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

Kathrin Muda; Daniela Bertinetti; Frank Gesellchen; Jennifer Sarah Hermann; Felix von Zweydorf; Arie Geerlof; Anette Jacob; Marius Ueffing; Christian Johannes Gloeckner; Friedrich W. Herberg

doi:10.1073/pnas.1312701111

New Research In

Edited* by Susan S. Taylor, University of California, San Diego, La Jolla, CA, and approved November 19, 2013 (received for review July 7, 2013)

Significance

Leucine-rich repeat kinase 2 (LRRK2) is a multidomain protein implicated in Parkinson disease, and cAMP-dependent protein kinase (PKA) has been suggested to act as an upstream kinase phosphorylating LRRK2. Using a phosphoproteomics approach, we identified several novel PKA phosphorylation sites on LRRK2. We could demonstrate that one PKA phosphosite comprises the second most common mutation in LRRK2 (R1441C/G/H) attributed to Parkinson disease. Our findings reveal that this site is mandatory for subsequent 14-3-3 binding and affects LRRK2 kinase activity. These data provide a mechanistic insight into the regulation of LRRK2 kinase activity and its perturbation by disease-associated mutations.

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.

Footnotes

↵¹C.J.G. and F.W.H. contributed equally to this work.
↵²To whom correspondence should be addressed. E-mail: herberg{at}uni-kassel.de.

Author contributions: K.M., D.B., M.U., C.J.G., and F.W.H. designed research; K.M., D.B., F.v.Z., and C.J.G. performed research; F.G., J.S.H., A.G., and A.J. contributed new reagents/analytic tools; K.M., D.B., and C.J.G. analyzed data; and K.M., D.B., C.J.G., and F.W.H. wrote the paper.
The authors declare no conflict of interest.
↵*This Direct Submission article had a prearranged editor.
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