Author(s) : J. Brockdorff, M. Nielsen, K. Kaltoft, T. Mustelin, C. Röpke, A. Svejaard, C. Geisler, N. Odum, Institute for Medical Microbiology and Immunology, Panum Institute, Building 22.5, Blegdamsvej 3,
DK-2200 Copenhagen N, Denmark..
Summary : The role of Lck in IL-2-induced proliferation and cell survival is still controversial. Here, we show that the Src family kinase inhibitor, PP1, reduced the IL-2-induced proliferation of human T cells significantly without inhibiting the anti-apoptotic effect of IL-2. As Lck is the only Src family kinase activated upon IL-2 stimulation in T cells, this indicates that Lck is involved in IL-2-induced proliferation but not survival. IL-2-induced MAP kinase activation was only slightly inhibited by PP1, suggesting that Lck is not essential for IL-2-induced MAP kinase activation in human T cells. We found that an IL-2-sensitive, human mycosis fungoides-derived tumor T cell line is Lck negative, and that the IL-2-induced MAP kinase activation is comparable to non-cancerous T cells, although a little delayed in kinetics. An Lck expressing clone was established by transfecting Lck into mycosis fungoides tumor T cells, but Lck had no influence on the delayed kinetics of MAP kinase activation, indicating that Lck is not essential for MAP kinase activation in mycosis fungoides tumor T cells or in non-cancerous T cells. Taken together, this indicates that Lck is involved in IL-2-induced proliferation, but not cell survival, through a pathway not involving MAP kinase.
Figure 1 PP1 inhibits proliferation but not survival signals. (A) Triplicates of 104 CD4+ T cells/sample
were incubated for 1, 3, or 5 days with or without 10 muM PP1 and with or
without IL-2. After the indicated days of culture [3H]thymidine
was added, and the cells were harvested twelve hours later followed by measurement
of the incorporated amount of [3H]thymidine. The results are
representative of 5 different proliferation assays. (B) 106
CD4+ T cells/sample were incubated with the indicated amount
of PP1 with or without IL-2. After 3 days of culture, the cells were harvested
and 7-ADD was added (see methods). Thepercentage of cells undergoing apoptosis
was measured by FACS analysis. (C) 106 CD4+
T cells/sample were incubated with the indicated amount of IL-2 with or
without 10 muM PP1. After 3 days of culture the cells were harvested and
7-ADD was added (see methods). The percentage of cells undergoing apoptosis
was measured by FACS analysis.
Figure 2 Effect of PP1 on IL-2-induced MAPK activation.
CD4+ T cells were incubated overnight with the indicated amount
of PP1, followed by 5 min of medium or IL-2 stimulation. After cell lysis,
the samples were analysed for activated Erk proteins by Western blotting,
using phospho-specific p44/42 mAb (top panel). The membrane was stripped
and reprobed with anti-Erk pAb (middel panel), stripped again and reprobed
with anti-phosphotyrosine mAb (bottom panel).
Figure 3 MyLa2059 is Lck negative. (A) 3 x 106 cells were lysed and analysed for Lck proteins
by Western blotting. (B) Total cellular RNA was isolated from MyLa2059
and Jurkat cells, followed by RT-PCR with primer pairs specific for Lck
(lanes 1 and 2 for Jurkat cells and lanes 4 and 5 for MyLa2059 cells) or
CD3-gamma chain (lane 3 for Jurkat cells, lane 6 for MyLa2059 cells, and
lane 7 for a control sample with no templates). After cDNA amplification
by PCR, the samples were analysed in a 1% agarose gel.
Figure 4 Timecourse of IL-2-induced MAPK activation. (A) CD4+ T cells and MyLa2059 cells were stimulated with
IL-2 for the indicated time, followed by cell lysis and analysis for activated
Erk proteins by Western blotting, using phospho-specific p44/42 mAb (top
panel). The membrane was stripped and reprobed with anti-Erk pAb (bottom
panel). (B) MyLa2059 cells were incubated with the indicated amount
of PP1 overnight, followed by stimulation with medium or IL-2 for 5 min,
cell lysis, and analysis for activated Erk proteins by Western blotting
(top panel). The membrane was stripped and reprobed with anti-Erk pAb (middle
panel), stripped again and reprobed with anti-phosphotyrosine mAb (bottom
panel).
Figure 5 Effect of PP1 on MAPK activation in MyLa2059-Lck cells.
MyLa2059 cells and MyLa2059 cells stably transfected with Lck (MyLa2059-Lck)
were stimulated with medium or IL-2 for the indicated time, followed by
cell lysis and analysis for either Lck (A) or activated Erk (B, top
panel) by Western blotting, using anti-Lck mAb or phospho-specific p44/42
mAb respectively. (B) The membrane was stripped and reprobed with
anti-Erk enpAb (bottom panel).
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