14 3 3 protein masks the nuclear localization sequence of caspase 2

Caspase‐2 is an apical protease responsible for the proteolysis of cellular substrates directly involved in mediating apoptotic signaling cascades. Caspase‐2 activation is inhibited by phosphorylation followed by binding to the scaffolding protein 14‐3‐3, which recognizes two phosphoserines located in the linker between the caspase recruitment domain and the p19 domains of the caspase‐2 zymogen. However, the structural details of this interaction and the exact role of 14‐3‐3 in the regulation of caspase‐2 activation remain unclear. Moreover, the caspase‐2 region with both 14‐3‐3‐binding motifs also contains the nuclear localization sequence (NLS), thus suggesting that 14‐3‐3 binding may regulate the subcellular localization of caspase‐2. Here, we report a structural analysis of the 14‐3‐3ζ:caspase‐2 complex using a combined approach based on small angle X‐ray scattering, NMR, chemical cross‐linking, and fluorescence spectroscopy. The structural model proposed in this study suggests that phosphorylated caspase‐2 and 14‐3‐3ζ form a compact and rigid complex in which the p19 and the p12 domains of caspase‐2 are positioned within the central channel of the 14‐3‐3 dimer and stabilized through interactions with the C‐terminal helices of both 14‐3‐3ζ protomers. In this conformation, the surface of the p12 domain, which is involved in caspase‐2 activation by dimerization, is sterically occluded by the 14‐3‐3 dimer, thereby likely preventing caspase‐2 activation. In addition, 14‐3‐3 protein binding to caspase‐2 masks its NLS. Therefore, our results suggest that 14‐3‐3 protein binding to caspase‐2 may play a key role in regulating caspase‐2 activation

14 3 3 protein directly interacts with the kinase domain of calcium calmodulin dependent protein kinase kinase CaMKK2

BackgroundCalcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca$^{2+}$/calmodulin-dependent kinase (CaMK) family involved in adiposity regulation, glucose homeostasis and cancer. This upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase is inhibited by phosphorylation, which also triggers an association with the scaffolding protein 14-3-3. However, the role of 14-3-3 in the regulation of CaMKK2 remains unknown.MethodsThe interaction between phosphorylated CaMKK2 and the 14-3-3γ protein, as well as the architecture of their complex, were studied using enzyme activity measurements, small-angle x-ray scattering (SAXS), time-resolved fluorescence spectroscopy and protein crystallography.ResultsOur data suggest that the 14-3-3 protein binding does not inhibit the catalytic activity of phosphorylated CaMKK2 but rather slows down its dephosphorylation. Structural analysis indicated that the complex is flexible and that CaMKK2 is located outside the phosphopeptide-binding central channel of the 14-3-3γ dimer. Furthermore, 14-3-3γ appears to interact with and affect the structure of several regions of CaMKK2 outside the 14-3-3 binding motifs. In addition, the structural basis of interactions between 14‐3-3 and the 14-3-3 binding motifs of CaMKK2 were elucidated by determining the crystal structures of phosphopeptides containing these motifs bound to 14-3-3.Conclusions14-3-3γ protein directly interacts with the kinase domain of CaMKK2 and the region containing the inhibitory phosphorylation site Thr145 within the N-terminal extension.General significanceOur results suggested that CaMKK isoforms differ in their 14-3-3-mediated regulations and that the interaction between 14-3-3 protein and the N-terminal 14-3-3-binding motif of CaMKK2 might be stabilized by small-molecule compounds