Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Feb 12;478(3):553-578.
doi: 10.1042/BCJ20200937.

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

Asad U Malik  1 , Athanasios Karapetsas  1 , Raja S Nirujogi  1 , Sebastian Mathea  2 , Deep Chatterjee  2 , Prosenjit Pal  1 , Pawel Lis  1 , Matthew Taylor  1 , Elena Purlyte  1 , Robert Gourlay  1 , Mark Dorward  1 , Simone Weidlich  1 , Rachel Toth  1 , Nicole K Polinski  3 , Stefan Knapp  2 , Francesca Tonelli  1 , Dario R Alessi  1
Affiliations

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

Asad U Malik et al. Biochem J. .

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 homolog 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, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.

Keywords: Rab GTPase; kinase; leucine rich repeat kinase; phosphorylation.

PubMed Disclaimer

Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1.
Figure 1.. LRRK1 phosphorylates endogenous Rab7A at Ser72.
Wild type and homozygous LRRK1 knock-out primary MEFs cultured in 10% (by vol) serum were lysed. (1A, upper panel) 50 µg of extracts from two independent clones were subjected to SDS–polyacrylamide gel electrophoresis and the region encompassing Rab proteins (20–30 kDa) was excised and subjected to in-gel digestion using trypsin. The extracted peptides were spiked with 25 femto moles of heavy phosphorylated Rab3, Rab7a, Rab8, Rab10, Rab35 and Rab43. Samples were analyzed using Parallel reaction monitoring (PRM) acquisition mode on a QE-HFX mass spectrometer. The raw data was processed using Skyline software and the relative expression of each pRab was determined between LRRK1 KO and wt. n = 4, Each biological replicate analyzed as technical duplicate of each sample and the individual data marked with a black circle and the data presented as mean ± SEM. (1A, lower panel) The MEF extracts (20 µg) from three independent clones for wild type and homozygous knock-out clones were subjected to immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different clone. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. (B) As in A, except that three independent clones of littermate-matched wild type, heterozygous LRRK1 knock-out (+/−) and homozygous knock-out (−/−) primary MEFs were treated ± 200 nM MLi-2 for 30 min.
Figure 2.
Figure 2.. Further evidence that LRRK1 specifically phosphorylates Rab7A.
(A and B) HEK293 cells were transiently transfected with the indicated plasmids encoding for wild type (wt) or kinase inactive (KI, D1409A) LRRK1 or LRRK2 and the indicated wild type or mutant Rab protein. Twenty-four hours post-transfections the cells were lysed and extracts (20 µg) from a triplicate experiment in which cells cultured in separate dishes were subjected to immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different replicate. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system.
Figure 3.
Figure 3.. Recombinant LRRK1 phosphorylates Rab7A at Ser-72 in vitro.
(A) Wild type (wt) Rab7A or Rab7A[S72A] was incubated in a phosphorylation reaction with recombinant LRRK1 as described in the materials and methods. Reactions were terminated after 60 min with SDS-Sample buffer. Samples subjected phos-tag electrophoresis followed by Coomassie staining (upper panel) or conventional immunoblot analysis with the indicated antibodies (all at 1 µg/ml) (lower panel). The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. A Coomassie gel of the recombinant LRRK1 utilized in this experiment is shown in Supplementary Figure S1. (B) As in (A) except that [γ32P]-ATP was employed for the phosphorylation reaction. Samples were resolved by SDS–PAGE and stained using Coomassie blue (upper band) and subjected to autoradiography (lower band). (C) Bands corresponding to 32P-Rab7A were excised from the gel and subjected to trypsin digestion. Resulting peptides were separated by reverse-phase C18 chromatography and 32P radioactivity was detected using an online radioactivity detector. (D) The major peak of 32P-radioactivity was analyzed by Edman degradation as well mass spectrometry. Release of 32P radioactivity after each cycle of Edman degradation was determined and the amino acid sequence of the peptide determined by mass spectrometry analysis is shown. (E) Wild type Rab10 (upper panel) or Rab7A (lower panel) were incubated in a phosphorylation reaction with either recombinant LRRK1 or LRRK2 described in the materials and methods. Reactions were terminated at the indicated time-points with SDS-Sample buffer and analyzed by immunoblot analysis with the indicated antibodies (all at 1 µg/ml).
