C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca 2؉ -binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKC␣-C2 domain in complex with Ca 2؉ , 1,2-dihexanoyl-sn-glycero-3-[phospho-L-serine] (PtdSer), and 1,2-diayl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P2] shows that PtdSer binds specifically to the calcium-binding region, whereas PtdIns(4,5)P2 occupies the concave surface of strands ␤3 and ␤4. Strikingly, the structure reveals a PtdIns(4,5)P2-C2 domain-binding mode in which the aromatic residues Tyr-195 and Trp-245 establish direct interactions with the phosphate moieties of the inositol ring. Mutations that abrogate Tyr-195 and Trp-245 recognition of PtdIns(4,5)P2 severely impaired the ability of PKC␣ to localize to the plasma membrane. Notably, these residues are highly conserved among C2 domains of topology I, and a general mechanism of C2 domain-membrane docking mediated by PtdIns(4,5)P2 is presented.calcium phosphoinositides ͉ peripheral membrane proteins T he C2 domains are considered peripheral proteins that are water-soluble and associate reversibly with lipid bilayers. Recently, evidence has demonstrated that some of these domains are able to interact with the inositol phospholipid 1,2-diacyl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P 2 ] (1-4), which is able to directly participate in a myriad of functions, including cell signaling at the plasma membrane, regulation of membrane traffic and transport, cytoskeleton dynamics, and nuclear events (5, 6). Despite the number of C2 domain 3D structures currently available, questions about how they interact with the different target phospholipids, their precise spatial position in the lipid bilayer, and their role in transmitting signals downstream have yet to be explored.The main role of the C2 domain in classical PKCs (cPKCs) is to act as the Ca 2ϩ -activated membrane-targeting motif (7, 8). The 3D structure of these C2 domains comprises 8 antiparallel ␤-strands assembled in a ␤-sandwich architecture, with flexible loops on top and at the bottom (9-12). This C2 domain displays 2 functional regions: the Ca 2ϩ -binding region and the polybasic cluster. The former is located in the flexible top loops, binds 2 or 3 Ca 2ϩ ions, depending on the isoenzyme (10,11,13,14), and interacts with 1,2-diacyl-sn-glycero-3-[phospho-L-serine] (PtdSer) (11,15,16). The second region is a polybasic cluster that is located at the concave surface of the C2 domain formed by strands ␤3 and ␤4. Recent studies indicate that this region might bind specifically to PtdIns(4,5)P 2 in a Ca 2ϩ -dependent manner (1,(17)(18)(19)(20)(21).To gain insight into the structural and functional basis for the PtdIns(4,5)P 2 -dependent membrane targeting of the PKC␣-C2 domain, we determined the 3D structures of the ternary and quaternary complexes of the C2 domain of PKC␣, crystallized in presence of Ca 2ϩ and PtdIns(4,5...

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Signal transduction through protein kinase Cs (PKCs) strongly depends on their subcellular localization. Here, we investigate the molecular determinants of PKC␣ localization by using a model system of neural growth factor (NGF)-differentiated pheochromocytoma (PC12) cells and extracellular stimulation with ATP. Strikingly, the Ca 2؉ influx, initiated by the ATP stimulation of P2X receptors, rather than the Ca 2؉ released from the intracellular stores, was the driving force behind the translocation of PKC␣ to the plasma membrane. Furthermore, the localization process depended on two regions of the C2 domain: the Ca 2؉ -binding region and the lysine-rich cluster, which bind Ca 2؉ and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ], respectively. It was demonstrated that diacylglycerol was not involved in the localization of PKC␣ through its C1 domain, and in lieu, the presence of PtdIns(4,5)P 2 increased the permanence of PKC␣ in the plasma membrane. Finally, it also was shown that ATP cooperated with NGF during the differentiation process of PC12 cells by increasing the length of the neurites, an effect that was inhibited when the cells were incubated in the presence of a specific inhibitor of PKC␣, suggesting a possible role for this isoenzyme in the neural differentiation process. Overall, these results show a novel mechanism of PKC␣ activation in differentiated PC12 cells, where Ca 2؉ influx, together with the endogenous PtdIns(4,5)P 2 , anchor PKC␣ to the plasma membrane through two distinct motifs of its C2 domain, leading to enzyme activation.

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The exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome factor RRP6 of Drosophila melanogaster and its human ortholog EXOSC10 play a role in DNA repair. Here, we show that RRP6 and EXOSC10 are recruited to DNA double-strand breaks (DSBs) in S2 cells and HeLa cells, respectively. Depletion of RRP6/ EXOSC10 does not interfere with the phosphorylation of the histone variant H2Av (Drosophila) or H2AX (humans), but impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A-V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway. Taken together, our results suggest that the ribonucleolytic activity of RRP6/EXOSC10 is required for the recruitment of RAD51 to DSBs.