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LRRK2 dephosphorylation increases its ubiquitination.

Zhao J, Molitor TP, Langston JW, Nichols RJ - Biochem. J. (2015)

Bottom Line: In the present study, we found that potent and selective inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(935) then ubiquitination and degradation of a significant fraction of LRRK2.To investigate the link between dephosphorylation induced by inhibitor treatment and LRRK2 ubiquitination, we studied LRRK2 in conditions where it is dephosphorylated such as expression of PD mutants [R1441G, Y1699C and I2020T] or by blocking 14-3-3 binding to LRRK2 via difopein expression, and found LRRK2 is hyper-ubiquitinated.This dynamic dephosphorylation-ubiquitination cycle could explain detrimental loss-of-function phenotypes found in peripheral tissues of LRRK2 kinase inactive mutants, LRRK2 KO (knockout) animals and following LRRK2 inhibitor administration.

View Article: PubMed Central - PubMed

Affiliation: The Parkinson's Institute, 675 Almanor Ave, Sunnyvale, CA 94085, U.S.A.

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LRRK2 ubiquitination linkage analysis(A) HA vector or HA-ubiquitin or the indicated HA-ubiquitin mutants were transfected into HEK293 T-REx–GFP or GFP–LRRK2 expressing cells. Forty-eight hours after transfection, cells were treated with DMSO or 2 μM GNE1023 for 90 min and lysed in buffer containing 10 μM NEM. GFP–Trap-A immunoprecipitates and cell lysate samples were analysed by immunoblot. Anti-HA antibody indicates the conjugation of ubiquitin mutants on LRRK2. Anti-GFP antibody shows equal loading of samples. (B) A549 cells transfected with GFP–LRRK2 and HA vector, HA-ubiquitin or the indicated HA-ubiquitin mutants for 24 h and then treated with 2 mM GNE1023 for 24 h. Paraformaldehyde fixed cells were stained with HA (Alexa 594). Cells were imaged for GFP (LRRK2) green and HA (ubiquitin) red and DNA (DAPI) blue. Scale bar is 20 μm. (larger images provided in Supplementary Figure 3). (C) Percentage of co-transfected (GFP positive and HA positive) cells with LRRK2 cytoplasmic accumulations, mean ± S.E.M, chi-squared test *P≤0.05, **P≤0.005, ***P≤0.0005. (n=4, with at least 25 cells counted per experiment).
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Figure 3: LRRK2 ubiquitination linkage analysis(A) HA vector or HA-ubiquitin or the indicated HA-ubiquitin mutants were transfected into HEK293 T-REx–GFP or GFP–LRRK2 expressing cells. Forty-eight hours after transfection, cells were treated with DMSO or 2 μM GNE1023 for 90 min and lysed in buffer containing 10 μM NEM. GFP–Trap-A immunoprecipitates and cell lysate samples were analysed by immunoblot. Anti-HA antibody indicates the conjugation of ubiquitin mutants on LRRK2. Anti-GFP antibody shows equal loading of samples. (B) A549 cells transfected with GFP–LRRK2 and HA vector, HA-ubiquitin or the indicated HA-ubiquitin mutants for 24 h and then treated with 2 mM GNE1023 for 24 h. Paraformaldehyde fixed cells were stained with HA (Alexa 594). Cells were imaged for GFP (LRRK2) green and HA (ubiquitin) red and DNA (DAPI) blue. Scale bar is 20 μm. (larger images provided in Supplementary Figure 3). (C) Percentage of co-transfected (GFP positive and HA positive) cells with LRRK2 cytoplasmic accumulations, mean ± S.E.M, chi-squared test *P≤0.05, **P≤0.005, ***P≤0.0005. (n=4, with at least 25 cells counted per experiment).

Mentions: Ubiquitin is a diverse signalling molecule in which specific linkages can encode different downstream biological repercussions. The roles of Lys48 and Lys63 linkages in driving protein degradation and signal transduction are well characterized, whereas the roles of other atypical linkages are now being unravelled [63]. In Figure 2, we observed immunoreactivity of anti-Lys48 and -Lys63 ubiquitin antibodies on endogenous linkages in LRRK2 immunoprecipitates. To provide support for these linkages on LRRK2, we used an expression system with ubiquitin mutants that allow conjugation through only one lysine residue, Lys48 or Lys63, whereas all other lysines are mutated to arginine. We found that HA-tagged WT and both Lys48 and Lys63 ubiquitin linkages could be detected in GFP–LRRK2 immunoprecipitates and both of these linkages increased with inhibitor treatment (Figure 3A). Mutation of all ubiquitin lysines to arginine (Lys0) still resulted in ubiquitin conjugation to LRRK2, which was further reduced by mutation of the initiating methionine to leucine (Lys0/M1L). Introduction of this mutant into Lys48 and Lys63 mutants (Lys48/M1L and Lys63/M1L) reduced the conjugation of ubiquitin to LRRK2, but not other proteins in the cell lysate.