Figure 4.
Figure 4.. PMA stimulates LRRK1-dependent Rab7A phosphorylation.
(A) Wild type MEFs were deprived of serum for 16 h and incubated for 1 h in the presence or absence of the dose of indicated inhibitor. Cells were next stimulated with the dose of indicated agonists for the times shown. Control blots for all agonists are included in Supplementary Figure S2. Extracts (20 µg) from duplicate independent clones were subjected to immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different cell clone. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. (B) Wild type, heterozygous (+/−) and homozygous LRRK1 knock-out (−/−) primary MEFs were deprived of serum for 16 h and incubated in the presence or absence of 100 ng/ml PMA for 30 min and immunoblotted as in (A). (C) As in (A) except that wild type MEFs were treated with 100 ng/ml PMA for the indicated time-points. (D) As in (A), except that wild type MEFs were deprived of serum for 16 h and then incubated for 1 h in the presence or absence of the dose of indicated inhibitor. Cells were then stimulated with PMA (100 ng/ml) for 30 min. Control blots for all inhibitors are shown in Supplementary Figure S3. (E) As in (A) except that cells were treated in the presence or absence of 2 µM MRT67307 for 1 h and then stimulated with either 5 ng/ml interleukin-1a (IL-1A) for 15 min or 100 ng/ml PMA for 30 min. (F) As in (A) except that the previously described [47] wild type and homozygous PINK1 knock-out MEFs were treated in the presence or absence of 1 µM oligomycin and 10 µM antimycin A for 24 h. For measurement of PINK1 stabilization, PINK1 was immunoprecipitated from extracts using PINK1 antibody (DA039, MRC PPU reagents and Services) coupled to Protein A/G beads (see materials and methods). For measurement of phosphorylation of ubiquitin at Ser65, the ubiquitin in the extracts was enriched by performing HALO-MultiDsk affinity purification (see materials and methods).
Figure 5.
Figure 5.. Identification of mutations that enhance LRRK1 activity.
(A) Sequence alignment of LRRK2 and LRRK1. Residues corresponding to Parkinson's mutations in LRRK2 are highlighted in blue. Tyr971 was reported to be an inhibitory phosphorylation site targeted by EGF receptor is indicated in green. (B) Domain arrangement of LRRK2 and LRRK1, with pathogenic and corresponding residues highlighted in black. Reported variants of LRRK1 are indicated in blue. (C and D) HEK293 cells were transiently transfected with the indicated plasmids encoding for wild type (wt) and indicated mutant of LRRK1 and wild type Rab7A. The Kinase inactive (KI) mutant corresponds to LRRK1[D1409A]. Twenty-four hours post-transfection the cells were lysed and extracts (20 µg) from a duplicate experiment in which cells cultured in separate dishes were subjected to immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different replicate. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. Subsequent quantification presents the mean with error bars representing SEM. Similar results were obtained in three independent experiments. In Supplementary Figure S5 data is shown that the double LRRK1[Y971F + K746G] mutant does not stimulate LRRK1 activity beyond the single mutants.
Figure 6.
Figure 6.. LRRK1 is inhibited by GZD-824 but not MLi-2.
