P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity

Alexia F. Kalogeropulou, Jing Zhao, Marc F. Bolliger, Anna Memou, Shreya Narasimha, Tyler P. Molitor, William H. Wilson, Hardy J. Rideout, R. Jeremy Nichols

Research output: Contribution to journalArticle

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Abstract

Autosomal-dominant, missense mutations in the leucine-rich repeat protein kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson’s disease (PD). LRRK2 kinase activity is increased in several pathogenic mutations (N1437H, R1441C/G/H, Y1699C, G2019S), implicating hyperphosphorylation of a substrate in the pathogenesis of the disease. Identification of the downstream targets of LRRK2 is a crucial endeavor in the field to understand LRRK2 pathway dysfunction in the disease. We have identified the signaling adapter protein p62/SQSTM1 as a novel endogenous interacting partner and a substrate of LRRK2. Using mass spectrometry and phospho-specific antibodies, we found that LRRK2 phosphorylates p62 on Thr138 in vitro and in cells. We found that the pathogenic LRRK2 PD-associated mutations (N1437H, R1441C/G/H, Y1699C, G2019S) increase phosphorylation of p62 similar to previously reported substrate Rab proteins. Notably, we found that the pathogenic I2020T mutation and the risk factor mutation G2385R displayed decreased phosphorylation of p62. p62 phosphorylation by LRRK2 is blocked by treatment with selective LRRK2 inhibitors in cells. We also found that the amino-terminus of LRRK2 is crucial for optimal phosphorylation of Rab7L1 and p62 in cells. LRRK2 phosphorylation of Thr138 is dependent on a p62 functional ubiquitin-binding domain at its carboxy-terminus. Co-expression of p62 with LRRK2 G2019S increases the neurotoxicity of this mutation in a manner dependent on Thr138. p62 is an additional novel substrate of LRRK2 that regulates its toxic biology, reveals novel signaling nodes and can be used as a pharmacodynamic marker for LRRK2 kinase activity.

Original languageEnglish
Pages (from-to)1271-1293
Number of pages23
JournalBiochemical Journal
Volume475
Issue number7
DOIs
Publication statusPublished - 9 Apr 2018

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Leucine
Toxicity
Phosphotransferases
Substrates
Phosphorylation
Mutation
Parkinson Disease
Phospho-Specific Antibodies
Pharmacodynamics
Poisons
Missense Mutation
Genetic Predisposition to Disease
Ubiquitin
Protein Kinases
Mass spectrometry
Mass Spectrometry
Proteins
Genes

Cite this

Kalogeropulou, A. F., Zhao, J., Bolliger, M. F., Memou, A., Narasimha, S., Molitor, T. P., ... Jeremy Nichols, R. (2018). P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity. Biochemical Journal, 475(7), 1271-1293. https://doi.org/10.1042/BCJ20170699
Kalogeropulou, Alexia F. ; Zhao, Jing ; Bolliger, Marc F. ; Memou, Anna ; Narasimha, Shreya ; Molitor, Tyler P. ; Wilson, William H. ; Rideout, Hardy J. ; Jeremy Nichols, R. / P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity. In: Biochemical Journal. 2018 ; Vol. 475, No. 7. pp. 1271-1293.
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title = "P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity",
abstract = "Autosomal-dominant, missense mutations in the leucine-rich repeat protein kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson’s disease (PD). LRRK2 kinase activity is increased in several pathogenic mutations (N1437H, R1441C/G/H, Y1699C, G2019S), implicating hyperphosphorylation of a substrate in the pathogenesis of the disease. Identification of the downstream targets of LRRK2 is a crucial endeavor in the field to understand LRRK2 pathway dysfunction in the disease. We have identified the signaling adapter protein p62/SQSTM1 as a novel endogenous interacting partner and a substrate of LRRK2. Using mass spectrometry and phospho-specific antibodies, we found that LRRK2 phosphorylates p62 on Thr138 in vitro and in cells. We found that the pathogenic LRRK2 PD-associated mutations (N1437H, R1441C/G/H, Y1699C, G2019S) increase phosphorylation of p62 similar to previously reported substrate Rab proteins. Notably, we found that the pathogenic I2020T mutation and the risk factor mutation G2385R displayed decreased phosphorylation of p62. p62 phosphorylation by LRRK2 is blocked by treatment with selective LRRK2 inhibitors in cells. We also found that the amino-terminus of LRRK2 is crucial for optimal phosphorylation of Rab7L1 and p62 in cells. LRRK2 phosphorylation of Thr138 is dependent on a p62 functional ubiquitin-binding domain at its carboxy-terminus. Co-expression of p62 with LRRK2 G2019S increases the neurotoxicity of this mutation in a manner dependent on Thr138. p62 is an additional novel substrate of LRRK2 that regulates its toxic biology, reveals novel signaling nodes and can be used as a pharmacodynamic marker for LRRK2 kinase activity.",
author = "Kalogeropulou, {Alexia F.} and Jing Zhao and Bolliger, {Marc F.} and Anna Memou and Shreya Narasimha and Molitor, {Tyler P.} and Wilson, {William H.} and Rideout, {Hardy J.} and {Jeremy Nichols}, R.",
note = "The authors thank the Brin/Wojcicki Foundation and the Michael J. Fox Foundation for funding this work.",
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Kalogeropulou, AF, Zhao, J, Bolliger, MF, Memou, A, Narasimha, S, Molitor, TP, Wilson, WH, Rideout, HJ & Jeremy Nichols, R 2018, 'P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity', Biochemical Journal, vol. 475, no. 7, pp. 1271-1293. https://doi.org/10.1042/BCJ20170699

