CaMKK2 is inactivated by cAMP-PKA signaling and 14-3-3 adaptor proteins

The calcium-calmodulin (Ca protein kinase kinase-2 (CaMKK2) is a key of mechanisms that inactivate CaMKK2 has important therapeutic implications. Here we show that stimulation of cyclic AMP (cAMP)-dependent protein kinase (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. of Ser495 directly impairs Ca hold provide detailed molecular insights into how cAMP-PKA signaling


INTRODUCTION
The calcium ion (Ca 2+ ) is a dynamic second messenger that relays signals from ligandactivated receptors and voltage-stimulated ion channels at the cell membrane, to regulate a wide array of physiological functions (1). A key transducer of Ca 2+ signaling is calmodulin (CaM), a ubiquitous Ca 2+ -binding protein that regulates the activity of numerous downstream effectors in response to elevations in intracellular Ca 2+ (2). Important to the actions of the Ca 2+ -CaM complex is the Ca 2+ -CaM dependent protein kinase kinase-2 (CaMKK2), which is the core component of a phosphorylation signaling pathway that regulates appetite and whole-body energy metabolism (3). CaMKK2 inhibition protects against prostate cancer development, hepatocellular carcinoma and high-fat diet induced obesity, glucose intolerance and insulin resistance (4)(5)(6), therefore identifying the intracellular mechanisms that inactivate CaMKK2 has important implications for understanding and treating human diseases.
CaMKK2 regulation involves a complex interplay between allosteric activation by Ca 2+ -CaM and multi-site phosphorylation. It has a modular structure composed of an internal catalytic domain and a regulatory module made up of overlapping autoinhibitory and CaM binding sequences, flanked by N-and C-terminal sequences of unknown function (7). The autoinhibitory sequence obstructs the catalytic site by an intra-steric mechanism that is relieved by Ca 2+ -CaM binding, allowing for maximal kinase activity (8). In human CaMKK2, activation by Ca 2+ -CaM induces Thr85 autophosphorylation, which creates a molecular memory of the Ca 2+ -signal that keeps CaMKK2 in the activated state after the stimulus has diminished (9,10).
CaMKK2 knockout mice are protected against high-fat diet induced weight gain, insulin resistance and glucose intolerance (4). Likewise, deletion of the RIIa regulatory subunit of cAMP-dependent protein kinase (PKA) in mice (which results in activation of the PKA catalytic subunit) causes a similar phenotype (15,16), therefore the physiological regulation of whole-body energy metabolism by PKA may involve some inhibitory crosstalk with the CaMKK2 pathway. Consistent with this idea, it was reported recently that CaMKK2 is inactivated by cAMP-dependent protein kinase (PKA) in cell free assays (17). However, it is unclear to what extent this regulatory mechanism occurs in intact cells. Here, we report that CaMKK2 is inactivated in cells by agonists that stimulate cAMP-PKA signaling including the appetite-suppressant, Liraglutide. PKA inactivates CaMKK2 by a direct mechanism that impairs Ca 2+ -CaM activation, and an indirect mechanism involving recruitment of 14-3-3 adaptor proteins. Our data reveal how cAMP-PKA signaling negatively regulates CaMKK2 and provides a molecular rationale for using PKA-activating drugs for human diseases associated with inappropriate activation of the CaMKK2 pathway.

RESULTS
cAMP-PKA signaling inactivates CaMKK2 by impairing Ca 2+ -CaM activation COS7 cells expressing recombinant human CaMKK2 were treated with forskolin and 3isobutyl-1-methylxanthine (IBMX) to increase intracellular cAMP, after which we immunoprecipitated CaMKK2 and measured kinase activity over a range of CaM concentrations. Forskolin/IBMX treatment increased intracellular cAMP (680-fold over control) and impaired CaMKK2 activation by Ca 2+ -CaM ( Fig. 1. A and B), whereas the concentration of CaM required for halfmaximal activation, and kinase activity in the absence of Ca 2+ -CaM, was not significantly affected (Table 1). Co-expression with a small-hairpin RNA that decreased expression of the PKA a catalytic subunit ( Fig. 1. C and D) or pre-treating cells with the PKA inhibitor H89 (Fig. 1E), completely prevented forskolin/IBMX-induced inactivation of CaMKK2.
