Autophosphorylation in the Leucine-Rich Repeat Kinase 2 (LRRK2) GTPase Domain Modifies Kinase and GTP-Binding Activities

Webber, Philip J.; Smith, Anita; Sen, Satyabrata; Renfrow, Matthew B.; Mobley, James A.; West, Andrew B.

doi:10.1016/j.jmb.2011.07.033

Cited by 120 publications

(169 citation statements)

References 39 publications

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“…Other regions of the protein, such as the COR domain, display reduced accessibility, in agreement with previous work showing that the COR domain of Roco proteins is involved in dimerization and is also likely surrounded by other domains in the full-length protein (27). Additional lysine residues with high accessibility to the cross-linking reagent were observed in the neighborhood of known phosphorylation sites (19,28). In contrast, the N terminus exhibits a low coverage with monolink peptides.…”

Section: Resultssupporting

confidence: 89%

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

Guaitoli

Raimondi

Gilsbach

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

140

163

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Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson's disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity-together with the LRR domain-to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD.LRRK2 | Parkinson's disease | structural modeling | EM | CL-MS

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Section: Resultssupporting

confidence: 89%

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

Guaitoli

Raimondi

Gilsbach

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

140

163

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“…6B, autophosphorylation of LRRK2 was increased 1.6-fold in LRRK2 R1441H and S1444A compared with LRRK2 WT, as determined by liquid scintillation counting. For LRRK2 G2019S, we observed enhanced protein autophosphorylation activity of approximately threefold, as described earlier by others (19,33). To exclude that the observed increase in activity was caused by the amino acid substitution itself rather than by PKA-mediated phosphorylation and to test whether LRRK2 kinase activity is regulated by 14-3-3 binding on S1444 in vitro, we performed kinase assays monitoring phosphorylation of the previously identified LRRK2 autophosphorylation site T2483 (7) in the presence or absence of PKA, 14-3-3, or both (Fig.…”

Section: Mutation Of a Pka Consensus Motif Largely Reduces Phosphatesupporting

confidence: 88%

Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3

Muda

Bertinetti

Gesellchen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

107

140

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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 (1441RASpS1444). 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.

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“…LRRK2 has been found to autophosphorylate > 20 serine and threonine residues in vitro 37, 50, 56, 57, 58, 59, 60, 61. The majority of the autophosphorylation sites reside in the ROC domain, with only a few in the COR and kinase domains (Fig.…”

Section: Importance Of Lrrk2 Autophosphorylation and Constitutive Phomentioning

confidence: 99%

Cellular processes associated with LRRK2 function and dysfunction

Wallings¹,

2015

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Mutations in the leucine‐rich repeat kinase 2 (LRRK2)‐encoding gene are the most common cause of monogenic Parkinson's disease. The identification of LRRK2 polymorphisms associated with increased risk for sporadic Parkinson's disease, as well as the observation that LRRK2‐Parkinson's disease has a pathological phenotype that is almost indistinguishable from the sporadic form of disease, suggested LRRK2 as the culprit to provide understanding for both familial and sporadic Parkinson's disease cases. LRRK2 is a large protein with both GTPase and kinase functions. Mutations segregating with Parkinson's disease reside within the enzymatic core of LRRK2, suggesting that modification of its activity impacts greatly on disease onset and progression. Although progress has been made since its discovery in 2004, there is still much to be understood regarding LRRK2′s physiological and neurotoxic properties. Unsurprisingly, given the presence of multiple enzymatic domains, LRRK2 has been associated with a diverse set of cellular functions and signalling pathways including mitochondrial function, vesicle trafficking together with endocytosis, retromer complex modulation and autophagy. This review discusses the state of current knowledge on the role of LRRK2 in health and disease with discussion of potential substrates of phosphorylation and functional partners with particular emphasis on signalling mechanisms. In addition, the use of immune cells in LRRK2 research and the role of oxidative stress as a regulator of LRRK2 activity and cellular function are also discussed.

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Autophosphorylation in the Leucine-Rich Repeat Kinase 2 (LRRK2) GTPase Domain Modifies Kinase and GTP-Binding Activities

Cited by 120 publications

References 39 publications

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3

Cellular processes associated with LRRK2 function and dysfunction

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