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Molecular and Cellular Biology, September 1998, p. 5263-5271, Vol. 18, No. 9
Laboratories of Molecular
Pharmacology1 and
Biochemistry,2 Biosignal Research
Center, Kobe University, Nada-ku, Kobe 657-8501, Japan
Received 16 March 1998/Returned for modification 13 April
1998/Accepted 12 June 1998
We expressed
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Three Distinct Mechanisms for Translocation and
Activation of the
Subspecies of Protein Kinase C
subspecies of protein kinase C (
-PKC) fused
with green fluorescent protein (GFP) in CHO-K1 cells and observed the movement of this fusion protein in living cells after three different stimulations. The
-PKC-GFP fusion protein had
enzymological characteristics very similar to those of the native
-PKC and was present throughout the cytoplasm in CHO-K1 cells.
ATP at 1 mM caused a transient translocation of
-PKC-GFP to
the plasma membrane approximately 30 s after the stimulation and a
sequent retranslocation to the cytoplasm within 3 min. A
tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol
13-acetate (TPA; 1 µM), induced a slower translocation of
-PKC-GFP, and the translocation was unidirectional.
Concomitantly, the kinase activity of
-PKC-GFP was increased by
these two stimulations, when the kinase activity of the
immunoprecipitated
-PKC-GFP was measured in vitro in the absence of PKC activators such as phosphatidylserine and
diacylglycerol. Hydrogen peroxide (H2O2; 5 mM)
failed to translocate
-PKC-GFP but increased its kinase
activity more than threefold.
-PKC-GFP was strongly tyrosine
phosphorylated when treated with H2O2 but was tyrosine phosphorylated not at all by ATP stimulation and only slightly by TPA treatment. Both TPA and ATP induced the
translocation of
-PKC-GFP even after treatment with
H2O2. Simultaneous treatment with TPA and
H2O2 further activated
-PKC-GFP up to
more than fivefold. TPA treatment of cells overexpressing
-PKC-GFP led to an increase in the number of cells in
G2/M phase and of dikaryons, while stimulation with
H2O2 increased the number of cells in S phase
and induced no significant change in cell morphology. These results
indicate that at least three different mechanisms are involved in the
translocation and activation of
-PKC.
*
Corresponding author. Mailing address: Laboratory of
Molecular Pharmacology, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan. Phone:
81-78-803-1251. Fax: 81-78-803-0993. E-mail:
naosaito{at}kobe-u.ac.jp.
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