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targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca2+]i
Medizinische Hochschule Hannover, Hannover, Germany;
* Institute of Biochemistry, Free University, Berlin, Germany; and
Franz Volhard Clinic and the Max-Delbrück Center for Molecular Medicine, Medizinische Fakultät der Charité, Humboldt University of Berlin, Germany
1Correspondence: Medizinische Hochschule Hannover, OE 6840, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. E-mail: haller.hermann{at}mh-hannover.de
Protein kinase C (PKC) isoforms exert specific intracellular functions,
but the different isoforms display little substrate specificity
in vitro. Selective PKC isoform targeting may be a
mechanism to achieve specificity. We used a green fluorescent fusion
protein (GFP) to test the hypothesis that local changes in
[Ca2+]i regulate translocation of PKC
and
that different modes of Ca2+ and Ca2+ release
play a role in PKC targeting. We constructed deletion mutants of
PKC to analyze the Ca2+-sensitive domains and their role
in targeting. Confocal microscopy was used and
[Ca2+]i was measured by fluo-3. The fusion
protein PKC-GFP was expressed in vascular smooth muscle cells and
showed a cytosolic distribution similar to the wild-type PKC
protein. The Ca2+ ionophore ionomycin induced a speckled
cytosolic PKC-GFP distribution, followed by membrane translocation,
while depolarization by KCl induced primarily membrane translocation.
Selective voltage-operated Ca2+ channel opening led to a
localized accumulation of PKC-GFP near the plasma membrane. Opening
Ca2+ stores with InsP3, thapsigargin, or
ryanodine induced a specific PKC-GFP targeting to distinct
intracellular areas. The G-protein-coupled receptor agonist thrombin
induced a rapid translocation of the fusion protein to focal domains.
The tyrosine kinase receptor agonist PDGF induced Ca2+
influx and led to a linear PKC-GFP membrane association. PKC-GFP
deletion mutants demonstrated that the C2 domain, but not the catalytic
subunit, is necessary for Ca2+-induced PKC targeting.
Targeting was also abolished when the ATP binding site was deleted. We
conclude that PKC can rapidly be translocated to distinct
intracellular or membrane domains by local increases in
[Ca2+]i. The targeting mechanism is dependent
on the C2 and ATP binding site of the enzyme. Localized
[Ca2+]i changes determine the spatial and
temporal targeting of PKC.—Maasch, C., Wagner, S., Lindschau, C.,
Alexander, G., Buchner, K., Gollasch, M., Luft, F. C., Haller, H.
Protein kinase C targeting is regulated by temporal and
spatial changes in intracellular free calcium concentration
[Ca2+]i.
Key Words: isoforms • green fluorescent protein • vascular smooth muscle cells • receptor coupling • translocation • mutation
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