Journal: The Journal of Biological Chemistry

Article Title: Allosteric regulation of the Golgi-localized PPM1H phosphatase by Rab GTPases modulates LRRK2 substrate dephosphorylation in Parkinson’s disease

doi: 10.1016/j.jbc.2025.110679

Figure Lengend Snippet: PPM1H binds nonphosphorylated Rab8A and Rab10 but not Rab12 via Leu66. Microscale thermophoresis of mNeon PPM1H and mNeon L66R PPM1H with Rab10 Q68L ( A and D ), Rab8A Q67L ( B , E ), or Rab12 Q101L ( C and F ). Purified Rab proteins were serially diluted, and mNeon PPM1H or mNeon L66R PPM1H was added (final concentration of 100 nM). Graphs show the mean ± SD from three independent measurements, each using different protein preparations. Values are summarized in .

Article Snippet: Recombinant DNA , pET14b His Rab8A (Q67L) full length , Addgene , 186014; RRID: Addgene_1860014 , Reuse , .

Techniques: Microscale Thermophoresis, Purification, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Allosteric regulation of the Golgi-localized PPM1H phosphatase by Rab GTPases modulates LRRK2 substrate dephosphorylation in Parkinson’s disease

doi: 10.1016/j.jbc.2025.110679

Figure Lengend Snippet: Binding properties of Rab8A and thiophosphorylated Rab8A to PPM1H. A and B , thiophosphorylated Rab8A Q67L does not rely on L66 for PPM1H binding. C and D , thio-phosphorylated Q67L Rab8A but not nonphosphorylated Rab8A binds much less tightly to a PPM1H FLAP domain mutated PPM1H R338A. E and F , GTP-bound WT Rab8A binds more strongly than GDP-bound WT Rab8A to PPM1H. Various Rab8A or pRab8A proteins were serially diluted and incubated with 100 nM mNeon PPM1H variants as in . Graphs represent mean ± SD from three independent experiments using separate protein preparations. pRab, phosphoRab.

Article Snippet: Recombinant DNA , pET14b His Rab8A (Q67L) full length , Addgene , 186014; RRID: Addgene_1860014 , Reuse , .

Techniques: Binding Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Allosteric regulation of the Golgi-localized PPM1H phosphatase by Rab GTPases modulates LRRK2 substrate dephosphorylation in Parkinson’s disease

doi: 10.1016/j.jbc.2025.110679

Figure Lengend Snippet: Rab8A associates with liposome-bound PPM1H. Sucrose gradient coflotation of His-Rab8A Q67L (full length, nonprenylated) with mNeon PPM1H ( A , B ) or mNeon L66R PPM1H ( C , D ) in the presence and absence of 50 nm liposomes. The distribution of PPM1H and Rab8A across the gradient was determined by immunoblot; fractions were collected from the top . Quantification of three independent experiments is shown (±SD).

Article Snippet: Recombinant DNA , pET14b His Rab8A (Q67L) full length , Addgene , 186014; RRID: Addgene_1860014 , Reuse , .

Techniques: Liposomes, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Allosteric regulation of the Golgi-localized PPM1H phosphatase by Rab GTPases modulates LRRK2 substrate dephosphorylation in Parkinson’s disease

doi: 10.1016/j.jbc.2025.110679

Figure Lengend Snippet: PPM1H’s N-terminal residues contribute to Rab8A and Rab10 binding. Microscale thermophoresis of mNeon Δ37 PPM1H with His Rab8A Q67L ( A ) or Rab10 Q68L ( B ). C , sucrose gradient coflotation of His Rab8A Q67L with mNeon Δ37 PPM1H in the presence and absence of 50 nm diameter NTA(Ni) containing liposomes. D , quantification of three independent experiments is shown (±SD). Ni, nickel; NTA, nitrilotriacetic acid.

Article Snippet: Recombinant DNA , pET14b His Rab8A (Q67L) full length , Addgene , 186014; RRID: Addgene_1860014 , Reuse , .

Techniques: Binding Assay, Microscale Thermophoresis, Liposomes

Journal: The Journal of Biological Chemistry

Article Title: Allosteric regulation of the Golgi-localized PPM1H phosphatase by Rab GTPases modulates LRRK2 substrate dephosphorylation in Parkinson’s disease

doi: 10.1016/j.jbc.2025.110679

Figure Lengend Snippet: Rab8A, but not Rab12, inhibits PPM1H activity. A , anti-pRab10 immunoblot analysis of dephosphorylation by PPM1H ( upper gel ) or L66R PPM1H ( lower gel ). B and C , data were plotted as PPM1H activity, with maximum activity equal to the amount of pRab10 dephosphorylation seen in the absence of non-pRab inhibitor (lanes 3 and 4 of each gel) compared with reactions lacking PPM1H (lanes 1 and 2). D , rate of pRab10 phosphatase activity (monitored at t = 0, 8, 15, and 30 min) for mNeon-PPM1H, mNeon-L66R PPM1H, and mNeon Δ37 PPM1H. E , quantification of pRab10 intensity from ( D ), with maximum intensity detected at 0 min normalized to a value of 1. Error bars represent SD from three independent experiments; representative gels are shown. Black line , mNeon L66R PPM1H activity; red line , mNeon PPM1H activity; and blue line , mNeon Δ37 PPM1H activity. pRab, phosphoRab.

Article Snippet: Recombinant DNA , pET14b His Rab8A (Q67L) full length , Addgene , 186014; RRID: Addgene_1860014 , Reuse , .

Techniques: Activity Assay, Western Blot, De-Phosphorylation Assay