One of the features of ROCO proteins controlled
One of the features of ROCO proteins, controlled by their domain architecture is their oligomerization status. By size-exclusion chromatography and Western blot analysis, DAPK1 was proved to form homodimers via its GTPase and kinase domains . Similarly, 77 8 microscopy studies conducted on purified LRRK1 and LRRK2 proteins showed that also these two ROCO kinase family members can form homodimers . By using a combination of several techniques, including computing modeling, electron microscopy and mass spectrometry, a first 3D model, in which homodimers are formed with the two LRRK2 proteins organized in the same direction was proposed [5,6]. Their ROC/COR domains occupy the central position, enabling regulation of GTP binding. Importantly in this model, the kinase domain is clustered together with the WD40, Ank and LRR motifs, suggesting that they play a role in the regulation of the kinase activity, including autophosphorylation events such as phosphorylation at S1292, which is widely used as a biomarker of LRRK2 activity . The 3D model also supports cross-regulation of the LRRK2 kinase domain activity by the binding of GTP to the ROC/COR domains. This hypothesis was already highlighted following experimental data showing that the kinase activity of LRRK2 is amplified upon binding of non-hydrolysable GTP as well as by demonstrating that the R1441C mutation within the ROC domain leads to an increased kinase but a decreased GTPase activity . In principle, the GTPase domain functions as a molecular switch for the kinase domain and enables kinase activity when GTP is bound to the ROC domain, while the GDP-bound state decreases the kinase activity of LRRK2 [5,6,56]. Moreover, very recently, cryo-electron microscopy and single particle analysis allowed investigating the structure of full-length dimeric LRRK2 and LRRK1 at the higher resolution of respectively 16 and 25 Å . The latter study indicated that the LRRK2 proteins are likely arranged in a two-fold symmetric orientation whereby the Ank and the LRR domains of one monomer interact with the kinase and WD40 domains of the second, with the Arm domains extending outside the dimer core. Such orientation is fully compatible with the fact that the LRRK2 ROC domain is its primary target for autophosphorylation. Additionally, this study demonstrated that LRRK2 and LRRK1 both dimerize via a very similar mechanism. Unlike LRRK2 and LRRK1, the kinase and the ROC/COR domains of DAPK1 are separated by the Ca2+/CaM autoregulatory unit and the Ank region (Fig. 1). Therefore, a slightly different mechanism of GTPase and kinase domain regulation has been proposed [50,53]. DAPK1 kinase activity is stimulated upon protein phosphatase 2A-mediated dephosphorylation of S308, which forms an inhibitory autophosphorylation site within the CaM-binding domain [, , ]. Moreover, based on crystallography and biochemical data, it was shown that CaM plays a crucial role in DAPK1 activation. Binding of CaM to DAPK1 prevents S308 autophosphorylation and fulfills DAPK1 activation by removing the Ca2+/CaM domain from the catalytic cleft and enabling docking of the substrate [53,61,62]. Another level of catalytic DAPK1 regulation depends on the binding of GTP to the P-loop motif present in the ROC domain. GTP-bound DAPK1 is characterized by increased levels of phospho-S308 and therefore decreased kinase activity, which occurred irrespectively of the presence of protein phosphatase 2A [50,53]. In HEK293T cells transfected with LRRK1, LRRK2 and DAPK1, all of the ROCO kinases exhibited the potential to form homo- and heterodimers including LRRK2-DAPK1 and LRRK1-DAPK1 heterodimers as observed by gel filtration and co-immunoprecipitation experiments . In mammalian cell lysates, the majority of LRRK2 was found to exist in dimeric form [63,64]. Co-immunoprecipitation experiments revealed the importance of the ROC/COR domains and possibly the WD40 region in this process, while Arm, Ank and LRR motifs did not demonstrate self-interaction properties . The ability of full length LRRK2 to form dimers was not affected by the G2019S, R1441C/G/H and Y1699C mutations [54,63]. Dimerization of ROC/COR LRRK2 domain fragments also was not affected by the presence of a non-hydrolysable GTP analog or when a GTP-binding deficient mutant was introduced , however, dimerization events were suggested to regulate GTPase function itself. Dimerization of the ROC/COR domains was shown to be required for binding and efficient GTP hydrolysis [65,66].