Activation of the glucagon-like peptide-1 (GLP-1) receptor decreases food intake and promotes weight loss via stimulation of PKA activity (18). Therefore, we examined the effect of the GLP-1 receptor agonist, Liraglutide, on CaMKK2 activity in SH-SY5Y neuroblastoma cells that endogenously express the GLP-1 receptor (19).
Similar to the effects of forskolin/IBMX, Liraglutide diminished Ca 2+ -CaM activation but had no effect on CaMKK2 activity in the absence of Ca 2+ -CaM (Fig. 1F).

PKA-mediated inactivation of CaMKK2 is dependent on Ser495 phosphorylation
To investigate the mechanism by which cAMP-PKA signaling inactivates CaMKK2, we determined the phosphorylation profile of CaMKK2 in cells under control and PKAactivated conditions. Cells expressing human CaMKK2 were treated with or without forskolin/IBMX, after which CaMKK2 was purified and analyzed by whole-protein timeof-flight (TOF) mass spectrometry. Under control conditions, the primary mass peak corresponded to a triply phosphorylated CaMKK2 species ( Fig. 2A) as previously reported (14,20).
In contrast, CaMKK2 purified from forskolin/IBMX treated cells showed a higher range of mass peaks, consistent with increased phosphorylation on at least three additional sites (Fig. 2B). Using tandem mass spectrometry, we compared the spectra of tryptic peptides from CaMKK2 purified from control and forskolin/IBMX treated cells, and found that Ser100, Ser495 (which resides within the CaM binding sequence) and Ser511 were phosphorylated in CaMKK2 from the forskolin/IBMX treated cells but not from the control cells (Fig. S1). All three sites match the PKA phosphorylation consensus motif (-R/K-R/K-X-S/T; where X is any amino acid) (21) and are conserved across a diverse range of species (Fig. S2). Using validated phosphospecific antibodies (Fig. S3), we confirmed PKA-dependent phosphorylation of all three serine residues by immunoblot (Fig. 2C).
The function of each site was determined by treating cells expressing nonphosphorylatable mutants (S100A, S495C and S511A) with forskolin/IBMX, after which we measured kinase activity in the presence and absence of Ca 2+ -CaM. We substituted Ser495 for cysteine rather than alanine as the S495A mutation impaired binding of Ca 2+ -CaM (Fig.  S4). The S495C mutant was activated by Ca 2+ -CaM with similar kinetic parameters to wild-type CaMKK2 (Fig S5). Wild-type CaMKK2 and the S100A and S511A mutants from forskolin/IBMX treated cells displayed decreased Ca 2+ -CaM activation, however the S495C mutant was unaffected (Fig. 2D). This indicates that phosphorylation of Ser495 is entirely responsible for suppressing Ca 2+ -CaM activation in response to PKA signaling. Mirroring the impaired Ca 2+ -CaM activation, wild-type CaMKK2 and the S100A and S511A mutants from forskolin/IBMX treated cells displayed decreased Ca 2+ -CaM binding, but not the S495C mutant (Fig. 2E).
PKA-dependent phosphorylation of CaMKK2 promotes binding to 14-3-3 adaptor proteins Analysis of the phosphorylation sites indicated the sequence surrounding Ser511 conforms to a canonical 14-3-3 protein binding motif (mode-1 motif; RSXpSXP; where X is any amino acid) (22). We therefore immunoprecipitated CaMKK2 from cells treated with forskolin/IBMX and tested for co-purification of endogenous 14-3-3 proteins by immunoblot. Figure 3A shows that forskolin/IBMX treatment induced binding of 14-3-3 proteins to CaMKK2 but not in cells pre-treated with H89. We detected binding of all seven isoforms of 14-3-3 by tandem mass spectrometry (Table S1). 14-3-3 binding is dependent upon phosphorylation of both Ser100 and Ser511 (but not Ser495), as the S100A and S511A mutations were individually sufficient to abolish co-purification of 14-3-3 proteins with CaMKK2 (Fig. 3B).
