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  • Polinski, Nicole K.  (3)
  • Tonelli, Francesca  (3)
  • 1
    Online Resource
    Online Resource
    Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms
    Portland Press Ltd. ; 2021
    In:  Biochemical Journal Vol. 478, No. 3 ( 2021-02-12), p. 553-578
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 478, No. 3 ( 2021-02-12), p. 553-578
    Abstract: Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BCJ20200937
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2021
    detail.hit.zdb_id: 1473095-9
    SSG: 12
    Location Call Number Limitation Availability
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    Online Resource
  • 2
    Online Resource
    Online Resource
    The Parkinson's disease VPS35[D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human
    Portland Press Ltd. ; 2018
    In:  Biochemical Journal Vol. 475, No. 11 ( 2018-06-15), p. 1861-1883
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 475, No. 11 ( 2018-06-15), p. 1861-1883
    Abstract: Missense mutations in the LRRK2 (Leucine-rich repeat protein kinase-2) and VPS35 genes result in autosomal dominant Parkinson's disease. The VPS35 gene encodes for the cargo-binding component of the retromer complex, while LRRK2 modulates vesicular trafficking by phosphorylating a subgroup of Rab proteins. Pathogenic mutations in LRRK2 increase its kinase activity. It is not known how the only thus far described pathogenic VPS35 mutation, [p.D620N] exerts its effects. We reveal that the VPS35[D620N] knock-in mutation strikingly elevates LRRK2-mediated phosphorylation of Rab8A, Rab10, and Rab12 in mouse embryonic fibroblasts. The VPS35[D620N] mutation also increases Rab10 phosphorylation in mouse tissues (the lung, kidney, spleen, and brain). Furthermore, LRRK2-mediated Rab10 phosphorylation is increased in neutrophils as well as monocytes isolated from three Parkinson's patients with a heterozygous VPS35[D620N] mutation compared with healthy donors and idiopathic Parkinson's patients. LRRK2-mediated Rab10 phosphorylation is significantly suppressed by knock-out or knock-down of VPS35 in wild-type, LRRK2[R1441C], or VPS35[D620N] cells. Finally, VPS35[D620N] mutation promotes Rab10 phosphorylation more potently than LRRK2 pathogenic mutations. Available data suggest that Parkinson's patients with VPS35[D620N] develop the disease at a younger age than those with LRRK2 mutations. Our observations indicate that VPS35 controls LRRK2 activity and that the VPS35[D620N] mutation results in a gain of function, potentially causing PD through hyperactivation of the LRRK2 kinase. Our findings suggest that it may be possible to elaborate compounds that target the retromer complex to suppress LRRK2 activity. Moreover, patients with VPS35[D620N] associated Parkinson's might benefit from LRRK2 inhibitor treatment that have entered clinical trials in humans.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BCJ20180248
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2018
    detail.hit.zdb_id: 1473095-9
    SSG: 12
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 3
    Online Resource
    Online Resource
    Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson's disease kinase
    Portland Press Ltd. ; 2018
    In:  Biochemical Journal Vol. 475, No. 1 ( 2018-01-15), p. 1-22
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 475, No. 1 ( 2018-01-15), p. 1-22
    Abstract: Mutations that activate the LRRK2 (leucine-rich repeat protein kinase 2) protein kinase predispose to Parkinson's disease, suggesting that LRRK2 inhibitors might have therapeutic benefit. Recent work has revealed that LRRK2 phosphorylates a subgroup of 14 Rab proteins, including Rab10, at a specific residue located at the centre of its effector-binding switch-II motif. In the present study, we analyse the selectivity and sensitivity of polyclonal and monoclonal phospho-specific antibodies raised against nine different LRRK2-phosphorylated Rab proteins (Rab3A/3B/3C/3D, Rab5A/5B/5C, Rab8A/8B, Rab10, Rab12, Rab29[T71], Rab29[S72] , Rab35 and Rab43). We identify rabbit monoclonal phospho-specific antibodies (MJFF-pRAB10) that are exquisitely selective for LRRK2-phosphorylated Rab10, detecting endogenous phosphorylated Rab10 in all analysed cell lines and tissues, including human brain cingulate cortex. We demonstrate that the MJFF-pRAB10 antibodies can be deployed to assess enhanced Rab10 phosphorylation resulting from pathogenic (R1441C/G or G2019S) LRRK2 knock-in mutations as well as the impact of LRRK2 inhibitor treatment. We also identify rabbit monoclonal antibodies displaying broad specificity (MJFF-pRAB8) that can be utilised to assess LRRK2-controlled phosphorylation of a range of endogenous Rab proteins, including Rab8A, Rab10 and Rab35. The antibodies described in the present study will help with the assessment of LRRK2 activity and examination of which Rab proteins are phosphorylated in vivo. These antibodies could also be used to assess the impact of LRRK2 inhibitors in future clinical trials.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BCJ20170802
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2018
    detail.hit.zdb_id: 1473095-9
    SSG: 12
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
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