Phosphopeptide Analysis Reveals Two Discrete Clusters of Phosphorylation in the N-Terminus and the Roc Domain of the Parkinson-Disease Associated Protein Kinase LRRK2

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) that increase its kinase activity associate with familial forms of Parkinson disease (PD). As phosphorylation determines the functional state of most protein kinases, we systematically mapped LRRK2 phosphorylation sites by mass spectrometry. Our analysis revealed a high degree of constitutive phosphorylation in a narrow serine-rich region preceding the LRR-domain. Allowing de novo autophosphorylation of purified LRRK2 in an in vitro autokinase assay prior to mass spectrometric analysis, we discovered multiple sites of autophosphorylation. Solely serine and threonine residues were found phosphorylated suggesting LRRK2 as a true serine threonine kinase. Autophosphorylation mainly targets the ROC GTPase domain and its clustering around the GTP binding pocket of ROC suggests cross-regulatory activity between kinase and Roc domain. In conclusion, the phosphoprotein LRRK2 functions as an autocatalytically active serine threonine kinase. Clustering of phosphosites within two discrete domains suggest that phosphorylation may regulate its biological functions in a yet unknown fashion.

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

“…Other regions of the protein, such as the COR domain, display reduced accessibility, in agreement with previous work showing that the COR domain of Roco proteins is involved in dimerization and is also likely surrounded by other domains in the full-length protein (27). Additional lysine residues with high accessibility to the cross-linking reagent were observed in the neighborhood of known phosphorylation sites (19,28). In contrast, the N terminus exhibits a low coverage with monolink peptides.…”

“…To improve the yield and purity of LRRK2 necessary for structural studies, the expression and purification procedure for recombinant full-length LRRK2 was optimized from previously published protocols (19). Following transient transfection of HEK293T cells, N-terminal Strep/FLAG (SF)-tagged LRRK2 was purified via the tandem Strep-tag II moiety.…”

“…The generation of N-terminal, SFtagged (NSF), full-length LRRK2 expression constructs has been previously described (19). Similar methods were used to create an N-terminal deletion construct, 1,280-end (Δ1,279-LRRK2) using the pDEST-NSF-tandem affinity purification (47) Gateway cloning vector.…”

“…Purification of SF-tagged LRRK2 purified from 4 × 14-cm dishes (600 cm 2 ) of confluent HEK293T cells was performed as described previously (19,49). Briefly, after removal of the medium, cells were lysed in 1 mL of lysis buffer and washing buffer [50 mM Hepes (pH 8.0), 100 mM NaCl, 1 mM DTT, 5 mM MgCl 2 , 0.5 mM EDTA, 10% (vol/vol) glycerol, 0.01% Triton X-100], supplemented with 0.55% Nonidet P-40 and Complete Protease Inhibitors (Roche), per 14-cm dish.…”

Cellular processes associated with LRRK2 function and dysfunction

“…LRRK2 has been found to autophosphorylate > 20 serine and threonine residues in vitro 37, 50, 56, 57, 58, 59, 60, 61. The majority of the autophosphorylation sites reside in the ROC domain, with only a few in the COR and kinase domains (Fig.…”