P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity. / Kalogeropulou, Alexia F.; Zhao, Jing; Bolliger, Marc F.; Memou, Anna; Narasimha, Shreya; Molitor, Tyler P.; Wilson, William H.; Rideout, Hardy J.; Jeremy Nichols, R.

In: Biochemical Journal, Vol. 475, No. 7, 09.04.2018, p. 1271-1293.

Research output: Contribution to journalArticle

TY - JOUR

T1 - P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity

AU - Kalogeropulou, Alexia F.

AU - Zhao, Jing

AU - Bolliger, Marc F.

AU - Memou, Anna

AU - Narasimha, Shreya

AU - Molitor, Tyler P.

AU - Wilson, William H.

AU - Rideout, Hardy J.

AU - Jeremy Nichols, R.

N1 - The authors thank the Brin/Wojcicki Foundation and the Michael J. Fox Foundation for funding this work.

PY - 2018/4/9

Y1 - 2018/4/9

N2 - Autosomal-dominant, missense mutations in the leucine-rich repeat protein kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson’s disease (PD). LRRK2 kinase activity is increased in several pathogenic mutations (N1437H, R1441C/G/H, Y1699C, G2019S), implicating hyperphosphorylation of a substrate in the pathogenesis of the disease. Identification of the downstream targets of LRRK2 is a crucial endeavor in the field to understand LRRK2 pathway dysfunction in the disease. We have identified the signaling adapter protein p62/SQSTM1 as a novel endogenous interacting partner and a substrate of LRRK2. Using mass spectrometry and phospho-specific antibodies, we found that LRRK2 phosphorylates p62 on Thr138 in vitro and in cells. We found that the pathogenic LRRK2 PD-associated mutations (N1437H, R1441C/G/H, Y1699C, G2019S) increase phosphorylation of p62 similar to previously reported substrate Rab proteins. Notably, we found that the pathogenic I2020T mutation and the risk factor mutation G2385R displayed decreased phosphorylation of p62. p62 phosphorylation by LRRK2 is blocked by treatment with selective LRRK2 inhibitors in cells. We also found that the amino-terminus of LRRK2 is crucial for optimal phosphorylation of Rab7L1 and p62 in cells. LRRK2 phosphorylation of Thr138 is dependent on a p62 functional ubiquitin-binding domain at its carboxy-terminus. Co-expression of p62 with LRRK2 G2019S increases the neurotoxicity of this mutation in a manner dependent on Thr138. p62 is an additional novel substrate of LRRK2 that regulates its toxic biology, reveals novel signaling nodes and can be used as a pharmacodynamic marker for LRRK2 kinase activity.

AB - Autosomal-dominant, missense mutations in the leucine-rich repeat protein kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson’s disease (PD). LRRK2 kinase activity is increased in several pathogenic mutations (N1437H, R1441C/G/H, Y1699C, G2019S), implicating hyperphosphorylation of a substrate in the pathogenesis of the disease. Identification of the downstream targets of LRRK2 is a crucial endeavor in the field to understand LRRK2 pathway dysfunction in the disease. We have identified the signaling adapter protein p62/SQSTM1 as a novel endogenous interacting partner and a substrate of LRRK2. Using mass spectrometry and phospho-specific antibodies, we found that LRRK2 phosphorylates p62 on Thr138 in vitro and in cells. We found that the pathogenic LRRK2 PD-associated mutations (N1437H, R1441C/G/H, Y1699C, G2019S) increase phosphorylation of p62 similar to previously reported substrate Rab proteins. Notably, we found that the pathogenic I2020T mutation and the risk factor mutation G2385R displayed decreased phosphorylation of p62. p62 phosphorylation by LRRK2 is blocked by treatment with selective LRRK2 inhibitors in cells. We also found that the amino-terminus of LRRK2 is crucial for optimal phosphorylation of Rab7L1 and p62 in cells. LRRK2 phosphorylation of Thr138 is dependent on a p62 functional ubiquitin-binding domain at its carboxy-terminus. Co-expression of p62 with LRRK2 G2019S increases the neurotoxicity of this mutation in a manner dependent on Thr138. p62 is an additional novel substrate of LRRK2 that regulates its toxic biology, reveals novel signaling nodes and can be used as a pharmacodynamic marker for LRRK2 kinase activity.

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