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 (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.

Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR

“…and threonines interact with 14-3-3, for example LRRK2, p53, and BAD (30,(33)(34)(35)(36). However, the CFTR R-domain interaction with 14-3-3 is exceptional for the large number of interaction sites (nine).…”

Cross-talk between LRRK2 and PKA: implication for Parkinson's disease?

“…Subsequent studies confirmed that PKA is capable of phosphorylating LRRK2 in vitro and in cells, providing indications on the specific phosphorylation sites [43,44]. Li et al [43] showed that phosphorylation of S910 and S935 at the N-terminus of LRRK2 is increased upon forskolin (an adenylyl cyclase activator) treatment and is reduced upon PKA pharmacological inhibition.…”

“…Li et al [43] showed that phosphorylation of S910 and S935 at the N-terminus of LRRK2 is increased upon forskolin (an adenylyl cyclase activator) treatment and is reduced upon PKA pharmacological inhibition. A different study reported that PKA directly phosphorylates LRRK2 S1444 in the ROC domain [44]. It is interesting to note that phosphorylation of both S910/ S935 and S1444 promotes LRRK2 binding to 14-3-3 proteins [43][44][45], chaperones that are crucial to control the activity of different signaling molecules [46].…”

“…A different study reported that PKA directly phosphorylates LRRK2 S1444 in the ROC domain [44]. It is interesting to note that phosphorylation of both S910/ S935 and S1444 promotes LRRK2 binding to 14-3-3 proteins [43][44][45], chaperones that are crucial to control the activity of different signaling molecules [46]. Accordingly, multiple lines of evidence indicate that 14-3-3 binding regulates LRRK2 kinase activity [44,47] and/or subcellular localization [48].…”

“…It is interesting to note that phosphorylation of both S910/ S935 and S1444 promotes LRRK2 binding to 14-3-3 proteins [43][44][45], chaperones that are crucial to control the activity of different signaling molecules [46]. Accordingly, multiple lines of evidence indicate that 14-3-3 binding regulates LRRK2 kinase activity [44,47] and/or subcellular localization [48]. In this context, Muda et al [44] found that LRRK2 R1441C/G/H pathogenic mutations prevent phosphorylation of S1444 by PKA, indicating that a substitution of the nearby arginine at position 1441 may disrupt the PKA recognition motif.…”