A recent study reported that 14-3-3 binding to CaMKK2 impaired dephosphorylation of phospho-Ser495 (pSer495) by protein phosphatase-1 in a cellfree assay (17), indicating that a potential biological function of 14-3-3 binding is to maintain CaMKK2 in the inactivated state by protecting pSer495 from dephosphorylation by protein phosphatases. To test whether this mechanism occurs in a cellular context, cells expressing wild-type CaMKK2 or the S100A and S511A mutants were treated with forskolin/IBMX, after which we measured Ser495 phosphorylation and Ca 2+ -CaM activation at various time points following incubation with H89.
Forskolin/IBMX stimulated Ser495 phosphorylation and suppressed Ca 2+ -CaM activation of wild-type CaMKK2, which was sustained over the treatment period even after the addition of H89.
In contrast, pSer495 was dephosphorylated and maximal activation was restored within 5 mins for the S100A and S511A mutants in response to H89 treatment ( Fig. 3. C and D). The simplest interpretation of these data is that 14-3-3 binding protects against pSer495 dephosphorylation in cells.

Structure of a 14-3-3-diphosphopeptide complex
To further investigate the interaction between CaMKK2 and 14-3-3, we determined the crystal structure of 14-3-3z in complex with the diphosphorylated pSer100-pSer511 peptide to a resolution of 2.44 Å (Table S2). The unit cell contained four molecules of 14-3-3, arranged in a back to back dimer of dimers, with a 180˚ rotation with respect to the other dimer. Each dimer contained two phosphopeptide binding sites, both of which showed strong electron density for the critical pSer100 and pSer511 residues (Fig. 5A). We did not observe an admixture of the pSer100-pSer511 peptide bound in both possible orientations to the 14-3-3 dimer, therefore we were able to unambiguously place pSer100 and pSer511 into the phosphopeptide binding sites of discrete monomers within the dimeric complex.

DISCUSSION
Herein, we report that stimulation of cAMP-PKA signaling in cells inactivates CaMKK2 by a mechanism involving tripartite phosphorylation of conserved serine residues and recruitment of 14-3-3 adaptor proteins. We found that phosphorylation of Ser495, a highly conserved site located within the CaM binding sequence, directly impairs Ca 2+ -CaM binding and activation of CaMKK2. A similar mechanism of inhibitory crosstalk by the cAMP-PKA pathway, involving direct phosphorylation of CaM binding sites, has been reported for other Ca 2+ -CaM regulated proteins including CaMKK1, b-adducin and the Ca 2+ -dependent K + channel KCa3.1 (25)(26)(27). This provides a multi-level system for fine-tuning Ca 2+ -signaling, by allowing the magnitude and duration of signal transmission to be modulated (28).
As well as impairing Ca 2+ -CaM activation via Ser495 phosphorylation, PKAdependent phosphorylation of Ser100 and Ser511 mediates binding of 14-3-3 proteins, which keep CaMKK2 in the inactivated state by protecting against pSer495 dephosphorylation by cellular protein phosphatases. The absolute requirement for phosphorylation on both sites for 14-3-3 binding and phosphatase protection is consistent with the 'gatekeeper' model of 14-3-3 engagement with target proteins (24). In this model, 14-3-3 target proteins that contain two phospho-binding sites generally have a primary high affinity site that functions as the gatekeeper, and a secondary lower affinity site that is not only required to stabilize the overall interaction but is also essential for the biological effect of 14-3-3 binding. Our data indicates that pSer511 is the gatekeeper site on CaMKK2 due to its greater 14-3-3 binding affinity compared to the pSer100 site, which is in accordance with the pSer511 site matching the canonical 14-3-3 consensus binding motif (23).
Our crystal structure of 14-3-3 complexed with a diphosphorylated peptide that simulates simultaneous binding of the pSer100 and pSer511 sites, revealed that each site occupies the phosphopeptide-binding groove of discrete 14-3-3 monomers within the dimeric complex. Strong electron density was observed for the residues directly adjacent to the phosphoserine residues (five residues surrounding pSer100 and seven residues surrounding pSer511), while no electron density was visible for the flexible glycine linker region of the peptide. We also observed distinct binding of the diphosphorylated peptide to specific 14-3-3 monomers within the dimer, such that the gatekeeper pSer511 was always bound to 14-3-3 monomers B and D, whereas pSer100 was always bound to monomers A and C. This peculiarity is common to all tandem phosphopeptides/14-3-3 complex structures solved to date (29)(30)(31)(32)(33).
