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Noriyuki Matsuda - One of the best experts on this subject based on the ideXlab platform.
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Parkin catalyzed ubiquitin ester transfer is triggered by pink1 dependent phosphorylation
Journal of Biological Chemistry, 2013Co-Authors: Masahiro Iguchi, Kei Okatsu, Fumika Koyano, Hidetaka Kosako, Mayumi Kimura, Keiji Tanaka, Yuki Kujuro, Norihiro Suzuki, Shinichiro Uchiyama, Noriyuki MatsudaAbstract:PINK1 and Parkin are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation. Background: Parkin is a ubiquitin ligase activated by a decrease in the mitochondrial membrane potential (ΔΨm). However, details regarding its mechanism remain limited. Results: PINK1-dependent phosphorylation of Parkin at Ser-65 following dissipation of ΔΨm triggers ubiquitin-ester transfer by the RING2 domain of Parkin to Cys-431. Conclusion: Parkin catalyzes trans- (ubiquitin-thioester)ification upon PINK1-dependent phosphorylation. Significance: The molecular process of Parkin-catalyzed ubiquitylation has been determined.
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Parkin catalyzed ubiquitin ester transfer is triggered by pink1 dependent phosphorylation
Journal of Biological Chemistry, 2013Co-Authors: Masahiro Iguchi, Kei Okatsu, Fumika Koyano, Hidetaka Kosako, Mayumi Kimura, Keiji Tanaka, Yuki Kujuro, Norihiro Suzuki, Shinichiro Uchiyama, Noriyuki MatsudaAbstract:Abstract PINK1 and Parkin are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation.
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mitochondrial hexokinase hki is a novel substrate of the Parkin ubiquitin ligase
Biochemical and Biophysical Research Communications, 2012Co-Authors: Kei Okatsu, Fumika Koyano, Etsu Go, Mayumi Kimura, Keiji Tanaka, Shunichiro Iemura, Tohru Natsume, Noriyuki MatsudaAbstract:Abstract Dysfunction of Parkin, a RING-IBR-RING motif containing protein, causes autosomal recessive familial Parkinsonism. Biochemically, Parkin is a ubiquitin-ligating enzyme (E3) that catalyzes ubiquitin transfer from ubiquitin-activating and -conjugating enzymes (E1/E2) to a substrate. Recent studies have revealed that Parkin localizes in the cytoplasm and its E3 activity is repressed under steady-state conditions. In contrast, Parkin moves to mitochondria with low membrane potential, thereby activating the latent enzymatic activity of the protein, which in turn triggers Parkin-mediated ubiquitylation of numerous mitochondrial substrates. However, the mechanism of how Parkin-catalyzed ubiquitylation maintains mitochondrial integrity has yet to be determined. To begin to address this, we screened for novel Parkin substrate(s) and identified mitochondrial hexokinase I (HKI) as a candidate. Following a decrease in membrane potential, Parkin ubiquitylation of HKI leads to its proteasomal degradation. Moreover, most disease-relevant mutations of Parkin hinder this event and endogenous HKI is ubiquitylated upon dissipation of mitochondrial membrane potential in genuine-Parkin expressing cells, suggesting its physiological importance.
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diverse effects of pathogenic mutations of Parkin that catalyze multiple monoubiquitylation in vitro
Journal of Biological Chemistry, 2006Co-Authors: Noriyuki Matsuda, Nobutaka Hattori, Yoshikuni Mizuno, Toshiaki Kitami, Toshiaki Suzuki, Keiji TanakaAbstract:Mutational dysfunction of Parkin gene, which encodes a double RING finger protein and has ubiquitin ligase E3 activity, is the major cause of autosomal recessive juvenile Parkinsonism. Although many studies explored the functions of Parkin, its biochemical character is poorly understood. To address this issue, we established an E3 assay system using maltose-binding protein-fused Parkin purified from Escherichia coli. Using this recombinant Parkin, we found that not the front but the rear RING finger motif is responsible for the E3 activity of Parkin, and it catalyzes multiple monoubiquitylation. Intriguingly, for autosomal recessive juvenile Parkinsonism-causing mutations of Parkin, whereas there was loss of E3 activity in the rear RING domain, other pathogenic mutants still exhibited E3 activity equivalent to that of the wild-type Parkin. The evidence presented allows us to reconsider the function of Parkin-catalyzed ubiquitylation and to conclude that autosomal recessive juvenile Parkinsonism is not solely attributable to catalytic impairment of the E3 activity of Parkin.