HEK293 cells were transiently transfected with the indicated plasmids encoding for wild type Rab7A and the indicated wild type and mutant forms of LRRK1 or wild type LRRK2. Twenty-four hours post-transfection, cells were treated with the indicated concentrations of MLi-2 (A) or GZD-824 (B) for 2 h. Cells were lysed and extracts (20 µg) from a duplicate experiment in which cells were cultured in separate dishes were subjected to immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different replicate. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. Similar results were obtained in three independent experiments. In Supplementary Figure S6 we present the equivalent data undertaken with GSK2578215A, HG10-102-01, LRRK2-IN1 and iN04 inhibitors. (C) Wild type Rab7A was incubated in a phosphorylation reaction with recombinant LRRK1 as described in the materials and methods in the presence of the indicated concentrations GZD-824. Quantifications of experiments performed in (B) and (C) are presented in Supplementary Figure S7. Reactions were terminated after 60 min with SDS-Sample buffer. Samples were subjected to immunoblot analysis with the indicated antibodies (all at 1 µg/ml). The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system.
Figure 7.
Figure 7.. LRRK1 is not regulated by VPS35[D620N] mutation and overexpression of Rab29.
(A) Wild type, and homozygous VPS35[D620N] knock-in primary MEFs were deprived of serum for 16 h and then incubated in the presence or absence of 100 ng/ml PMA for 30 min. Extracts (20 µg) from triplicate independent clones were subjected to immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different cell clone. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. Similar results were obtained in three independent experiments. (B, upper) HeLa cells were transfected with wild type GFP-LRRK1 or GFP-LRRK2 in the presence of absence of HA-Rab29. Twenty-four hours post-transfection cells were fixed in 4% (by vol) paraformaldehyde and stained with mouse anti-HA. Scale bar represents 30 µm. (B, lower) As in (B, upper) except that cell are lysed and analyzed by immunoblot analysis as described in (A).
Figure 8.
Figure 8.. Phosphorylation of Rab7A does not affect interaction with RILP.
HEK293 cells were transiently transfected with the indicated plasmids. Note that the Kinase inactive (KI) mutant corresponds to LRRK1[D1409A]. Twenty-four hours post-transfection cells were lysed either GFP-RILP (upper panel) or HA-Rab7A (middle panel) immunoprecipitated with GFP or HA antibody, respectively. The immunoprecipitates as well as cell extracts (20 µg, lower panel) were subjected to immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different replicate. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system.
Figure 9.
Figure 9.. PP1MH, but not PPM1J or PPM1M, dephosphorylates Rab7A at Ser72.
HEK293 cells were transiently transfected with the indicated plasmids encoding for wild type or catalytically inactive PPM1H (upper panel), PPM1J (middle panel) or PPM1M (lower panel), alongside LRRK1[K746G] or kinase inactive (KI) and wild type Rab7A. The Kinase inactive (KI) mutant corresponds to LRRK1[D1409A]. Twenty-four hours post-transfection the cells were lysed and extracts (20 µg) from a duplicate experiment in which cells cultured in separate dishes were subjected to immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different replicate. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Manning, G., Whyte, D.B., Martinez, R., Hunter, T. and Sudarsanam, S. (2002) The protein kinase complement of the human genome. Science 298, 1912–1934 10.1126/science.1075762 - DOI - PubMed
    1. Bosgraaf, L. and Van Haastert, P.J. (2003) Roc, a Ras/GTPase domain in complex proteins. Biochim. Biophys. Acta 1643, 5–10 10.1016/j.bbamcr.2003.08.008 - DOI - PubMed
    1. Sejwal, K., Chami, M., Rémigy, H., Vancraenenbroeck, R., Sibran, W., Sütterlin, R.et al. (2017) Cryo-EM analysis of homodimeric full-length LRRK2 and LRRK1 protein complexes. Sci. Rep. 7, 8667 10.1038/s41598-017-09126-z - DOI - PMC - PubMed
    1. Civiero, L., Vancraenenbroeck, R., Belluzzi, E., Beilina, A., Lobbestael, E., Reyniers, L.et al. (2012) Biochemical characterization of highly purified leucine-rich repeat kinases 1 and 2 demonstrates formation of homodimers. PLoS ONE 7, e43472 10.1371/journal.pone.0043472 - DOI - PMC - PubMed
    1. Nguyen, A.P. and Moore, D.J. (2017) Understanding the GTPase activity of LRRK2: regulation, function, and neurotoxicity. Adv. Neurobiol. 14, 71–88 10.1007/978-3-319-49969-7_4 - DOI - PMC - PubMed

Publication types

MeSH terms