An early endosome regulator, Rab5b, is an LRRK2 kinase substrate

Abstract

Leucine-rich repeat kinase 2 (LRRK2) has been identified as a causative gene for Parkinson's disease (PD). LRRK2 contains a kinase and a GTPase domain, both of which provide critical intracellular signal-transduction functions. We showed previously that Rab5b, a small GTPase protein that regulates the motility and fusion of early endosomes, interacts with LRRK2 and co-regulates synaptic vesicle endocytosis. Using recombinant proteins, we show here that LRRK2 phosphorylates Rab5b at its Thr6 residue in in vitro kinase assays with mass spectrophotometry analysis. Phosphorylation of Rab5b by LRRK2 on the threonine residue was confirmed by western analysis using cells stably expressing LRRK2 G2019S. The phosphomimetic T6D mutant exhibited stronger GTPase activity than that of the wild-type Rab5b. In addition, phosphorylation of Rab5b by LRRK2 also exhibited GTPase activity stronger than that of the unphosphorylated Rab5b protein. Two assays testing Rab5's activity, neurite outgrowth analysis and epidermal growth factor receptor degradation assays, showed that Rab5b T6D exhibited phenotypes that were expected to be observed in the inactive Rab5b, including longer neurite length and less degradation of EGFR. These results suggest that LRRK2 kinase activity functions as a Rab5b GTPase activating protein and thus, negatively regulates Rab5b signalling.

Leucine-Rich Repeat Kinase 2 (LRRK2) Stimulates IL-1β-Mediated Inflammatory Signaling through Phosphorylation of RCAN1

“…Next, we examined the phosphorylation efficiency of LRRK2 toward RCAN1 through comparison with RAB5B, another well-known target of LRRK2 (Yun et al, 2015). As shown in Figure 3J, in vitro kinase assays demonstrated that LRRK2 phosphorylated GST-RCAN1 more efficiently than RAB5B, confirming that RCAN1 is a novel substrate of LRRK2 kinase.…”

Leucine-rich repeat kinase 2 (LRRK2) regulates α-synuclein clearance in microglia

“…A recent study has reported that Rab5b, an isoform of Rab5, is phosphorylated by LRRK2 at Thr 6, resulting in a decreased endocytosis rate in Hela cells [55]. It is possible that up-regulation of the endocytosis rate in KO microglia could be due to the lack of phosphorylation of Rab5b.…”

α-Synuclein – Regulator of Exocytosis, Endocytosis, or Both?

“…Furthermore, inhibitors of LRRK2 kinase activity seem to reduce synaptic vesicle endocytosis [76]. Different mechanisms have been discussed, and LRRK2 has been found to phosphorylate mammalian endophilin A1 [75] as well as Rab5b [74,77]. However, although there is still controversy on the mechanism, a link to a-synuclein interaction with endophilin A or Rab proteins may reveal interesting new evidence related to Parkinson's pathology.…”