Protein kinase calpha targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca(2+)](i).
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 [Ca(2+)](i) regulate translocation of PKCalpha and that different modes of Ca(2+) and Ca(2+) release play a role in PKCalpha targeting. We constructed deletion mutants of PKCalpha to analyze the Ca(2+)-sensitive domains and their role in targeting. Confocal microscopy was used and [Ca(2+)](i) was measured by fluo-3. The fusion protein PKCalpha-GFP was expressed in vascular smooth muscle cells and showed a cytosolic distribution similar to the wild-type PKCalpha protein. The Ca(2+) ionophore ionomycin induced a speckled cytosolic PKCalpha-GFP distribution, followed by membrane translocation, while depolarization by KCl induced primarily membrane translocation. Selective voltage-operated Ca(2+) channel opening led to a localized accumulation of PKCalpha-GFP near the plasma membrane. Opening Ca(2+) stores with InsP(3), thapsigargin, or ryanodine induced a specific PKCalpha-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 Ca(2+) influx and led to a linear PKCalpha-GFP membrane association. PKCalpha-GFP deletion mutants demonstrated that the C2 domain, but not the catalytic subunit, is necessary for Ca(2+)-induced PKCalpha targeting. Targeting was also abolished when the ATP binding site was deleted. We conclude that PKCalpha can rapidly be translocated to distinct intracellular or membrane domains by local increases in [Ca(2+)](i). The targeting mechanism is dependent on the C2 and ATP binding site of the enzyme. Localized [Ca(2+)](i) changes determine the spatial and temporal targeting of PKCalpha.
Maasch
C
C
Medizinische Hochschule Hannover, Hannover, Germany.
Wagner
S
S
Lindschau
C
C
Alexander
G
G
Buchner
K
K
Gollasch
M
M
Luft
F C
FC
Haller
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001011:Aorta; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D015759:Ionomycin / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012433:Ryanodine / Q000494:pharmacology; D017384:Sequence Deletion / Q000235:genetics; D013379:Substrate Specificity; D019284:Thapsigargin / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection
D002121:Calcium Channel Blockers; D007527:Isoenzymes; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D012433:Ryanodine; D015759:Ionomycin; D011189:Potassium Chloride; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013917:Thrombin; D002118:Calcium