Rab29 fast exchange mutants: characterization of a challenging Rab GTPase v1

Gomez, Rachel C; Vides, Edmundo G; Pfeffer, Suzanne R

doi:10.17504/protocols.io.bffrjjm6

Cited by 3 publications

(3 citation statements)

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“…His Rab29, His Rab10 Q68L (1–181), His Rab10 Q68L (full length) His, His-Mst3, His-Rab8A Q67L, His-LRRK2 Armadillo (1–552), His-LRRK2 Armadillo (1–159), His-LRRK2 Armadillo (350–550), His-LRRK2 Armadillo K17A, His-LRRK2 Armadillo K18A, and GST-Rab8A Q67L were purified after expression in E. coli BL21 (DE3 pLys). Detailed protocols can be found in Gomez et al, 2020 ( https://dx.doi.org/10.17504/protocols.io.bffrjjm6 ) and Vides and Pfeffer, 2021 ( https://dx.doi.org/10.17504/protocols.io.bvvmn646 ). Bacterial cells were grown at 37°C in Luria Broth and induced at A600 nm = 0.6–0.7 by the addition of 0.3 mM isopropyl-1-thio-β- d -galactopyranoside (Gold Biotechnology) and harvested after 18 hr at 18°C.…”

Section: Methodsmentioning

confidence: 99%

A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation

Vides

Adhikari

Chiang

et al. 2022

eLife

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Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson's disease and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this domain, termed 'Site #1', can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed 'Site #2', that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity.

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Section: Methodsmentioning

confidence: 99%

A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation

Vides

Adhikari

Chiang

et al. 2022

eLife

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show abstract

“…His Rab29, His Rab10-Q68L (1–181), His-Mst3, His-Rab8A Q63L, His-LRRK2 Armadillo (1-552), His-LRRK2 Armadillo (1-159), His-LRRK2 Armadillo (350-550), His-LRRK2 Armadillo K17A, and His-LRRK2 Armadillo K18A were purified in E.coli BL21 (DE3 pLys). Detailed protocols can be found in Gomez et al, 2020 and Vides et al, 2021. Bacterial cells were grown at 37°C in Lucia Broth medium and induced at A600 nm = 0.6–0.7 by the addition of 0.3 mM isopropyl-1-thio-β-D-galactopyranoside (Gold Biotechnology) and harvested after 18 h at 18°C.…”

Section: Methodsmentioning

confidence: 99%

“…Rab GTPase, LRRK2 Armadillo domain, and LRRK2 purificationHis Rab29, His Rab10-Q68L (1-181), His-Mst3, His-Rab8A Q67L, His-LRRK2 Armadillo (1-552), His-LRRK2 Armadillo (1-159), His-LRRK2 Armadillo (350-550), His-LRRK2 Armadillo K17A, His-LRRK2 Armadillo K18A, and GST-Rab8A Q67L were purified in E.coli BL21 (DE3 pLys). Detailed protocols can be found inGomez et al, 2020 https://dx.doi.org/10.17504/protocols.io.bffrjjm6 and Vides et al, 2021 https://dx.doi.org/10.17504/protocols.io.bvvmn646. Bacterial cells were grown at (ThermoFischer).…”

mentioning

confidence: 99%

A Feed-forward Pathway Drives LRRK2 kinase Membrane Recruitment and Activation

Vides

Adhikari

Lis

et al. 2022

Preprint

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Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson’s disease and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this site, termed “Site #1”, can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed “Site #2”, that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments and mutation analysis demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab reaction products. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and apparent membrane activation of LRRK2 kinase activity.

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Genome-wide screen reveals Rab12 GTPase as a critical activator of Parkinson’s disease-linked LRRK2 kinase

Dhekne,

Tonelli,

Yeshaw

et al. 2023

eLife

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Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that these phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022). To learn more about LRRK2 pathway regulation, we carried out an unbiased, CRISPR-based genome-wide screen to identify modifiers of cellular phosphoRab10 levels. A flow cytometry assay was developed to detect changes in phosphoRab10 levels in pools of mouse NIH-3T3 cells harboring unique CRISPR guide sequences. Multiple negative and positive regulators were identified; surprisingly, knockout of the Rab12 gene was especially effective in decreasing phosphoRab10 levels in multiple cell types and knockout mouse tissues. Rab-driven increases in phosphoRab10 were specific for Rab12, LRRK2 dependent and PPM1H phosphatase reversible, and did not require Rab12 phosphorylation; they were seen with wild type and pathogenic G2019S and R1441C LRRK2. As expected for a protein that regulates LRRK2 activity, Rab12 also influenced primary cilia formation. Alphafold modeling revealed a novel Rab12 binding site in the LRRK2 Armadillo domain and we show that residues predicted to be essential for Rab12 interaction at this site influence phosphoRab10 and phosphoRab12 levels in a manner distinct from Rab29 activation of LRRK2. Our data show that Rab12 binding to a new site in the LRRK2 Armadillo domain activates LRRK2 kinase for Rab phosphorylation and could serve as a new therapeutic target for a novel class of LRRK2 inhibitors that do not target the kinase domain.

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Rab29 fast exchange mutants: characterization of a challenging Rab GTPase v1

Cited by 3 publications

References 0 publications

A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation

A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation

A Feed-forward Pathway Drives LRRK2 kinase Membrane Recruitment and Activation

Genome-wide screen reveals Rab12 GTPase as a critical activator of Parkinson’s disease-linked LRRK2 kinase

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