Although the structures of all four monomers are highly alike (r.m.s.d. <0.35 Å for monomers A, B C and D), there are perhaps subtle structural changes that allow the crystal to pack with the 14-3-3 dimer in the same orientation with respect to the phosphorylated peptide (pSer511 versus pSer100). The 14-3-3 dimer has been described as a molecular anvil as it maintains its overall structure upon ligand binding, while distorting the ligand to fit the phosphopeptide binding groove (24). Consequently, the 14-3-3 interaction forces a new conformation on the target protein to regulate activity, localization, post-translation modifications or protein-protein interactions. In the case of CaMKK2, 14-3-3 protein binding does not directly alter kinase activity since forskolin/IBMX treatment failed to impair Ca 2+ -CaM activation of the S495C mutant, despite the fact that 14-3-3 binding was unaffected. Rather, the effect of 14-3-3 binding is purely steric hindrance to protect pSer495 from dephosphorylation. CaMKK1 (the closest human homologue to CaMKK2) is inhibited by PKA by a mechanism similar to CaMKK2, but with some notable differences. Unlike CaMKK2, there are two inhibitory PKA phosphorylation sites on CaMKK1 i.e. Ser458 located within the CaM binding sequence and Thr108 (25,34).
Regarding the latter, the corresponding Thr145 residue in CaMKK2 has been reported to be phosphorylated in cells by AMPK rather than PKA, as part of an inhibitory feedback loop mechanism (35). This is consistent with our data, which shows that mutation of Ser495 is sufficient to prevent inactivation of CaMKK2 by PKA, reinforcing the view that Thr145 is not an inhibitory PKA phosphorylation site in cells. PKA also promotes binding of 14-3-3 proteins to CaMKK1 via phosphorylation of Ser74 and Ser475, which correspond to the Ser100 and Ser511 sites in CaMKK2, respectively. In contrast to CaMKK2, 14-3-3 protein binding directly inhibits CaMKK1 activity as well as blocking dephosphorylation of pThr108 (36,37).
The physiological function of the PKA-CaMKK2 signaling axis is unclear, however evidence in the literature hints at potential roles in metabolic control. For example, PKA RIIa regulatory subunit knockout mice that exhibit constitutive PKA catalytic subunit activation, are protected against high-fat diet induced weight gain, glucose intolerance and insulin resistance (15,16). This is similar to the phenotype displayed by CaMKK2 knockout mice (4), indicating that PKA-dependent regulation of whole-body energy metabolism may involve inactivation of CaMKK2. This concept is supported by our finding that CaMKK2 was inactivated in cells by the GLP-1 receptor agonist, Liraglutide, used clinically to treat obesity and Type 2 diabetes. The metabolic effects of Liraglutide (appetite suppression, weight loss, increased insulin sensitivity) are likewise similar to the metabolic phenotype observed in CaMKK2 knockout mice (4,18), raising the possibility that some of the therapeutic benefits of Liraglutide may also be mediated by the PKA-CaMKK2 pathway. A recent study found that PKA negatively regulates vascular endothelial growth factor (VEGF)-induced AMPK activation (a proangiogenic signaling pathway) via CaMKK2, indicating a potential role of the PKA-CaMKK2 axis in the control of angiogenesis (38).
Inappropriate activation of CaMKK2 plays a major role in the development of prostate cancer and hepatocellular carcinoma (5,39,40). CaMKK2 activation has also been implicated in acquired resistance of highgrade serous ovarian cancer to chemotherapy (41). Therefore, drugs that stimulate cAMP-PKA signaling may offer potential new treatment strategies to inactivate CaMKK2 in these cancers. Indeed, the GLP-1 receptor agonist Exendin-4 has been reported to decrease proliferation of prostate cancer cells by a PKA-dependent mechanism, and attenuate prostate cancer growth in a tumor xenograft mouse model (42). Exendin-4 was also shown to inhibit migration and promote apoptosis in ovarian cancer cells (43). Therefore, PKA-dependent inactivation of CaMKK2 may provide a potential mechanism for the anti-proliferative effects of Exendin-4 in both prostate and ovarian cancers.
