The second phase activation of protein kinase C delta at late G1 is required for DNA synthesis in serum-induced cell cycle progression.
BACKGROUND
Cell lines that stably over-express protein kinase C (PKC) delta frequently show a decrease in growth rate and saturation density, leading to the hypothesis that PKC delta has a negative effect on cell proliferation. However, the mode of PKC delta activation, the cell cycle stage requiring PKC delta activity, and the exact role of PKC delta at that stage remains unknown.


RESULTS
Here we show that the treatment of quiescent fibroblasts with serum activates PKC delta at two distinct time points, within 10 min after serum treatment, and for a longer duration between 6 and 10 h. This biphasic activation correlates with the phosphorylation of Thr-505 at the activation loop of PKC delta. Importantly, an inhibitor of PKC delta, rottlerin, suppresses the biphasic activation of PKC delta, and suppression of the second phase of PKC delta activation is sufficient for the suppression of DNA synthesis. Consistent with this, the transient over-expression of PKC delta mutant molecules lacking kinase activity suppresses serum-induced DNA synthesis. These results imply that PKC delta plays a positive role in cell cycle progression. While the over-expression of PKC delta enhances serum-induced DNA synthesis, this was not observed for PKC epsilon. Similar experiments using a series of PKCdelta/ epsilon chimeras showed that the carboxyl-terminal 51 amino acids of PKC delta are responsible for the stimulatory effect. On the other hand, the over-expression of PKC delta suppresses cell entry into M-phase, being consistent with the previous studies based on stable over-expressors.


CONCLUSIONS
We conclude that PKC delta plays a role in the late-G1 phase through the positive regulation of cell-cycle progression, in addition to negative regulation of the entry into M-phase.
Kitamura
Koichi
K
Department of Molecular Biology, Yokohama City University School of Medicine, Fuku-ura 3-9, Kanazawa-ku, Yokohama 236-0004, Japan.
Mizuno
Keiko
K
Etoh
Akiko
A
Akita
Yoshiko
Y
Miyamoto
Akitomo
A
Nakayama
Kei-Ichi
K
Ohno
Shigeo
S
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology; D002478:Cells, Cultured; D002678:Chimera; D004247:DNA / Q000096:biosynthesis*; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D016193:G1 Phase / Q000502:physiology*; D066298:In Vitro Techniques; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D051381:Rats; D013912:Threonine / Q000378:metabolism; D015854:Up-Regulation
D000098:Acetophenones; D001578:Benzopyrans; D013912:Threonine; D004247:DNA; C085746:rottlerin; C497260:Prkcd protein, rat; C497257:Prkce protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon