Calcium/calmodulin-dependent protein kinase
kinase 2 (CaMKK2) is a member of the Ca2+/calmodulin-dependent
kinase (CaMK) which is indispensable for cell function, gene expression and
apoptosis activation. Malfunction of this protein is often associated with
neuropathology, genital carcinogenesis and obesity, all making it a promising therapeutic
target. CaMKK2 is activated via binding Ca2+ sensor protein
calmodulin (CaM) and inhibited by 14-3-3 protein in a phosphorylation-dependent
manner. However, the precise molecular mechanisms by which these binding
interactions affect CaMKK2 are still unclear.
To elucidate the
structural basis of 14-3-3 and CaM binding on CaMKK2 we used analytical
ultracentrifugation, small-angle X-ray scattering and methods coupled with mass
spectrometry. Analytical
ultracentrifugation showed that phosphorylated CaMKK2 and 14-3-3 protein form a
stable complex with 1:2 molar stochiometry. Our data from SAXS suggest that the
complex has an asymmetric shape and CaMKK2 is located outside the central
channel of the 14-3-3g dimer.
Moreover, 14-3-3g appears to directly interact with several
regions of CaMKK2 outside the 14-3-3 binding motifs, including the kinase domain.
SAXS-based modeling of CaMKK2:CaM complex revealed multiple conformations,
where nearly half conformers adopts a compact structure. Simultaneously, hydrogen/deuterium
exchange and chemical crosslinking coupled with mass spectrometry suggested
that CaM also interacts with the kinase domain of CaMKK2. Direct interaction
between CaM and the kinase domain has been previously described only for one
related CaMK, Death-associated protein kinase. Altogether, it seems that
catalytic domain plays an important role in both activatory and inhibitory interactions.
However, these suggestions need to be supported by high-resolution structure of
both complexes.
This study was supported by the Czech
Science Foundation (Projects 16-02739S), the Czech Academy of Sciences
(Research Projects RVO: 67985823 of the Institute of Physiology),