Abstract
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.
Highlights
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)
The mechanism of protein kinase C (PKC) translocation was studied in living cells using green fluorescent protein (GFP)-tagged PKC, and it has been shown that each PKC subtype has a spatially and temporally different targeting mechanism that depends on the extracellular signals, contributing to the subspecies-specific functions of PKC [12,13,14]
Immunoblot Analysis of MARCKS-GFP Fusion Protein—We examined the effects of GFP fusion on the characteristics of MARCKS by immunoblotting
Summary
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). Phosphorylation of MARCKS and Its Mutant by ␦-PKC in Vitro— MARCKS-GFP and its mutants were prepared by immunoprecipitation using anti-GFP antibody from CHO-K1 cells transfected with the corresponding cDNA. The activation of PKC by 100 nM TPA induced a rapid translocation of MARCKS from the plasma membrane to the cytosol (Fig. 3A) within 20 s after the stimulation.
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