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Gene Synthesis> | The N-terminal GST-tagged kinase domain of ameba LRRK2 (GST-ROCO4, corresponding to residues 1–287 of PDB accession 4F0F_A) and the N-terminal His6-tagged human LRRK2 ROC domain (His6-ROC, corresponding to residues 1333–1516 of NP_940980.3) were created through custom gene synthesis (Genscript, Piscataway, NJ, USA), expressed in Escherichia coli, and purified (9, 22). | Get A Quote |
The leucine-rich repeat kinase (LRRK)-2 protein contains nonoverlapping GTPase and kinase domains, and mutation in either domain can cause Parkinson disease. GTPase proteins are critical upstream modulators of many effector protein kinases. In LRRK2, this paradigm may be reversed, as the kinase domain phosphorylates its own GTPase domain. In this study, we found that the ameba LRRK2 ortholog ROCO4 phosphorylates the GTPase domain [termed Ras-of-complex (ROC) domain in this family] of human LRRK2 on the same residues as the human LRRK2 kinase. Phosphorylation of ROC enhances its rate of GTP hydrolysis [from kcat (catalytic constant) 0.007 to 0.016 min(-1)], without affecting GTP or GDP dissociation kin... More