Fumika Koyano - One of the best experts on this subject based on the ideXlab platform.
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ubiquitin is phosphorylated by pink1 to activate Parkin
Nature, 2014Co-Authors: Fumika Koyano, Kei Okatsu, Etsu Go, Hidetaka Kosako, Hikaru Tsuchiya, Hidehito Yoshihara, Yasushi Tamura, Yoko Kimura, Mayumi Kimura, Takatsugu HirokawaAbstract: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. 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 Parkinsonism1,2. 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 substrates3,4,5. PINK1 acts as an upstream factor for Parkin6,7 and is essential both for the activation of latent E3 Parkin activity8 and for recruiting Parkin onto depolarized mitochondria8,9,10,11,12. Recently, mechanistic insights into mitochondrial quality control mediated by PINK1 and Parkin have been revealed3,4,5, and PINK1-dependent phosphorylation of Parkin has been reported13,14,15. However, the requirement of PINK1 for Parkin activation was not bypassed by phosphomimetic Parkin mutation15, 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.
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Parkin catalyzed ubiquitin ester transfer is triggered by pink1 dependent phosphorylation
Journal of Biological Chemistry, 2013Co-Authors: Masahiro Iguchi, Kei Okatsu, Fumika Koyano, Hidetaka Kosako, Mayumi Kimura, Keiji Tanaka, Yuki Kujuro, Norihiro Suzuki, Shinichiro Uchiyama, Noriyuki MatsudaAbstract:PINK1 and Parkin are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation. Background: Parkin is a ubiquitin ligase activated by a decrease in the mitochondrial membrane potential (ΔΨm). However, details regarding its mechanism remain limited. Results: PINK1-dependent phosphorylation of Parkin at Ser-65 following dissipation of ΔΨm triggers ubiquitin-ester transfer by the RING2 domain of Parkin to Cys-431. Conclusion: Parkin catalyzes trans- (ubiquitin-thioester)ification upon PINK1-dependent phosphorylation. Significance: The molecular process of Parkin-catalyzed ubiquitylation has been determined.
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Parkin catalyzed ubiquitin ester transfer is triggered by pink1 dependent phosphorylation
Journal of Biological Chemistry, 2013Co-Authors: Masahiro Iguchi, Kei Okatsu, Fumika Koyano, Hidetaka Kosako, Mayumi Kimura, Keiji Tanaka, Yuki Kujuro, Norihiro Suzuki, Shinichiro Uchiyama, Noriyuki MatsudaAbstract:Abstract PINK1 and Parkin are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation.
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mitochondrial hexokinase hki is a novel substrate of the Parkin ubiquitin ligase
Biochemical and Biophysical Research Communications, 2012Co-Authors: Kei Okatsu, Fumika Koyano, Etsu Go, Mayumi Kimura, Keiji Tanaka, Shunichiro Iemura, Tohru Natsume, Noriyuki MatsudaAbstract:Abstract Dysfunction of Parkin, a RING-IBR-RING motif containing protein, causes autosomal recessive familial Parkinsonism. Biochemically, Parkin is a ubiquitin-ligating enzyme (E3) that catalyzes ubiquitin transfer from ubiquitin-activating and -conjugating enzymes (E1/E2) to a substrate. Recent studies have revealed that Parkin localizes in the cytoplasm and its E3 activity is repressed under steady-state conditions. In contrast, Parkin moves to mitochondria with low membrane potential, thereby activating the latent enzymatic activity of the protein, which in turn triggers Parkin-mediated ubiquitylation of numerous mitochondrial substrates. However, the mechanism of how Parkin-catalyzed ubiquitylation maintains mitochondrial integrity has yet to be determined. To begin to address this, we screened for novel Parkin substrate(s) and identified mitochondrial hexokinase I (HKI) as a candidate. Following a decrease in membrane potential, Parkin ubiquitylation of HKI leads to its proteasomal degradation. Moreover, most disease-relevant mutations of Parkin hinder this event and endogenous HKI is ubiquitylated upon dissipation of mitochondrial membrane potential in genuine-Parkin expressing cells, suggesting its physiological importance.
Kalle Gehring - One of the best experts on this subject based on the ideXlab platform.
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a ubl ubiquitin switch in the activation of Parkin
The EMBO Journal, 2015Co-Authors: Véronique Sauvé, Asparouh Lilov, Marjan Seirafi, Marta Vranas, Shafqat Rasool, Guennadi Kozlov, Tara Sprules, Jimin Wang, Jean-françois Trempe, Kalle GehringAbstract:Mutations in Parkin and PINK1 cause an inherited early-onset form of Parkinson's disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto-inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho-ubiquitin (pUb) and the ubiquitin-like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86–130 at 2.54 A resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin-conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding.
