Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

Abstract

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homologue of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson’s mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we were unable to confirm previous data suggesting that Rab7A phosphorylation is regulated by the PINK1/TBK1 axis. Altogether, these finding reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.

R1441G but not G2019S mutation enhances LRRK2 mediated Rab10 phosphorylation in human peripheral blood neutrophils

“…Additionally, we show that MLi-2 at a concentration of up to 300 nM does not result in dephosphorylation of Rab7a, the endogenous phosphorylation substrate of the only known LRRK2 homologue LRRK1 [25] or dephosphorylation of AMPK at threonine 172. Serine 935 dephosphorylation of LRRK2 is seen at maximal levels starting at 100 nM MLi-2 and above.…”

“…The MJFF-total Rab10 mouse antibody was from nanoTools (Cat# 0680–100/Rab10-605B11). Rabbit monoclonal antibodies for total LRRK2 (N-terminus, residues 100–500, UDD3) and phospho-Ser935 LRRK2 (UDD2) as well as the sheep polyclonal antibodies for LRRK1 (sheep number S405C, 2nd bleed) were expressed and purified at the University of Dundee as described previously [ 7 , 10 , 25 ] and are available from MRC PPU Reagents and Services ( https://mrcppureagents.dundee.ac.uk/ ). The sheep polyclonal PPM1H antibodies against the full length PPM1H protein have been described before [ 2 ] and are also available from MRC PPU Reagents and Services (sheep number DA018, https://mrcppureagents.dundee.ac.uk/ ).…”

“…The rabbit polyclonal pSer72 Rab7A was recently described and was generated by The Michael J. Fox Foundation’s research tools program in partnership with Abcam [ 25 ]. Development of a monoclonal antibody is underway.…”

“…The MJFF-total Rab10 mouse antibody was from nanoTools (Cat# 0680-100/Rab10-605B11). Rabbit monoclonal antibodies for total LRRK2 (N-terminus, residues 100-500, UDD3) and phospho-Ser935 LRRK2 (UDD2) as well as the sheep polyclonal antibodies for LRRK1 (sheep number S405C, 2nd bleed) were expressed and purified at the University of Dundee as described previously [7,10,25] and are available from MRC PPU Reagents and Services (https:// mrcpp ureag ents. dundee.…”

LRRK2 Targeting Strategies as Potential Treatment of Parkinson’s Disease

“…These processes play a crucial role during immune response and several studies have linked LRRK2 signaling to inflammation in general and neuroinflammation specifically (reviewed in [25]. Interestingly, recently, it was shown that LRRK1, the human homolog of LRRK2, phosphorylates a different subset of Rab proteins than LRRK2, suggesting that LRRK enzymes are Rab specific kinases [26].…”