Autophosphorylation in the Leucine-Rich Repeat Kinase 2 (LRRK2) GTPase Domain Modifies Kinase and GTP-Binding Activities

The leucine-rich repeat kinase 2 (LRRK2) protein has both guanosine triphosphatase (GTPase) and kinase activities, and mutation in either enzymatic domain can cause late-onset Parkinson disease. Nucleotide binding in the GTPase domain may be required for kinase activity, and residues in the GTPase domain are potential sites for autophosphorylation, suggesting a complex mechanism of intrinsic regulation. To further define the effects of LRRK2 autophosphorylation, we applied a technique optimal for detection of protein phosphorylation, electron transfer dissociation, and identified autophosphorylation events exclusively nearby the nucleotide binding pocket in the GTPase domain. Parkinson-disease-linked mutations alter kinase activity but did not alter autophosphorylation site specificity or sites of phosphorylation in a robust in vitro substrate myelin basic protein. Amino acid substitutions in the GTPase domain have large effects on kinase activity, as insertion of the GTPase-associated R1441C pathogenic mutation together with the G2019S kinase domain mutation resulted in a multiplicative increase (∼ 7-fold) in activity. Removal of a conserved autophosphorylation site (T1503) by mutation to an alanine residue resulted in greatly decreased GTP-binding and kinase activities. While autophosphorylation likely serves to potentiate kinase activity, we find that oligomerization and loss of the active dimer species occur in an ATP- and autophosphorylation-independent manner. LRRK2 autophosphorylation sites are overall robustly protected from dephosphorylation in vitro, suggesting tight control over activity in vivo. We developed highly specific antibodies targeting pT1503 but failed to detect endogenous autophosphorylation in protein derived from transgenic mice and cell lines. LRRK2 activity in vivo is unlikely to be constitutive but rather refined to specific responses.

Graphical AbstractLRRK2 autophosphorylation (highlighted in red) occurs in the GTP-binding pocket of the LRRK2 GTPase domain. These modifications modify both GTP-binding and kinase activities and may be required for full activity of the protein.Figure optionsDownload full-size imageDownload high-quality image (212 K)Download as PowerPoint slide