LRRK2 dephosphorylation increases its ubiquitination.

Zhao J, Molitor TP, Langston JW, Nichols RJ - Biochem. J. (2015)

LRRK2 ubiquitination linkage analysis(A) HA vector or HA-ubiquitin or the indicated HA-ubiquitin mutants were transfected into HEK293 T-REx–GFP or GFP–LRRK2 expressing cells. Forty-eight hours after transfection, cells were treated with DMSO or 2 μM GNE1023 for 90 min and lysed in buffer containing 10 μM NEM. GFP–Trap-A immunoprecipitates and cell lysate samples were analysed by immunoblot. Anti-HA antibody indicates the conjugation of ubiquitin mutants on LRRK2. Anti-GFP antibody shows equal loading of samples. (B) A549 cells transfected with GFP–LRRK2 and HA vector, HA-ubiquitin or the indicated HA-ubiquitin mutants for 24 h and then treated with 2 mM GNE1023 for 24 h. Paraformaldehyde fixed cells were stained with HA (Alexa 594). Cells were imaged for GFP (LRRK2) green and HA (ubiquitin) red and DNA (DAPI) blue. Scale bar is 20 μm. (larger images provided in Supplementary Figure 3). (C) Percentage of co-transfected (GFP positive and HA positive) cells with LRRK2 cytoplasmic accumulations, mean ± S.E.M, chi-squared test *P≤0.05, **P≤0.005, ***P≤0.0005. (n=4, with at least 25 cells counted per experiment).
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Figure 3: LRRK2 ubiquitination linkage analysis(A) HA vector or HA-ubiquitin or the indicated HA-ubiquitin mutants were transfected into HEK293 T-REx–GFP or GFP–LRRK2 expressing cells. Forty-eight hours after transfection, cells were treated with DMSO or 2 μM GNE1023 for 90 min and lysed in buffer containing 10 μM NEM. GFP–Trap-A immunoprecipitates and cell lysate samples were analysed by immunoblot. Anti-HA antibody indicates the conjugation of ubiquitin mutants on LRRK2. Anti-GFP antibody shows equal loading of samples. (B) A549 cells transfected with GFP–LRRK2 and HA vector, HA-ubiquitin or the indicated HA-ubiquitin mutants for 24 h and then treated with 2 mM GNE1023 for 24 h. Paraformaldehyde fixed cells were stained with HA (Alexa 594). Cells were imaged for GFP (LRRK2) green and HA (ubiquitin) red and DNA (DAPI) blue. Scale bar is 20 μm. (larger images provided in Supplementary Figure 3). (C) Percentage of co-transfected (GFP positive and HA positive) cells with LRRK2 cytoplasmic accumulations, mean ± S.E.M, chi-squared test *P≤0.05, **P≤0.005, ***P≤0.0005. (n=4, with at least 25 cells counted per experiment).
Mentions: Ubiquitin is a diverse signalling molecule in which specific linkages can encode different downstream biological repercussions. The roles of Lys48 and Lys63 linkages in driving protein degradation and signal transduction are well characterized, whereas the roles of other atypical linkages are now being unravelled [63]. In Figure 2, we observed immunoreactivity of anti-Lys48 and -Lys63 ubiquitin antibodies on endogenous linkages in LRRK2 immunoprecipitates. To provide support for these linkages on LRRK2, we used an expression system with ubiquitin mutants that allow conjugation through only one lysine residue, Lys48 or Lys63, whereas all other lysines are mutated to arginine. We found that HA-tagged WT and both Lys48 and Lys63 ubiquitin linkages could be detected in GFP–LRRK2 immunoprecipitates and both of these linkages increased with inhibitor treatment (Figure 3A). Mutation of all ubiquitin lysines to arginine (Lys0) still resulted in ubiquitin conjugation to LRRK2, which was further reduced by mutation of the initiating methionine to leucine (Lys0/M1L). Introduction of this mutant into Lys48 and Lys63 mutants (Lys48/M1L and Lys63/M1L) reduced the conjugation of ubiquitin to LRRK2, but not other proteins in the cell lysate.

Bottom Line: In the present study, we found that potent and selective inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(935) then ubiquitination and degradation of a significant fraction of LRRK2.To investigate the link between dephosphorylation induced by inhibitor treatment and LRRK2 ubiquitination, we studied LRRK2 in conditions where it is dephosphorylated such as expression of PD mutants [R1441G, Y1699C and I2020T] or by blocking 14-3-3 binding to LRRK2 via difopein expression, and found LRRK2 is hyper-ubiquitinated.This dynamic dephosphorylation-ubiquitination cycle could explain detrimental loss-of-function phenotypes found in peripheral tissues of LRRK2 kinase inactive mutants, LRRK2 KO (knockout) animals and following LRRK2 inhibitor administration.

View Article: PubMed Central - PubMed

Affiliation: The Parkinson's Institute, 675 Almanor Ave, Sunnyvale, CA 94085, U.S.A.

Show MeSH
Related in: MedlinePlus