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Abstract
Signal transduction via protein kinase C (PKC) is closely regulated by its subcellular localization. In response to activation of cell-surface receptors, PKC is directed to the plasma membrane by two membrane-targeting domains, namely the C1 and C2 regions. This is followed by the return of the enzyme to the cytoplasm, a process shown recently to require PKC autophosphorylation (Feng, X., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 26870-26874). In the present study, we examined mechanisms of translocation and reverse translocation and the role of autophosphorylation in these processes. By visualizing the trafficking of wild-type as well as mutant PKCbetaII in live cells, we demonstrated that in response to cell-surface receptor activation, the function of the C1 region is required but not sufficient for recruitment of the enzyme to the plasma membrane. The C2 region is also critical in anchoring the enzyme to the plasma membrane. Furthermore, the inability of a kinase-deficient PKC to undergo reverse translocation was restored by the addition of intracellular calcium chelators, suggesting a role for the C2 region in the persistent phase of translocation. On the other hand, the inability of a C2 deletion mutant (C1 region intact) to translocate in response to agonist was reversed in mutants lacking kinase activity or by mutation of the Ser(660) autophosphorylation site to alanine, suggesting that autophosphorylation of this site is required for opposing the action of the C2 region. Therefore, the membrane-targeting function of the C1 region is facilitated by the C2 region and appears to be opposed by autophosphorylation. Taken together, these findings provide novel evidence of the functional regulation of reversible PKC membrane localization by autophosphorylation, and they show that the dynamic translocation of PKC in response to agonists is tightly regulated in a collaborative fashion by the C1 and C2 regions in balance with the effects of autophosphorylation.
Highlights
Extensive biochemical and immunocytochemical studies have indicated that the biological activity of protein kinase C (PKC) is intimately regulated by its subcellular localization [7, 8]
By visualizing real time cellular trafficking of PKCII conjugated to green fluorescent protein (GFP) in live cells, we showed that PKC undergoes a dynamic and reversible redistribution between the plasma membrane and cytoplasm in response to physiological stimuli such as activation of cell-surface G protein-coupled receptors (GPCRs) [21]
The inherent fluorescence of GFP allowed us to visualize the trafficking of GFP-PKCII in live Human embryonic kidney (HEK) 293 cells and to demonstrate that autophosphorylation is essential for the reversibility of PKC membrane translocation in response to GPCR activation [20]
Summary
Materials—Eagle’s minimal essential medium and HEPES were from Life Technologies, Inc. The expression of the hemagglutinin-tagged AT1AR in the presence of wild-type or various mutant GFP-PKCII constructs was assessed by flow cytometry following antibody staining. The levels of the AT1AR in cells transfected with various mutant GFP-PKCII constructs were either equivalent to or higher than that in the presence of wild-type GFP-PKCII. Protein Kinase C Assay—For assaying PKC activity in cells transfected with wild-type or mutant GFP-PKCII, the cell lysates (200 g) were first immunoprecipitated with 5 g of rabbit anti-PKCII antibody. Immunoblotting—Cell lysates from HEK 293 cells transfected with GFP-PKCII and its mutants were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose membranes. The cells expressing GFP-PKCII or its mutants were observed by confocal microscopy, which was performed on a Zeiss LSM-410 laser scanning microscope using a Zeiss 40ϫ 1.2 NA water immersion lens. The relative fluorescence intensity on the plasma membrane was calculated by the formula (Imb Ϫ Icyt)/Icyt, where Imb is the amplitude of the fluorescence signal on the plasma membrane and Icyt is the average cytosolic fluorescent intensity
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