14-3-3 directly interacts with the kinase domain of CaMKK1 and inhibits calmodulin binding

O. Petrvalska1, V. Obsilova1, T. Obsil2

1Department of Structural Biology of Signaling Proteins, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, 252 50 Vestec, Czech Republic

2Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic




The calcium/calmodulin-dependent kinases (CaMK) are of great interest due to their important functions in calcium signalling and especially in neuronal development [1]. Calcium/calmodulin-dependent kinase kinase 1 (CaMKK1) is a member of the CaMK family and it was shown that the phosphorylation at Thr108 and Ser458 residues by the cAMP-dependent protein kinase (PKA) directly inhibits its activity, whereas phosphorylation at Ser74 and Ser475 induces binding to the 14-3-3 protein [2,3]. The 14-3-3 is a family of regulatory proteins, which bind to other proteins in a phosphorylation dependent manner [4]. The binding of 14-3-3 to CaMKK1 via PKA phosphorylation is known to suppress CaMKK1 activity [2,3], but the underlying mechanism has not yet been described. Our research aimed to unravel this mechanism.


We analysed the structure of a complex formed between CaMKK1, phosphorylated at four different PKA sites, and a C-terminally-truncated version of the γ isoform of the 14-3-3 protein (14-3-3γ ΔC). Using hydrogen/deuterium exchange coupled to mass spectrometry, we show that 14-3-3γ ΔC binding alters the protein structure of the phosphorylated CaMKK1 in multiple regions. First, the the N- and C-terminal segments, which contain phosphorylation sites responsible for 14‑3‑3 binding. Second, the N‑lobe of the kinase domain, which is in close proximity to the CaMKK1 active site. Third, the region around the calcium binding domain. Additional small angle X-ray scattering (SAXS) and chemical cross-linking analyses revealed that the CaMKK1:14-3-3γ ΔC complex is dynamic and conformationally heterogeneous.


These findings suggest that the 14-3-3 protein-dependent inhibition of CaMKK1 may be explained by structural changes in both the catalytic and calcium binding domains. The 14-3-3:CaMKK1 complex formation might interfere with the access of Ca2+/CaM to the CaMKK1 calmodulin-binding domain and/or promote interaction between the autoinhibitory domain and the catalytic domain, thus inhibiting CaMKK1. To test the former hypothesis, we performed fluorescence anisotropy measurements using dansyl-tagged calmodulin. We revealed that while CaMKK1 binds to dansyl-calmodulin with a KD of ~mM, no binding is observed in the CaMKK1:14-3-3γ ΔC complex.


1. S. Takemoto-Kimura, K. Suzuki, S. Horigane, S. Kamijo, M. Inoue, M. Sakamoto, H. Fujii, H. Bito, J. Neurochem., 141, (2017), 808818.

2. M. A. Davare, T. Saneyoshi, E. S. Guire, S. C. Nygaard, T. R. Soderling, J. Biol. Chem., 279(50), (2004), 5219152199.

3. T. Ichimura, M. Taoka, Y. Hozumi, K. Goto, H. Tokumitsu, FEBS Letters, 582(5), (2008) 661/665.

4. T. Obsil, V. Obsilova, Semin. Cell Dev. Biol, 22(7), (2011), 663672.

This study was supported by Czech Science Foundation (Projects 19-00121S).