Ubiquitin is phosphorylated by PINK1 to activate parkin

• Published in: Nature (London) Vol. 510; no. 7503; pp. 162 - 166
• Main Authors: Koyano, Fumika, Okatsu, Kei, Kosako, Hidetaka, Tamura, Yasushi, Go, Etsu, Kimura, Mayumi, Kimura, Yoko, Tsuchiya, Hikaru, Yoshihara, Hidehito, Hirokawa, Takatsugu, Endo, Toshiya, Fon, Edward A., Trempe, Jean-François, Saeki, Yasushi, Tanaka, Keiji, Matsuda, Noriyuki
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 05-06-2014; Nature Publishing Group
• Subjects: 14; 14/19; 631/378/1689/1718; 631/45/474/2073; 631/80/304; 631/80/642/333; 82; 82/51; 82/58; Animals; Cell culture; Enzyme Activation; Fibroblasts; Genetic aspects; HeLa Cells; Humanities and Social Sciences; Humans; Kinases; letter; Membrane Potential, Mitochondrial; Mice; Mitochondria; Mitochondria - metabolism; multidisciplinary; Mutation; Mutation - genetics; Parkinson Disease; Parkinson's disease; Parkinsonism; Phosphorylation; Phosphoserine - metabolism; Phosphotransferases; Physiological aspects; Protein Kinases - metabolism; Proteins; Quality control; Science; Studies; Ubiquitin; Ubiquitin - chemistry; Ubiquitin - metabolism; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitination
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature13392

Ubiquitin, known for its role in post-translational modification of other proteins, undergoes post-translational modification itself; after a decrease in mitochondrial membrane potential, the kinase enzyme PINK1 phosphorylates ubiquitin at Ser 65, and the phosphorylated ubiquitin then interacts with ubiquitin ligase (E3) enzyme parkin, which is also phosphorylated by PINK1, and this process is sufficient for full activation of parkin enzymatic activity.
Phosphorylated ubiquitin is a parkin activator
The small protein ubiquitin, familiar for its role in post-translational modification of other proteins by binding to them and regulating their activity or stability, is shown here to be the substrate of the kinase PINK1, which together with the ubiquitin ligase parkin is a causal gene for hereditary recessive Parkinsonism. Noriyuki Matsuda and colleagues show that following a decrease in mitochondrial membrane potential, PINK1 phosphorylates ubiquitin at serine residue 65; the phosphorylated ubiquitin then interacts with parkin, which is also phosphorylated by PINK1. This interaction allows full activation of parkin enzymatic activity, which involves tagging mitochondrial substrates with ubiquitin.
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
1
,
2
. 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
3
,
4
,
5
. PINK1 acts as an upstream factor for parkin
6
,
7
and is essential both for the activation of latent E3 parkin activity
8
and for recruiting parkin onto depolarized mitochondria
8
,
9
,
10
,
11
,
12
. Recently, mechanistic insights into mitochondrial quality control mediated by PINK1 and parkin have been revealed
3
,
4
,
5
, and PINK1-dependent phosphorylation of parkin has been reported
13
,
14
,
15
. However, the requirement of PINK1 for parkin activation was not bypassed by phosphomimetic parkin mutation
15
, 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.