Abstract
The addition of phosphate groups to substrates allows protein kinases to regulate a myriad of biological processes, and contextual analysis of protein-bound phosphate is important for understanding how kinases contribute to physiology and disease. Leucine-rich repeat kinase 2 (LRRK2) is a Ser/Thr kinase linked to familial and sporadic cases of Parkinson's disease (PD). Recent work established that multiple Rab GTPases are physiological substrates of LRRK2, with Rab10 in particular emerging as a human substrate whose site-specific phosphorylation mirrors hyperactive LRRK2 lesions associated with PD. However, current assays to quantify Rab10 phosphorylation are expensive, time-consuming and technically challenging. In back-to-back studies reported in the Biochemical Journal, Alessi and colleagues teamed up with clinical colleagues and collaborators at the Michael J. Fox Foundation (MJFF) for Parkinson's research to develop, and validate, a panel of exquisitely sensitive phospho-specific Rab antibodies. Of particular interest, the monoclonal antibody-designated MJFF-pRAB10 detects phosphorylated Rab 10 on Thr73 in a variety of cells, brain extracts, PD-derived samples and human neutrophils, the latter representing a previously unrecognised biological resource for LRRK2 signalling analysis. In the future, these antibodies could become universal resources in the fight to understand and quantify connections between LRRK2 and Rab proteins, including those associated with clinical PD.
Highlights
Parkinson’s disease (PD) affects ∼1 in 100 people over the age of 60, with the number of sufferers predicted to increase to >10 million in the world’s most populated nations by 2030 [1]
Many of these reagents are, or will soon be, available either commercially or collaboratively. This published panel [13,16,19,23,24,35] can be employed alongside other tools to interrogate Leucinerich repeat kinase 2 (LRRK2) signalling and Rab protein phosphorylation via conventional immunological procedures. The utility of these reagents might extend well beyond this rather specific research area, and their general availability could influence current and future generations of research scientists seeking to uncover LRKK2 and Rab biology associated with health and disease
It is notable that the LRRK2-phosphorylated motif is very highly conserved in Rab proteins such as Rab10 from yeasts to worms, flies and vertebrates
Summary
Parkinson’s disease (PD) affects ∼1 in 100 people over the age of 60, with the number of sufferers predicted to increase to >10 million in the world’s most populated nations by 2030 [1]. Various cellular mechanisms have been proposed to contribute to the localised neuronal death that occurs in the substantia nigra of PD brains These include protein folding/aggregation defects generating α-synuclein-rich Lewy bodies, abnormal protein phosphorylation/ubiquitination, aberrant intracellular trafficking and oxidative stress coupled to mitochondrial dysfunction [3]. The evaluation of phosphosite preferences reveals that LRRK2-catalyzed phosphorylation occurs on a conserved Ser or Thr residue in 14 separate Rab proteins located within the nucleotide-sensing switch II region of the GTPase domain These amino acids (Glu-Arg-Phe/Tyr-Arg/His-Ser/ Thr-Hyd, where Hyd=hydrophobic, and Ser/Thr is the site of LRRK2 phosphorylation) conform to the broad LRRK2 substrate consensus motif previously defined with peptide substrates [32]. The identification of such a wide variety of Rab proteins as physiological LRRK2 substrates means that one obstacle for evaluating LRRK2 signalling output has essentially been overcome, with rapid, quantitative and reproducible Rab phosphorylation assays becoming a high priority on the research menu
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