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Molecular and Cellular Biology, September 1998, p. 5263-5271, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Three Distinct Mechanisms for Translocation and Activation of the delta  Subspecies of Protein Kinase C

Shiho Ohmori,1 Yasuhito Shirai,1 Norio Sakai,1 Motoko Fujii,1 Hiroaki Konishi,2 Ushio Kikkawa,2 and Naoaki Saito1,*

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 delta  subspecies of protein kinase C (delta -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 delta -PKC-GFP fusion protein had enzymological characteristics very similar to those of the native delta -PKC and was present throughout the cytoplasm in CHO-K1 cells. ATP at 1 mM caused a transient translocation of delta -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 delta -PKC-GFP, and the translocation was unidirectional. Concomitantly, the kinase activity of delta -PKC-GFP was increased by these two stimulations, when the kinase activity of the immunoprecipitated delta -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 delta -PKC-GFP but increased its kinase activity more than threefold. delta -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 delta -PKC-GFP even after treatment with H2O2. Simultaneous treatment with TPA and H2O2 further activated delta -PKC-GFP up to more than fivefold. TPA treatment of cells overexpressing delta -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 delta -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.

Molecular and Cellular Biology, September 1998, p. 5263-5271, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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