PKCalpha: a versatile key for decoding the cellular calcium toolkit.
Conventional protein kinases C (cPKCs) play an essential role in signal transduction and are believed to integrate both global Ca(2+) transients and diacylglycerol signals. We provide evidence that PKCalpha is a ubiquitous readout sensor for the cellular Ca(2+) toolkit, including highly restricted elementary Ca(2+) release. Threshold stimulations of cells with Ca(2+)-mobilizing agonists resulted in PKCalpha translocation events with limited spatial spreads (<4 microm) comprising two groups of lifetimes; brief events (400-1,500 ms) exclusively mediated by Ca(2+)-C2 domain membrane interactions and long-lasting events (>4 s) resulting from longer DAG-C1a domain-mediated membrane interactions. Although upon uncaging NP-EGTA, which is a caged Ca(2+) compound, WT-PKCalpha displayed rapid membrane translocations within <250 ms, PKCalpha constructs with C2 domains mutated in their Ca(2+)-binding region lacked any Ca(2+)-dependent translocation. Flash photolysis of diazo-2, a photosensitive caged Ca(2+) buffer, revealed a biphasic membrane dissociation (slow and fast period) of WT-PKCalpha. The slow phase was absent in cells expressing PKCalpha-constructs containing mutated C1a-domains with largely reduced DAG binding. Thus, two groups of PKCalpha membrane interactions coexist; C2- and C1a-mediated interactions with different lifetimes but rapid interconversion. We conclude that PKCalpha can readout very fast and, spatially and temporally, very complex cellular Ca(2+) signals. Therefore, cPKCs are important transducers for the ubiquitous cellular Ca(2+) signaling toolkit.
Reither
Gregor
G
Institute for Molecular Cell Biology, Medical Faculty of the Saarland University, D-66421 Homburg/Saar, Germany.
Schaefer
Michael
M
Lipp
Peter
P
D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D015374:Biosensing Techniques / Q000379:methods* / Q000639:trends; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D002522:Chlorocebus aethiops; D004075:Diglycerides / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D052139:Intracellular Calcium-Sensing Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007424:Intracellular Fluid / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D051571:Protein Kinase C-alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D002614:Chelating Agents; D004075:Diglycerides; D052139:Intracellular Calcium-Sensing Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D051571:Protein Kinase C-alpha; D002118:Calcium