In summary, we described a molecular mechanism by which intracellular activation of cAMP-PKA signaling inactivates CaMKK2. Further studies are required to uncover the physiological relevance of the PKA-CaMKK2 signaling node, however our data reveal a pathway that can potentially be targeted for human diseases associated with aberrant activation of CaMKK2.

EXPERIMENTAL PROCEDURES Cyclic-AMP Measurements
All cyclic-AMP measurements were acquired using liquid chromatography-mass spectrometry (LC-MS) from perchlorate extracts of forskolin and IBMX (Sigma-Aldrich) treated COS7 cells, using an ABSCIEX 5500 mass spectrometer operated with the turbo V ion source coupled to a Shimadzu Prominence LC-20AD UFLC pumps. LC conditions were optimised for a 50 mm (length) and 2.1 mm (inner diameter) C18 column (5 µm, Vydac). The LC solvent system was (A) 100% H2O and (B) 100% acetonitrile. cAMP was eluted at a flow rate of 300 µl/min in a gradient program consisting of 100% A (5 min), 0 to 70% B (10 min). Data was analysed with Multiquant 2.0.2 utilising the area under the LC chromatogram for the corresponding cAMP peak. Calibration curves were obtained by linear regression of the peak area ratio of a cAMP standard (Sigma-Aldrich). All data was acquired in negative mode. The chromatography was performed on Agilent 6220 ESI-TOF mass spectrometer coupled to an Agilent 1260 BinPump system. CaMKK2 was resolved on an Aeris 3.6 µm WIDEPORE C4 200 Å, LC Column (150 x 2.1 mm, Phenomenex) using an elution gradient of 5-90% acetonitrile at 200 μl/min for 30 min. Buffer A was 0.1% formic acid and buffer B was 100% acetonitrile/0.1% formic acid. The mass spectrometer was set to MS1 acquisition mode and operated in positive mode with a mass range of 100-3200 m/z and scan rate of 1.10. Source gas temperature was set to 325 °C, gas flow 8 litres/min and nebuliser 45 psi. Mass spectra were deconvoluted using Agilent MassHunter Qualitative Build 6 (v. 6.0.633.0) software.

Mass spectrometry
For tandem mass spectrometry, eluted CaMKK2 was pH adjusted using 50 mM triethyl ammonium bicarbonate (TEAB) solution, reduced with 10 mM TCEP for 45 min at 37 °C and alkylated with 55 mM iodoacetamide for 30 min at room temperature in the dark. The samples were then digested with trypsin (which cleaves on the C-terminal side of lysine and arginine residues) [1:50, w/w] overnight at 37°C. Digested tryptic peptides were cleaned up using Oasis HBL solid phase extraction (SPE) cartridges (Waters) and freeze-dried overnight.
Dried tryptic peptides were resuspended in 0.1% [v/v] formic acid and analysed by LC-MS/MS using a Q-Exactive plus mass spectrometer (Thermo Fisher Scientific) fitted with nanoflow reversedphase-HPLC (Ultimate 3000 RSLC, Dionex). The nano-LC system was equipped with an Acclaim Pepmap nano-trap column (Dionex -C18, 100 Å, 75 μm × 2 cm) and an Acclaim Pepmap RSLC analytical column (Dionex -C18, 100 Å, 75 μm × 50 cm). Typically for each LC-MS/MS experiment, 5 μl of the peptide mix was loaded onto the enrichment (trap) column at an isocratic flow of 5 μl/min of 3% [v/v] acetonitrile containing 0.1% [v/v] formic acid for 6 min before the enrichment column is switched in-line with the analytical column. The eluents used for the LC were 0.1% [v/v] formic acid (solvent A) and 100% acetonitrile/0.1% formic acid [v/v] (solvent B). The gradient used was 3% B to 25% B for 23 min, 25% B to 40% B in 2 min, 40% B to 80% B in 2 min and maintained at 85% B for the final 2 min before equilibration for 9 min at 3% B prior to the next analysis. All spectra were acquired in positive mode with full scan MS spectra scanning from m/z 375-1400 at 70000 resolution. Mass spectrometric raw data were converted to centroid peaklists using MSConvert (version 3.0.5047) and searched using Mascot (version 2.4) search algorithm against human SwissProt database (20282 sequences, November 2015). Trypsin was selected as the protease with two missed cleavages allowed. MS tolerance was set to 10 ppm, MS/MS tolerance at 0.2 Da, and ion score significance threshold was p<0.05. Cysteine carbamidomethylation was searched as a fixed modification, whereas oxidation of methionine, phosphorylation of serine, threonine and tyrosine were searched as variable modifications. The tandem mass spectrometry proteomics raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020133 (44).

