The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization, degradation, and toxicity

Abstract

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's Disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry-based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (Tripartite Motif Family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2 822-982, a flexible interdomain region we designate the "Regulatory Loop" (RL). Phosphorylation of LRRK2 Ser910/935 within LRRK2 RL serves as a molecular switch controlling LRRK2's association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 prevents upregulation of LRRK2 kinase activity by Rab29 and also rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, modulating its cytoskeletal recruitment, turnover, kinase activity, and cytotoxicity.

Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity

“…Because antibodies that recognize epitopes after denaturation may or may not recognize endogenous epitopes (i.e., in fixed cells), we first compared the performance of two different antibodies to total LRRK2 in the overexpression model. We also took advantage of prior observations that expression of some mutations (e.g., R1441C), or addition of kinase inhibitors, relocalizes tagged overexpressed LRRK2 to microtubules [28,[37][38][39], even though such localization has not been able to be confirmed for endogenous LRRK2 [31,40,41]. The mouse monoclonal antibody N241A/34 detected overexpressed LRRK2 as expected, but failed to detect LRRK2 bound to microtubules, whether a consequence of pathogenic mutation or after treatment with MLi-2 (Fig.…”

Endogenous Rab38 regulates LRRK2’s membrane recruitment and substrate Rab phosphorylation in melanocytes

“…Thus far, endogenous LRRK2 has been conclusively visualized by IF only in macrophages in which interferon- is utilized to augment LRRK2 expression. 3 For our studies in melanocytes, we therefore expressed exogenous LRRK2 and restricted all microscopy to cells expressing visible but low levels of LRRK2. We note that in B16 cells, highly overexpressed LRRK2 sometimes formed aggregates and skein-like structures of unclear physiologic relevance, identical to its behavior in other cell lines.…”

“…2 The remainder of the LRRK2 protein consists of protein-protein interaction domains (armadillo [ARM], ankyrin [ANK], LRR, and WD40 repeats) as well as a regulatory loop region between the ANK and LRR domains that can dictate LRRK2 binding partners and subcellular localization. 3 How LRRK2 drives PD is not known. However, important work suggests, first, that LRRK2 kinase activity is increased by PD-driving mutations, and second, that 14 Rab proteins (including Rab3, 5, 8, 10, 12, 29, 35, and 43) are LRRK2 kinase substrates and share a conserved Ser/Thr phosphorylation site.…”

“…2 The remainder of the LRRK2 protein consists of protein-protein interaction domains (armadillo [ARM], ankyrin [ANK], LRR, and WD40 repeats) as well as a regulatory loop region between the ANK and LRR domains that can dictate LRRK2 binding partners and subcellular localization. 3 How LRRK2 drives PD is not known.…”

“…23,24 All of these cell types additionally express high levels of LRRK2, potentially suggesting a functional interaction. 3,[25][26][27] Most importantly, LRRK2 knockout or LRRK2 kinase inhibition causes striking buildup of enlarged lamellar bodies in alveolar Type II pneumocytes in animal models, including non-human primates. 28,29 LRRK2 kinase inhibition appears to precisely phenocopy the effect of Rab38 mutation/knockout (cht mouse, Ruby rat), and both show enlarged and abundant lamellar bodies.…”