title: 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.
authors.affiliation: Institute for Brain Science and Technology, Inje University, Gaegumdong, Busanjingu, Busan, South Korea; InAm Neuroscience Research Center, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea; Department of Molecular and Life Sciences, Hanyang University, Ansanshi, Gyeonggido, South Korea; and Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea sonih@wku.ac.kr wseolha@gmail.com.
authors.forename: Hye Jin
authors.initials: HJ
authors.lastname: Yun
authors.affiliation: Institute for Brain Science and Technology, Inje University, Gaegumdong, Busanjingu, Busan, South Korea; InAm Neuroscience Research Center, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea; Department of Molecular and Life Sciences, Hanyang University, Ansanshi, Gyeonggido, South Korea; and Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea sonih@wku.ac.kr wseolha@gmail.com.
authors.forename: Hyejung
authors.initials: H
authors.lastname: Kim
authors.affiliation: Institute for Brain Science and Technology, Inje University, Gaegumdong, Busanjingu, Busan, South Korea; InAm Neuroscience Research Center, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea; Department of Molecular and Life Sciences, Hanyang University, Ansanshi, Gyeonggido, South Korea; and Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea.
authors.forename: Inhwa
authors.initials: I
authors.lastname: Ga
authors.affiliation: Institute for Brain Science and Technology, Inje University, Gaegumdong, Busanjingu, Busan, South Korea; InAm Neuroscience Research Center, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea; Department of Molecular and Life Sciences, Hanyang University, Ansanshi, Gyeonggido, South Korea; and Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea.
authors.forename: Hakjin
authors.initials: H
authors.lastname: Oh
authors.affiliation: Institute for Brain Science and Technology, Inje University, Gaegumdong, Busanjingu, Busan, South Korea; InAm Neuroscience Research Center, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea; Department of Molecular and Life Sciences, Hanyang University, Ansanshi, Gyeonggido, South Korea; and Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea Institute for Brain Science and Technology, Inje University, Gaegumdong, Busanjingu, Busan, South Korea; InAm Neuroscience Research Center, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea; Department of Molecular and Life Sciences, Hanyang University, Ansanshi, Gyeonggido, South Korea; and Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea.
authors.forename: Dong Hwan
authors.initials: DH
authors.lastname: Ho
authors.affiliation: Institute for Brain Science and Technology, Inje University, Gaegumdong, Busanjingu, Busan, South Korea; InAm Neuroscience Research Center, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea; Department of Molecular and Life Sciences, Hanyang University, Ansanshi, Gyeonggido, South Korea; and Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea.
authors.forename: Jiyoung
authors.initials: J
authors.lastname: Kim
authors.affiliation: Institute for Brain Science and Technology, Inje University, Gaegumdong, Busanjingu, Busan, South Korea; InAm Neuroscience Research Center, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea; Department of Molecular and Life Sciences, Hanyang University, Ansanshi, Gyeonggido, South Korea; and Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea.
authors.forename: Hyemyung
authors.initials: H
authors.lastname: Seo
authors.affiliation: Institute for Brain Science and Technology, Inje University, Gaegumdong, Busanjingu, Busan, South Korea; InAm Neuroscience Research Center, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea; Department of Molecular and Life Sciences, Hanyang University, Ansanshi, Gyeonggido, South Korea; and Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea Institute for Brain Science and Technology, Inje University, Gaegumdong, Busanjingu, Busan, South Korea; InAm Neuroscience Research Center, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea; Department of Molecular and Life Sciences, Hanyang University, Ansanshi, Gyeonggido, South Korea; and Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea sonih@wku.ac.kr.
authors.forename: Ilhong
authors.initials: I
authors.lastname: Son
authors.affiliation: Institute for Brain Science and Technology, Inje University, Gaegumdong, Busanjingu, Busan, South Korea; InAm Neuroscience Research Center, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea; Department of Molecular and Life Sciences, Hanyang University, Ansanshi, Gyeonggido, South Korea; and Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea Institute for Brain Science and Technology, Inje University, Gaegumdong, Busanjingu, Busan, South Korea; InAm Neuroscience Research Center, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea; Department of Molecular and Life Sciences, Hanyang University, Ansanshi, Gyeonggido, South Korea; and Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, Sanbondong, Gunposhi, Gyeonggido, South Korea wseolha@gmail.com.
authors.forename: Wongi
authors.initials: W
authors.lastname: Seol
mesh_terms: D011992:Endosomes / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D018716:Molecular Mimicry; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013379:Substrate Specificity; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D066246:ErbB Receptors; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases; D020558:GTP Phosphohydrolases; C427755:RAB5C protein, human; D020696:rab5 GTP-Binding Proteins
keywords: GTPase activity; LRRK2; Parkinson’s disease; Rab5; kinase substrate