Recombinant CaMKK2 expression and purification
Recombinant wild-type and mutant CaMKK2 was expressed in either COS7 cells or SH-SY5Y cells grown in Dulbecco Modified Eagle Medium or Eagles Minimum Essential Medium/F-12 medium (Sigma-Aldrich), respectively, supplemented with 10% fetal calf serum at 37 °C with 5% CO2. Cells were transfected at 60% confluency using FuGene HD (Roche Applied Science) with 2 µg of pcDNA3(-) plasmid containing N-terminal Flag-tagged human CaMKK2. For the PKA-Ca shRNA knockdown experiments, the cells were co-transfected with 2 µg of PKA-Ca targeted or scrambled shRNA plasmid (Santa Cruz). After 48 hrs, transfected cells were treated with or without forskolin and IBMX (or DMSO vehicle control) and harvested by rinsing with ice-cold phosphate-buffered saline (PBS), followed by rapid lysis in situ using 1 ml of lysis buffer (50 mM Tris.HCl [pH 7.4], 150 mM NaCl, 50 mM NaF, 1 mM NaPPi, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1% [v/v] Triton X-100) containing Complete protease inhibitor cocktail (Roche Applied Science).
Insoluble debris was removed by centrifugation and total protein content was quantified using the Bradford assay (Thermo Fisher Scientific). CaMKK2 was purified from 1.5 mg of total cell lysate using 100 µl of anti-Flag M2 agarose (50% v/v) pre-equilibrated in lysis buffer, followed by successive washes in lysis buffer containing 1 M NaCl, and finally into 50  Radioactivity was quantified by liquid scintillation counting. CaMKK2 activities were corrected for minor differences in expression levels by immunoblot analysis of cell lysates.
CaM overlay assay 200 ng of wild-type CaMKK2 and phosphorylation site mutants (S100A, S495C, S511A) purified from DMSO vehicle control and forskolin/IBMX treated COS7 cells were spotted onto nitrocellulose membrane (GE Lifesciences) and allowed to dry for 30 mins. The membrane was then blocked in PBS/1% Tween-20 (PBS-T) supplemented with 2% non-fat milk for 1 hr, after which it was incubated overnight at 4 °C with biotinylated CaM (500 nM; Millipore) and mouse anti-Flag antibody (100 ng/ml) in PBS-T containing 1% non-fat milk and 10 mM CaCl2. The membranes were briefly washed in PBS-T/10 mM CaCl2, and then incubated with IR680 dye labeled streptavidin and goat anti-mouse IgG IRDye 800 (Li-Cor) for 1 hr. After successive washing with PBS-T/10 mM CaCl2, membranes were scanned, and the images quantified with an Odyssey CLx Infrared Imager.

Surface plasmon resonance binding assays
The interaction between synthetic peptides corresponding to the Ser100 and Ser511 phosphorylation sites and 14-3-3z was measured using a BIAcore T200 instrument. N-terminal biotinylated peptides corresponding to unphosphorylated (Ser100-Ser511), monophosphorylated (pSer100-Ser511; Ser100-pSer511) and diphosphorylated (pSer100-pSer511) species of CaMKK2 (custom synthesized by Genscript) were immobilized on streptavidin sensor chips (SAHC30M; Xantec Bioanalytics, immobilization level ~ 0.8ng/mm 2 ). Various 14-3-3 concentrations (0-2 µM) diluted in HBS-P buffer (10 mM Hepes.NaOH [pH 7.35], 150 mM NaCl, 0.005% Tween-20) were injected over the sensor surface and binding profiles were recorded in real time, and sensorgrams generated by subtracting the signals from the blank streptavidin control flow cell. Following analyte injection phase completion (2 mins), the dissociation was monitored in HBS-P buffer for 5 mins. All flow-cells were washed with 10 mM glycine [pH 3.0] to ensure all remaining analytes had been dissociated prior to the next injection cycle. All binding experiments were conducted in triplicate with consistent results. Dissociation constants were calculated by fitting the data to a single site binding model, except for the diphosphorylated (pSer100-pSer511) peptide, which was fitted to a two-site model (BIAcore T200 evaluation software, version 2.0).
