High expression of c-kit in K562YO cells due to the prolonged half-life of its mRNA: the effects of modification with serine/threonine kinase signals.
We previously reported that the K562 cell line K562YO expressed a high level of the c-kit gene. In this study, we analyzed the mechanism of this expression and investigated the effects of the serine/threonine kinases such as protein kinase C (PKC) and cyclic adenosine 3',5'-monophosphate (cAMP)-dependent kinase (PKA) on it. The half-life of the c-kit mRNA in K562YO cells was greater than 10 hours, compared with 2 hours in the original K562 cells, which expressed a very low level of c-kit mRNA. This prolonged half-life can contribute to the high level of c-kit expression in K562YO cells. Cycloheximide (CHX), a protein synthesis inhibitor, caused increases in c-kit mRNA levels in K562YO cells. 12-O-tetradecanoylphorbol-13-acetate (TPA), by which PKC was activated at first and downregulated in a late phase, gradually decreased c-kit mRNA in K562YO cells until 9 hours and then returned to the control level 24 hours after treatment. TPA also rapidly decreased c-kit protein level on the membranes. In whole cells, c-kit protein was also decreased 6 hours after incubation with TPA. Calphostin C, a light-dependent PKC inhibitor, decreased c-kit mRNA levels within 30 minutes in a light-dependent manner. It also decreased c-kit protein in whole cells 2 hours after the addition. However, it increased the amount of c-kit protein on the cell surfaces. Dibutyryl cyclic AMP (dbc-AMP) increased c-kit mRNA as well as c-kit protein on membranes and in whole cells. Run-on transcriptional assay suggested that the agent (dbc-AMP) enhanced the transcription rate of the gene. These results suggest that c-kit protein on the membranes is downregulated by PKC activation and upregulated by PKC inhibition. In the whole cell lysate, c-kit proteins are decreased by PKC inhibition through downregulation of mRNA. On the other hand, the elevation of an intracellular cAMP level causes upregulation of both the mRNA and c-kit protein on membranes and in whole cells through enhanced transcription. Thus, c-kit gene expression is apparently modulated by PKC and PKA.
Ogawa
K
K
Department of Internal Medicine, Faculty of Medicine, Kyoto University, Japan.
Takeda
Y
Y
Tashima
M
M
Sawai
H
H
Toi
T
T
Okazaki
T
T
Sawada
H
H
Maruyama
Y
Y
Okuma
M
M
D003994:Bucladesine / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D006207:Half-Life; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D019009:Proto-Oncogene Proteins c-kit; D012333:RNA, Messenger / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D016184:Receptors, Colony-Stimulating Factor / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D016184:Receptors, Colony-Stimulating Factor; D003609:Dactinomycin; D003994:Bucladesine; D003513:Cycloheximide; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D013755:Tetradecanoylphorbol Acetate