title: Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3.
abstract: Leucine-rich repeat kinase 2 (LRRK2) is a multidomain protein implicated in Parkinson disease (PD); however, the molecular mechanism and mode of action of this protein remain elusive. cAMP-dependent protein kinase (PKA), along with other kinases, has been suggested to be an upstream kinase regulating LRRK2 function. Using MS, we detected several sites phosphorylated by PKA, including phosphorylation sites within the Ras of complex proteins (ROC) GTPase domain as well as some previously described sites (S910 and S935). We systematically mapped those sites within LRRK2 and investigated their functional consequences. S1444 in the ROC domain was confirmed as a target for PKA phosphorylation using ROC single-domain constructs and through site-directed mutagenesis. Phosphorylation at S1444 is strikingly reduced in the major PD-related LRRK2 mutations R1441C/G/H, which are part of a consensus PKA recognition site ((1441)RASpS(1444)). Furthermore, our work establishes S1444 as a PKA-regulated 14-3-3 docking site. Experiments of direct binding to the three 14-3-3 isotypes gamma, theta, and zeta with phosphopeptides encompassing pS910, pS935, or pS1444 demonstrated the highest affinities to phospho-S1444. Strikingly, 14-3-3 binding to phospho-S1444 decreased LRRK2 kinase activity in vitro. Moreover, substitution of S1444 by alanine or by introducing the mutations R1441C/G/H, abrogating PKA phosphorylation and 14-3-3 binding, resulted in increased LRRK2 kinase activity. In conclusion, these data clearly demonstrate that LRRK2 kinase activity is modulated by PKA-mediated binding of 14-3-3 to S1444 and suggest that 14-3-3 interaction with LRRK2 is hampered in R1441C/G/H-mediated PD pathogenesis.
authors.affiliation: Department of Biochemistry, University of Kassel, 34132 Kassel, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Muda
authors.forename: Daniela
authors.initials: D
authors.lastname: Bertinetti
authors.forename: Frank
authors.initials: F
authors.lastname: Gesellchen
authors.forename: Jennifer Sarah
authors.initials: JS
authors.lastname: Hermann
authors.forename: Felix
authors.initials: F
authors.lastname: von Zweydorf
authors.forename: Arie
authors.initials: A
authors.lastname: Geerlof
authors.forename: Anette
authors.initials: A
authors.lastname: Jacob
authors.forename: Marius
authors.initials: M
authors.lastname: Ueffing
authors.forename: Christian Johannes
authors.initials: CJ
authors.lastname: Gloeckner
authors.forename: Friedrich W
authors.initials: FW
authors.lastname: Herberg
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D000409:Alanine / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010300:Parkinson Disease / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D048948:14-3-3 Proteins; D011993:Recombinant Fusion Proteins; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D000409:Alanine
keywords: cAMP-dependent protein kinase; pathogenic mutation; protein–protein interaction