World Parkinson Congress edition:2 location:Glasgow, UK date:28 September-1 October 2010
Background: Leucine-rich repeat kinase 1 and 2 (LRRK1 and LRRK2) are large, complex multidomain proteins containing kinase and GTPase enzymatic activities and multiple protein-protein interaction domains. Mutations in LRRK2 are linked to autosomal dominant forms of Parkinson’s disease (PD) and result in amino acid changes within the catalytic core of the protein, with consequences on both its enzymatic and self-interaction properties. PD-associated LRRK1 mutations have not been reported yet and we have previously shown that pathological mutations in LRRK2 are more toxic than the equivalent mutations in LRRK1.
Aim: At present, little information on the normal function, the disease-inducing function of mutant LRRK2 and the 3D structures of the two proteins are available. Starting from the observation that only LRRK2 is associated with the disease, we decided to characterize the two proteins in parallel to single out LRRK2 specific properties.
Methods and Results: Most biochemical studies have been performed using suspensions of protein bound to affinity resin. Reports using recombinant LRRK2 in solution have employed truncated fragments or recombinant protein with large affinity tags (i.e. GST). Here, we produced highly pure recombinant LRRK1 and LRRK2 soluble proteins with a small affinity tag. Using a lentiviral expression vector, we have been able to express 3xFLAG tagged full-length LRRK1 and LRRK2 in HEK293T cells and purify LRRK2 to >90% purity. We also expressed and purified soluble full-length 2xMyc tagged LRRK1 and LRRK2 using mammalian expression plasmids. Proteins obtained in this way show activity in autophosphorylation, phosphorylation of a moesin based peptide (ERM peptide, aka LRRKtide) as well as low GTPase activity.
Discussion: We are currently identifying the autophosphorylation and heterologous phosphorylation sites of both proteins by mass spectrometry. In addition, we are using cryoelectron-microscopy to define the quaternary structure of the proteins, which we, and others, have shown to be dimeric in solution. We expect that comparing these biochemical and structural properties of LRRK2 versus LRRK1 in the context of full-length molecules could provide additional clues on the pathogenic mechanism of LRRK2.