shows the expression of the LYP proteins as detected by IB. B, Erk activation was assayed in Jurkat cells transfected with different versions of LYP, as indicated, and stimulated with anti-CD3 Ab for 5 min. Erk was immunoprecipitated from lysates of these cells and its phosphorylation was detected by IB. Expression was verified in total lysates (TL) by IB. Phospho-ERK (P-Erk) blot was measured by densitometry and the data were expressed as arbitrary units under the blot.C, As in B, p38 activity was evaluated in Jurkat T cells stimulated with anti-CD3 and anti-CD28 Ab for 30 min by IP of HA-p38 and IB with a specific antibody for dually phosphorylated p38. Phospho-p38 (P-p38) blot was measured by densitometry scanning and the data were expressed as arbitrary units under the blot. D, Activation of a luciferase reporter gene driven by the NF-AT/AP1 site of IL-2 promoter in Jurkat cells co-transfected with LYPR, LYPTW, and CSK-W47A plasmids, as indicated. Expression of LYP and CSK proteins as detected by IB is shown in the insert. E, Activation of a luciferase reporter gene driven by the IL-2 minimal promoter in Jurkat cells transfected with LYPR, LYPW, and CSK-W47A plasmids. The insert shows the IB of LYP and CSK proteins. R, LYPR; W, LYPW. F, Expression of CD25 in Jurkat cells transfected with the plasmids indicated was measured by flow cytometry upon stimulation with anti-CD3 plus anti-CD28 antibodies for 24 hours. Expression of LYP and CSK proteins as detected by IB is shown in the insert.
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.