Fig. 6: Control of PYK2 function through fuzzy CaM-binding and dimerisation of KFL.

a CaM antagonist calmidazolium (Cz, 50 µM) blocks PYK2 Y402 phosphorylation induced by membrane depolarisation in PC12 cells. Depolarisation was induced by isosmotic replacement of 40 mM NaCl by 40 mM KCl in the extracellular medium for 3 min. Graphical representation of PYK2 pY402 over total PYK 2 densitometry for transfected and endogenous PYK2 bands (mean values of 2 replicates). b Dimerisation of PYK2-KFL_728–839 in the presence of 10 µM CaM. Experiments were carried out by MST (top) and FA (bottom) in the presence of Ca²⁺ or EGTA. The fluorescently labelled protein (indicated by a red star) was at a concentration of 50 nM, and the unlabelled protein was serially diluted from a concentration of 100 µM. The condition (Ca²⁺ or EGTA), and the derived K_d values are noted in the figures. c MST analysis of fluorescently labelled PYK2 KFL_728–839 was kept at 50 nM (where it is predominantly monomeric) and titrated with unlabelled PYK2 FERM–kinase. The presence of Ca²⁺ or EGTA and the K_d are stated. Binding experiments are represented as (mean ± SD, n = 3). d Proposed molecular mechanism. Left: Theoretical model of the closed monomeric PYK2, adopted in the absence of Ca²⁺ and other specific stimuli. Molecular surfaces of the FERM (green), kinase (cyan) and FAT (magenta) domains are shown. The FERM–kinase association was modelled based on the corresponding FAK fragment (PDB accession 2j0k). PYK2 FAT was docked onto the FERM domain as modelled for FAK in ref. ²³. The flexible linker regions are shown as dashed lines. The position of Y402 is indicated. Right: Theoretical model of the pre-activation Ca²⁺/CaM-bound PYK2 dimer, modelled on PDB accessions 2j0k and 4ny0. CaM is shown as grey rectangle with the four red dots illustrating the bound Ca²⁺ ions. Note that the CaM–KFL complex is highly disordered, and not symmetric as shown in this illustration. Colours as in the left panel, but FERM, kinase and FAT domains of the second molecule are coloured in grey.