Abstract
Conventional protein kinase Cs (cPKCs) are key signaling proteins for transducing intracellular Ca2+ signals into downstream phosphorylation events. However, the lifetime of individual membrane-bound activated cPKCs is an order of magnitude shorter than the average time needed for target-protein phosphorylation. Here, we employed intermolecular Förster resonance energy transfer (FRET) in living cells combined with computational analysis to study the spatial organization of cPKCs bound to the plasma membrane. We discovered Ca2+-dependent cPKC nano-clusters that significantly extend cPKC’s plasma-membrane residence time. These protein patterns resulted from self-assembly mediated by Ca2+-binding C2-domains, which are widely used for membrane-targeting of Ca2+-sensing proteins. We also established clustering of other unrelated C2-domain containing proteins, suggesting that nano-cluster formation is a key step for efficient cellular Ca2+-signaling.
Highlights
We employed intermolecular Förster resonance energy transfer (FRET) in living cells combined with computational analysis to study the spatial organization of conventional protein kinase Cs (cPKCs) binding to the plasma membrane, a prerequisite for cPKC activation
We discovered transient Ca2+-dependent cPKC nano-clusters that significantly extend the plasma-membrane residence time of cPKC molecules
Stochastic simulations pointed to a 3-fold increased signaling efficiency of cPKCs in nano-clusters. These protein arrays resulted from cPKC self-assembly through their Ca2+-binding C2-domain, a molecular motif widely used for membrane targeting of Ca2+-sensing proteins[27]
Summary
We employed intermolecular FRET in living cells combined with computational analysis to study the spatial organization of cPKCs binding to the plasma membrane, a prerequisite for cPKC activation. We discovered transient Ca2+-dependent cPKC nano-clusters that significantly extend the plasma-membrane residence time of cPKC molecules. Such increases in the membrane residence time overcome the inherently slow phosphorylation rates of the PKCskinase domain and result in more efficient downstream signaling. Stochastic simulations pointed to a 3-fold increased signaling efficiency of cPKCs in nano-clusters. These protein arrays resulted from cPKC self-assembly through their Ca2+-binding C2-domain, a molecular motif widely used for membrane targeting of Ca2+-sensing proteins[27]. We established clustering of other unrelated C2-domain containing proteins and even of isolated C2 domains, suggesting that in living cells nano cluster formation is a general feature of Ca2+-dependent membrane-binding proteins utilizing C2-domains. Our findings strongly indicate that nano-cluster formation of C2-domain containing proteins constitutes an essential step in Ca2+ readout during cellular signaling and emphasize the importance and versatility of such cooperative effects for the cellular signaling toolkit
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