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The Parkinson's disease (PD)-linked protein Leucine-Rich Repeat Kinase 2 (LRRK2) consists of seven domains, including a kinase and a Roc G domain. Despite the availability of several high-resolution structures, the dynamic regulation of its unique intramolecular domain stack is nevertheless still not well understood. By in-depth biochemical analysis, assessing the Michaelis-Menten kinetics of the Roc G domain, we have confirmed that LRRK2 has, similar to other Roco protein family members, a KM value of LRRK2 that lies within the range of the physiological GTP concentrations within the cell. Furthermore, the R1441G PD variant located within a mutational hotspot in the Roc domain showed an increased catalytic efficiency. In contrast, the most common PD variant G2019S, located in the kinase domain, showed an increased KM and reduced catalytic efficiency, suggesting a negative feedback mechanism from the kinase domain to the G domain. Autophosphorylation of the G1+2 residue (T1343) in the Roc P-loop motif is critical for this phosphoregulation of both the KM and the kcat values of the Roc-catalyzed GTP hydrolysis, most likely by changing the monomer-dimer equilibrium. The LRRK2 T1343A variant has a similar increased kinase activity in cells compared to G2019S and the double mutant T1343A/G2019S has no further increased activity, suggesting that T1343 is crucial for the negative feedback in the LRRK2 signaling cascade. Together, our data reveal a novel intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase-dependent mechanism. Interestingly, PD mutants differently change the kinetics of the GTPase cycle, which might in part explain the difference in penetrance of these mutations in PD patients.
Pubmed ID: 39699947
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A freely accessible on-line systems biology resource devoted to all aspects of protein modification, as well as other post-translational modifications. It provides valuable and unique tools for both cell biologists and mass spectroscopists. PhosphoSite is a human- and mouse-centric database. It includes features such as: viewing the locations of modified residues on molecular models; browsing and searching MS2 records by disease, tissue, and cell line; submitting lists of peptides to identify previously reported genes; searching by sub-cellular localization, treatment, tissues, cell types, cell lines and diseases, and protein types and protein domains; searching for experimentally-verified kinase substrates and viewing preferred substrate motifs; and viewing MS2 spectra for peptides and sites not previously published.
View all literature mentionsNon-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.
View all literature mentionsAmerican chemical, life science and biotechnology company owned by Merck KGaA. Merger of Sigma Chemical Company and Aldrich Chemical Company. Provides organic and inorganic chemicals, building blocks, reagents, advanced materials and stable isotopes for chemical synthesis, medicinal chemistry and materials science, antibiotics, buffers, carbohydrates, enzymes, forensic tools, hematology and histology, nucleotides, proteins, peptides, amino acids and their derivatives.
View all literature mentionsA commercial antibody supplier which supplies primary and secondary antibodies, biochemicals, proteins, peptides, lysates, immunoassays and other kits.
View all literature mentionsDatabase of all cell lines used in biomedical research which include immortalized cell lines, naturally immortal cell lines (stem cells), widely used and distributed finite life cell lines, vertebrate cell lines (majority being human, mouse, and rat), and invertebrate (insects and ticks) cell lines, as well as cell line synonyms. Each cell line is provided with the following information: the recommended name (the name which appears in the original publication), a list of synonyms, a unique accession number, comments on a number of topics including misspellings and gene transfection, information on the tissue/organ origin with the UBERON code, the NCI Thesaurus or Orphanet ORDO code for the disease(s) the individual suffered from (for cancer and human genetic disease lines only), the species of origin, the parent cell line, cross-references of sister cell lines, the sex of the individual, the category in which the cell line belongs (Adult stem cell; Cancer cell line; Embryonic stem cell; Factor-dependent cell line; Finite cell line; Hybrid cell line; Hybridoma; Induced pluripotent stem cell; Spontaneously immortalized cell line; Stromal cell line; Telomerase immortalized cell line; Transformed cell line; Undefined cell line type), web links, publication references, and/or cross-references to cell line catalogs/collections, ontologies, cell lines databases/resources, and to databases that list cell lines as samples.
View all literature mentionsDrosophila melanogaster with name cm[1] from BDSC.
View all literature mentionsCell line HEK293T is a Transformed cell line with a species of origin Homo sapiens (Human)
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