Phosphorylation of 4E-BP1 in the Mammalian Brain Is Not Altered by LRRK2 Expression or Pathogenic Mutations

Elpida Tsika,Rina Bandopadhyay,Alexander J Whitworth,Darren J Moore,Alzbeta Trancikova,Klodjan Stafa,Philip J Webber,Liliane Glauser,Adamantios Mamais,Andrew B West

doi:10.1371/journal.pone.0047784

Abstract

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of autosomal dominant familial Parkinson's disease (PD). LRRK2 encodes a multi-domain protein containing GTPase and kinase enzymatic domains. Disease-associated mutations in LRRK2 variably influence enzymatic activity with the common G2019S variant leading to enhanced kinase activity. Mutant LRRK2 induces neuronal toxicity through a kinase-dependent mechanism suggesting that kinase activity is important for mediating the pathogenic effects of LRRK2 mutations. A number of LRRK2 kinase substrates have been identified in vitro but whether they represent authentic physiological substrates in mammalian cells or tissues is not yet clear. The eukaryotic initiation factor 4E (eIF4E)-binding protein, 4E-BP1, was recently identified as a potential substrate of LRRK2 kinase activity in vitro and in Drosophila with phosphorylation occurring at Thr37 and Thr46. Here, we explore a potential interaction of LRRK2 and 4E-BP1 in mammalian cells and brain. We find that LRRK2 can weakly phosphorylate 4E-BP1 in vitro but LRRK2 overexpression is not able to alter endogenous 4E-BP1 phosphorylation in mammalian cells. In mammalian neurons LRRK2 and 4E-BP1 display minimal co-localization, whereas the subcellular distribution, protein complex formation and covalent post-translational modification of endogenous 4E-BP1 are not altered in the brains of LRRK2 knockout or mutant LRRK2 transgenic mice. In the brain, the phosphorylation of 4E-BP1 at Thr37 and Thr46 does not change in LRRK2 knockout or mutant LRRK2 transgenic mice, nor is 4E-BP1 phosphorylation altered in idiopathic or G2019S mutant PD brains. Collectively, our results suggest that 4E-BP1 is neither a major nor robust physiological substrate of LRRK2 in mammalian cells or brain.

Highlights

Mutations in the leucine-rich repeat kinase 2 (LRRK2, PARK8) gene cause late-onset, autosomal dominant Parkinson’s disease (PD), and represent the most common cause of inherited PD [1,2,3]
To better define a potentially important interaction between LRRK2 and 4E-BP1, we have explored the effects of LRRK2 expression and pathogenic mutations on the phosphorylation status of 4E-BP1 in the mammalian brain using transgenic and knockout mice that are available
The identification of physiological substrates for LRRK2 kinase activity is of major importance for understanding the pathogenic effects of disease-causing mutations, for understanding the molecular signaling pathways downstream of LRRK2 activity but upstream of LRRK2-dependent neuronal toxicity, and as potential surrogate markers of LRRK2 kinase activity in vivo for monitoring the actions of kinase inhibitors

Summary

Introduction

Mutations in the leucine-rich repeat kinase 2 (LRRK2, PARK8) gene cause late-onset, autosomal dominant Parkinson’s disease (PD), and represent the most common cause of inherited PD [1,2,3]. LRRK2 plays an important role in the development of familial and sporadic PD. The LRRK2 gene encodes a large multi-domain protein belonging to the ROCO protein family [9]. Mutations known to cause PD are clustered within the central catalytic region including the GTPase (N1437H, R1441C, R1441G and R1441H), COR (Y1699C) and kinase (G2019S and I2020T) domains [9]. Mutations alter enzymatic activities that include enhanced kinase activity (i.e. G2019S and N1437H) [10,11,12], reduced GTPase activity (i.e. R1441C/G/H and Y1699C) [13,14,15,16] or enhanced GTP-binding (i.e. N1437H, R1441C/G/H and Y1699C) [17] of LRRK2. Alterations in the enzymatic activity of LRRK2 due to pathogenic mutations are most likely important for the development of PD

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Conclusion