Knockdown of C-terminal Src kinase by siRNA-mediated RNA interference augments T cell receptor signaling in mature T cells.
C-terminal Src kinase (Csk) controls the Src family kinase Lck, which is essential for T cell antigen receptor (TCR)-mediated signaling. For the first time, we here report the effects of acute elimination of Csk in Jurkat T cells and primary T cells using short interfering (si) RNA. In both cell types, 70-85% knockdown of Csk was achieved within 48 h. No alterations in surface expression of CD3, CD4 or CD8, or in Lck protein level were observed. Phosphorylation of Y505 in Lck was markedly reduced and a concomitant 4-5-fold increase in Lck Y394 phosphorylation was observed both in normal and Jurkat T cells. Kinase assays revealed 2-3-fold higher Lck activity. In Jurkat cells, basal levels of zeta chain phosphorylation were elevated, and spontaneous NFAT-AP-1 activation occurred, indicating aberrant Lck kinase activity. After TCR triggering, Csk knockdown cells revealed faster and stronger, but not sustained, phosphorylation of Lck Y394 and zeta chains compared to control. TCR-induced activation of NFAT-AP-1 and TCR/CD28-stimulated IL-2 secretion occurred at weaker stimuli and with augmented responses in Csk knockdown Jurkat and primary T cells, respectively. Altogether, these data suggest that acute elimination of Csk in T cells without evolution of compensatory mechanisms results in aberrant Lck activity and augmented TCR-stimulated responses.
Vang
Torkel
T
The Biotechnology Center of Oslo, University of Oslo, Blindern, N-0317 Oslo, Norway.
Abrahamsen
Hilde
H
Myklebust
Sondre
S
Enserink
Jorrit
J
Prydz
Hans
H
Mustelin
Tomas
T
Amarzguioui
Mohammed
M
Tasken
Kjetil
K
D000081247:CSK Tyrosine-Protein Kinase; D006801:Humans; D019169:Jurkat Cells; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013601:T-Lymphocytes / Q000201:enzymology / Q000378:metabolism*; D019061:src-Family Kinases
D034741:RNA, Small Interfering; D011948:Receptors, Antigen, T-Cell; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases; C000631346:CSK protein, human