Importance of protein kinase C targeting for the phosphorylation of its substrate, myristoylated alanine-rich C-kinase substrate.
We visualized the translocation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in living Chinese hamster ovary-K1 cells using MARCKS tagged to green fluorescent protein (MARCKS-GFP). MARCKS-GFP was rapidly translocated from the plasma membrane to the cytoplasm after the treatment with phorbol ester, which translocates protein kinase C (PKC) to the plasma membrane. In contrast, PKC activation by hydrogen peroxide, which was not accompanied by PKC translocation, did not alter the intracellular localization of MARCKS-GFP. Non-myristoylated mutant of MARCKS-GFP was distributed throughout the cytoplasm, including the nucleoplasm, and was not translocated by phorbol ester or by hydrogen peroxide. Phosphorylation of wild-type MARCKS-GFP was observed in cells treated with phorbol ester but not with hydrogen peroxide, whereas non-myristoylated mutant of MARCKS-GFP was phosphorylated in cells treated with hydrogen peroxide but not with phorbol ester. Phosphorylation of both MARCKS-GFPs reduced the amount of F-actin. These findings revealed that PKC targeting to the plasma membrane is required for the phosphorylation of membrane-associated MARCKS and that a mutant MARCKS existing in the cytoplasm can be phosphorylated by PKC activated in the cytoplasm without translocation but not by PKC targeted to the membrane.
Ohmori
S
S
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501, Japan.
Sakai
N
N
Shirai
Y
Y
Yamamoto
H
H
Miyamoto
E
E
Shimizu
N
N
Saito
N
N
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007202:Indicators and Reagents; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007202:Indicators and Reagents; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate