Ubiquitin is phosphorylated by PINK1 to activate parkin.
PINK1 (PTEN induced putative kinase 1) and PARKIN (also known as PARK2) have been identified as the causal genes responsible for hereditary recessive early-onset Parkinsonism. PINK1 is a Ser/Thr kinase that specifically accumulates on depolarized mitochondria, whereas parkin is an E3 ubiquitin ligase that catalyses ubiquitin transfer to mitochondrial substrates. PINK1 acts as an upstream factor for parkin and is essential both for the activation of latent E3 parkin activity and for recruiting parkin onto depolarized mitochondria. Recently, mechanistic insights into mitochondrial quality control mediated by PINK1 and parkin have been revealed, and PINK1-dependent phosphorylation of parkin has been reported. However, the requirement of PINK1 for parkin activation was not bypassed by phosphomimetic parkin mutation, and how PINK1 accelerates the E3 activity of parkin on damaged mitochondria is still obscure. Here we report that ubiquitin is the genuine substrate of PINK1. PINK1 phosphorylated ubiquitin at Ser 65 both in vitro and in cells, and a Ser 65 phosphopeptide derived from endogenous ubiquitin was only detected in cells in the presence of PINK1 and following a decrease in mitochondrial membrane potential. Unexpectedly, phosphomimetic ubiquitin bypassed PINK1-dependent activation of a phosphomimetic parkin mutant in cells. Furthermore, phosphomimetic ubiquitin accelerates discharge of the thioester conjugate formed by UBCH7 (also known as UBE2L3) and ubiquitin (UBCH7∼ubiquitin) in the presence of parkin in vitro, indicating that it acts allosterically. The phosphorylation-dependent interaction between ubiquitin and parkin suggests that phosphorylated ubiquitin unlocks autoinhibition of the catalytic cysteine. Our results show that PINK1-dependent phosphorylation of both parkin and ubiquitin is sufficient for full activation of parkin E3 activity. These findings demonstrate that phosphorylated ubiquitin is a parkin activator.
1] Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan [2] Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8561, Japan.
Fumika
F
Koyano
1] Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan [2] Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8561, Japan.
Kei
K
Okatsu
Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, The University of Tokushima, Tokushima 770-8503, Japan.
Hidetaka
H
Kosako
Research Center for Materials Science, Nagoya University, Nagoya, Aichi 464-8602, Japan.
Yasushi
Y
Tamura
Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
Etsu
E
Go
Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
Mayumi
M
Kimura
1] Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan [2] Graduate School of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan.
Yoko
Y
Kimura
Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
Hikaru
H
Tsuchiya
Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
Hidehito
H
Yoshihara
Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
Takatsugu
T
Hirokawa
1] JST-CREST/Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan [2] JST-CREST/Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto 603-8555, Japan.
Toshiya
T
Endo
McGill Parkinson Program, Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montréal, Québec H3A 2B4, Canada.
Edward A
EA
Fon
Department of Pharmacology & Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada.
Jean-François
JF
Trempe
Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
Yasushi
Y
Saeki
Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
Keiji
K
Tanaka
1] Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan [2] Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
Noriyuki
N
Matsuda
D000818:Animals; D004789:Enzyme Activation; D005347:Fibroblasts; D006367:HeLa Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010300:Parkinson Disease; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D000099202:PTEN-Induced Putative Kinase
D010768:Phosphoserine; D011494:Protein Kinases; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D000099202:PTEN-Induced Putative Kinase; C111567:parkin protein