14 3 3 protein regulates Nedd4 2 by modulating interactions between HECT and WW domains

Pohl et al. investigated the structural basis of Nedd4-2 regulation by 14-3-3 and found that phosphorylated Ser342 and Ser448 are the main residues that facilitate 14-3-3 binding to Nedd4-2. The authors propose that the Nedd4-2:14-3-3 complex then stimulates a structural rearrangement of Nedd4-2 through inhibiting interaction of its structured domains

Stabilization of Protein Protein Interactions between CaMKK2 and 14 3 3 by Fusicoccins

Ca2 calmodulin dependent protein kinase kinase 2 CaMKK2 regulates several key physiological and pathophysiological processes, and its dysregulation has been implicated in obesity, diabetes, and cancer. CaMKK2 is inhibited through phosphorylation in a process involving binding to the scaffolding 14 3 3 protein, which maintains CaMKK2 in the phosphorylation mediated inhibited state. The previously reported structure of the N terminal CaMKK2 14 3 3 binding motif bound to 14 3 3 suggested that the interaction between 14 3 3 and CaMKK2 could be stabilized by small molecule compounds. Thus, we investigated the stabilization of interactions between CaMKK2 and 14 3 3 amp; 947; by Fusicoccin A and other fusicoccanes diterpene glycosides that bind at the interface between the 14 3 3 ligand binding groove and the 14 3 3 binding motif of the client protein. Our data reveal that two of five tested fusicoccanes considerably increase the binding of phosphopeptide representing the 14 3 3 binding motif of CaMKK2 to 14 3 3 amp; 947;. Crystal structures of two ternary complexes suggest that the steric contacts between the C terminal part of the CaMKK2 14 3 3 binding motif and the adjacent fusicoccane molecule are responsible for differences in stabilization potency between the study compounds. Moreover, our data also show that fusicoccanes enhance the binding affinity of phosphorylated full length CaMKK2 to 14 3 3 amp; 947;, which in turn slows down CaMKK2 dephosphorylation, thus keeping this protein in its phosphorylation mediated inhibited state. Therefore, targeting the fusicoccin binding cavity of 14 3 3 by small molecule compounds may offer an alternative strategy to suppress CaMKK2 activity by stabilizing its phosphorylation mediated inhibited stat

The activity of Saccharomyces cerevisiae Na , K H antiporter Nha1 is negatively regulated by 14 3 3 protein binding at serine 481

Na+/H+ antiporters are involved in ensuring optimal intracellular concentrations of alkali-metal cations and protons in most organisms. In Saccharomyces cerevisiae, the plasma-membrane Na+, K+/H+ antiporter Nha1 mediates Na+ and K+ efflux, which is important for cell growth in the presence of salts. Nha1 belongs among housekeeping proteins and, due to its ability to export K+, it has many physiological functions. The Nha1 transport activity is regulated through its long, hydrophilic and unstructured C-terminus (554 of 985 aa). Although Nha1 has been previously shown to interact with the yeast 14-3-3 isoform (Bmh2), the binding site remains unknown. In this work, we identified the residues through which Nha1 interacts with the 14-3-3 protein. Biophysical characterization of the interaction between the C-terminal polypeptide of Nha1 and Bmh proteins in vitro revealed that the 14-3-3 protein binds to phosphorylated Ser481 of Nha1, and the crystal structure of the phosphopeptide containing Ser481 bound to Bmh1 provided the structural basis of this interaction. Our data indicate that 14-3-3 binding induces a disorder-to-order transition of the C-terminus of Nha1, and in vivo experiments showed that the mutation of Ser481 to Ala significantly increases cation efflux activity via Nha1, which renders cells sensitive to low K+ concentrations. Hence, 14-3-3 binding is apparently essential for the negative regulation of Nha1 activity, which should be low under standard growth conditions, when low amounts of toxic salts are present and yeast cells need to accumulate high amounts of K+

14 3 3 protein binding blocks the dimerization interface of caspase 2

Among all species, caspase 2 C2 is the most evolutionarily conserved caspase required for effective initiation of apoptosis following death stimuli. C2 is activated through dimerization and autoproteolytic cleavage and inhibited through phosphorylation at Ser139 and Ser164, within the linker between the caspase recruitment and p19 domains of the zymogen, followed by association with the adaptor protein 14 3 3, which maintains C2 in its immature form procaspase proC2 . However, the mechanism of 14 3 3 dependent inhibition of C2 activation remains unclear. Here, we report the structural characterization of the complex between proC2 and 14 3 3 by hydrogen deuterium mass spectrometry and protein crystallography to determine the molecular basis for 14 3 3 mediated inhibition of C2 activation. Our data reveal that the 14 3 3 dimer interacts with proC2 not only through ligand binding grooves but also through other regions outside the central channel, thus explaining the isoform dependent specificity of 14 3 3 protein binding to proC2 and the substantially higher binding affinity of 14 3 3 protein to proC2 than to the doubly phosphorylated peptide. The formation of the complex between 14 3 3 protein and proC2 does not induce any large conformational change in proC2. Furthermore, 14 3 3 protein interacts with and masks both the nuclear localization sequence and the C terminal region of the p12 domain of proC2 through transient interactions in which both the p19 and p12 domains of proC2 are not firmly docked onto the surface of 14 3 3. This masked region of p12 domain is involved in C2 dimerization. Therefore, 14 3 3 protein likely inhibits proC2 activation by blocking its dimerization surfac

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