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1.  14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization 
Biochemical Journal  2010;430(Pt 3):393-404.
LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients, but still little is understood about how it is regulated or functions. In the present study we have demonstrated that 14-3-3 protein isoforms interact with LRRK2. Consistent with this, endogenous LRRK2 isolated from Swiss 3T3 cells or various mouse tissues is associated with endogenous 14-3-3 isoforms. We have established that 14-3-3 binding is mediated by phosphorylation of LRRK2 at two conserved residues (Ser910 and Ser935) located before the leucine-rich repeat domain. Our results suggests that mutation of Ser910 and/or Ser935 to disrupt 14-3-3 binding does not affect intrinsic protein kinase activity, but induces LRRK2 to accumulate within discrete cytoplasmic pools, perhaps resembling inclusion bodies. To investigate links between 14-3-3 binding and Parkinson's disease, we studied how 41 reported mutations of LRRK2 affected 14-3-3 binding and cellular localization. Strikingly, we found that five of the six most common pathogenic mutations (R1441C, R1441G, R1441H, Y1699C and I2020T) display markedly reduced phosphorylation of Ser910/Ser935 thereby disrupting interaction with 14-3-3. We have also demonstrated that Ser910/Ser935 phosphorylation and 14-3-3 binding to endogenous LRRK2 is significantly reduced in tissues of homozygous LRRK2(R1441C) knock-in mice. Consistent with 14-3-3 regulating localization, all of the common pathogenic mutations displaying reduced 14-3-3-binding accumulated within inclusion bodies. We also found that three of the 41 LRRK2 mutations analysed displayed elevated protein kinase activity (R1728H, ~2-fold; G2019S, ~3-fold; and T2031S, ~4-fold). These results provide the first evidence suggesting that 14-3-3 regulates LRRK2 and that disruption of the interaction of LRRK2 with 14-3-3 may be linked to Parkinson's disease.
doi:10.1042/BJ20100483
PMCID: PMC2932554  PMID: 20642453
cytoplasmic localization; 14-3-3 protein; leucine-rich repeat protein kinase 2 (LRRK2); Parkinson's disease; pathogenic mutation; phosphorylation; CDC, cell division cycle; DIG, digoxigenin; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; FBS, fetal bovine serum; GFP, green fluorescent protein; HEK-293, human embryonic kidney; Hsp90, heat-shock protein 90; IPI, International Protein Index; KLH, keyhole-limpet haemocyanin; LRRK2, leucine-rich repeat protein kinase 2; MARK3, microtubule affinity-regulating kinase 3; PD, Parkinson's disease; ROC, Ras of complex GTPase domain; COR, C-terminal of ROC; SILAC, stable isotope labelling of amino acids; TBST, Tris-buffered saline with Tween 20

Results 1-1 (1)