Data were processed using XDS and scaled in Aimless (47,48). The structure was solved by the molecular replacement method using a 14-3-3 dimer (PDB code 1QJB) as the search model (49). The asymmetric unit cell consists of a dimer of dimers, with one dimer represented by chains A and chain B and the second dimer represented by chains C and D. The two dimers are almost identical with an r.m.s.d of 0.49 Å. In the final structure there are 230 residues in chains A, B, D and 299 residues in chain C of 14-3-3. An extra N-terminal histidine residue is present in all the chains as a result of cloning of the 14-3-3 gene (denoted as residue 0). Residues 70-74 from chain C, residues 206-211 from chain A, residues 69-72 from chain B, residues 70-72 from chain A and residues 203-210 from chain C were not modelled due to poor electron density in these regions. The structure was refined using REFMAC and Phenix (50,51). Of the 27 residues in the diphosphopeptide, 12-14 residues were built into the 2Fo-Fc map in each dimer. The final Rwork and Rfree for the 14-3-3diphosphopeptide complex was 0.18 and 0.23 respectively. The model had a Molprobity clash score of 5.89 (99th percentile) (52), a protein geometry score of 1.95 (95th percentile), and favored Ramachandran of 97.32% with no outliers.

Statistical analysis
The data are presented as mean values ± standard deviation (SD) for at least three independent experiments. Statistical analyses were performed using GraphPad Prism (version 8.4.3). T-tests, one-way and twoway ANOVA, and extra sum-of-squares F tests were used for statistical appraisal, where indicated, in the figure legends.

DATA AVAILABILITY
Co-ordinates and structure factors have been deposited in the protein databank (PDB) with the accession code 6EF5. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (https://www.ebi.ac.uk/pride/archive) with the dataset identifier PXD020133.   Binding of endogenous 14-3-3 proteins to wild-type CaMKK2 purified from transfected COS7 cells pre-treated with 20 µM H89 for 60 mins, followed by treatment with DMSO (vehicle control) or 50 µM forskolin/500 µM IBMX for 20 mins. 14-3-3 protein and total CaMKK2 were detected using rabbit anti-pan-14-3-3 and mouse anti-Flag antibodies, respectively. A representative immunoblot is shown. (B) Binding of endogenous 14-3-3 proteins to wild-type CaMKK2 and phosphorylation site mutants purified from transfected COS7 cells treated with DMSO (vehicle control) or 50 µM forskolin/500 µM IBMX for 20 mins. 14-3-3 proteins and total CaMKK2 were detected using rabbit anti-pan-14-3-3 and mouse anti-Flag antibodies, respectively. A representative immunoblot is shown. (C) Ser495 phosphorylation of wild-type CaMKK2 and the S100A and S511A mutants purified from transfected COS7 cells treated with 50 µM forskolin/500 µM IBMX over a 60 min time course, with addition of 20 µM H89 to the cells at the 20 min time point. Ser495 phosphorylation and total CaMKK2 were detected using rabbit phospho-specific and mouse anti-Flag antibodies, respectively. A representative immunoblot is shown. (D) Ca 2+ -CaM activation of wild-type CaMKK2 and the S100A and S511A mutants from transfected COS7 cells treated with 50 µM forskolin/500 µM IBMX over a 60 min time course, with addition of 20 µM H89 to the cells at the 20 min time point. CaMKK2 was immunoprecipitated and activity measured in the presence of 100 µM Ca 2+ and 1 µM CaM. n=3 independent experiments. The area under the curve is displayed as mean ± SD and was statistically appraised by one-way ANOVA (F=122.7, P<0.0001) using Tukey's post-hoc multiple comparisons test. **** P<0.0001.