The calcium-independent protein kinase C participates in an early process of CD3/CD28-mediated induction of thymocyte apoptosis.
Thymocyte negative selection eliminates self-reactive clones and involves both a T-cell receptor (TCR)/CD3-mediated signal and a costimulatory signal, which can be delivered via CD28. Anti-CD3/anti-CD28-triggered apoptosis in isolated CD4+CD8+ thymocytes in vitro provides a basic model for negative selection. Effects of isoform-selective and non-isoform-selective inhibitors of protein kinase C (PKC) on this apoptotic process suggest that activation of Ca2+-independent PKC isoforms during the first 2-3 hr of culture is essential for inducing apoptosis, and that Ca2+-dependent PKC isoforms may be influential, but not essential, for apoptosis. To assess the CD3/CD28-mediated activation of PKC in the apoptotic process, we prepared CD4+CD8+ thymocytes (without contamination with cells that had received negative or positive selection signals in vivo) by establishing TCR-transgenic mice with RAG-2-deficient and non-selecting major histocompatibility complex (MHC) backgrounds, in addition to a CD4+CD8+ thymocyte-enriched population from normal mice. Translocation of Ca2+-independent PKC from the cytosolic fraction to the particulate fraction of CD4+CD8+ thymocytes was induced by CD3/CD28-mediated stimulation, but not by CD3- or CD28-mediated stimulation alone, and peaked 2 hr after the start of culture. The kinase activity of the translocated Ca2+-independent PKC was dependent on cofactors in vitro, indicating that novel (n)PKC, but not atypical (a)PKC or a proteolytic PKC fragment, was responsible for the activity. Immunoblotting analysis indicated that the nPKC-theta isoform was the major contributor among nPKC isoforms, and that the classical (c)PKC-alpha isoform was the major contributor among cPKC isoforms. These results suggest that activation of nPKC (especially the theta isoform) in CD4+CD8+ thymocytes is involved in a pathway for negative selection.
Asada
A
A
Integrative Projects Center, Peptide Research and Reproductive Engineering, Mitsubishi Kasei Institute of Life Sciences, Machida-shi, Tokyo, Japan.
Zhao
Y
Y
Komano
H
H
Kuwata
T
T
Mukai
M
M
Fujita
K
K
Tozawa
Y
Y
Iseki
R
R
Tian
H
H
Sato
K
K
Motegi
Y
Y
Suzuki
R
R
Yokoyama
M
M
Iwata
M
M
D000818:Animals; D017209:Apoptosis / Q000276:immunology*; D018106:CD28 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002118:Calcium / Q000502:physiology; D018929:Cell Culture Techniques; D017635:Clonal Anergy / Q000276:immunology; D007527:Isoenzymes / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D011493:Protein Kinase C / Q000276:immunology* / Q000378:metabolism; D016176:T-Lymphocyte Subsets / Q000201:enzymology / Q000276:immunology*
D018106:CD28 Antigens; D017252:CD3 Complex; D007527:Isoenzymes; D011493:Protein Kinase C; D002118:Calcium