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A Ubl/ubiquitin switch in the activation of Parkin
The EMBO journal, 2015Co-Authors: Véronique Sauvé, Asparouh Lilov, Marjan Seirafi, Marta Vranas, Shafqat Rasool, Guennadi Kozlov, Tara Sprules, Jimin Wang, Jean-françois Trempe, Kalle GehringAbstract:Mutations in Parkin and PINK1 cause an inherited early-onset form of Parkinson's disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto-inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho-ubiquitin (pUb) and the ubiquitin-like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86–130 at 2.54 A resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin-conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding.
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Deciphering the activation of the E3 ubiquitin ligase Parkin
Acta Crystallographica Section A Foundations and Advances, 2014Co-Authors: Véronique Sauvé, Kalle GehringAbstract:Parkin is an E3 ubiquitin ligase responsible for some autosomal recessive forms of Parkinson's disease. Even though Parkin is a RING-type E3 ligase, it uses a hybrid RING/HECT mechanism for its activity. The crystal structures of full-length and the RING0-RING1-In-Between-RING-RING2 module of Parkin reveal a conformation of Parkin in which its E2 binding site is too far from its catalytic cysteine for the transfer of ubiquitin [1]. Many intramolecular interactions occur between the different RING domains, as well as with a repressor element, which, with RING0, are unique to Parkin. Mutations of residues involved in those interactions lead to an increase of Parkin activity. This suggests that Parkin adopts an auto-inhibited state in basal conditions. Therefore, under stress-response conditions, Parkin needs to undergo molecular rearrangements, modulated by post-translational modification and/or interactions with other proteins, to become active. The phosphorylation of serine 65 in the Ubl domain of Parkin by Pink1, a kinase also found mutated in some Parkinson's patient, was shown to increase the activity of Parkin. Recent publications have demonstrated that ubiquitin is also phosphorylated by Pink1 and, furthermore, that phosphorylated ubiquitin could activate Parkin [2,3]. We have used different techniques of structural biology and protein-protein interactions to further characterize the interaction of phosphorylated ubiquitin with Parkin. This work provides insight into the mechanism of activation of Parkin and that causes Parkinson's disease.
Kei Okatsu - One of the best experts on this subject based on the ideXlab platform.
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ubiquitin is phosphorylated by pink1 to activate Parkin
Nature, 2014Co-Authors: Fumika Koyano, Kei Okatsu, Etsu Go, Hidetaka Kosako, Hikaru Tsuchiya, Hidehito Yoshihara, Yasushi Tamura, Yoko Kimura, Mayumi Kimura, Takatsugu HirokawaAbstract: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. 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 Parkinsonism1,2. 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 substrates3,4,5. PINK1 acts as an upstream factor for Parkin6,7 and is essential both for the activation of latent E3 Parkin activity8 and for recruiting Parkin onto depolarized mitochondria8,9,10,11,12. Recently, mechanistic insights into mitochondrial quality control mediated by PINK1 and Parkin have been revealed3,4,5, and PINK1-dependent phosphorylation of Parkin has been reported13,14,15. However, the requirement of PINK1 for Parkin activation was not bypassed by phosphomimetic Parkin mutation15, 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.
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Parkin catalyzed ubiquitin ester transfer is triggered by pink1 dependent phosphorylation
Journal of Biological Chemistry, 2013Co-Authors: Masahiro Iguchi, Kei Okatsu, Fumika Koyano, Hidetaka Kosako, Mayumi Kimura, Keiji Tanaka, Yuki Kujuro, Norihiro Suzuki, Shinichiro Uchiyama, Noriyuki MatsudaAbstract:PINK1 and Parkin are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation. Background: Parkin is a ubiquitin ligase activated by a decrease in the mitochondrial membrane potential (ΔΨm). However, details regarding its mechanism remain limited. Results: PINK1-dependent phosphorylation of Parkin at Ser-65 following dissipation of ΔΨm triggers ubiquitin-ester transfer by the RING2 domain of Parkin to Cys-431. Conclusion: Parkin catalyzes trans- (ubiquitin-thioester)ification upon PINK1-dependent phosphorylation. Significance: The molecular process of Parkin-catalyzed ubiquitylation has been determined.
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Parkin catalyzed ubiquitin ester transfer is triggered by pink1 dependent phosphorylation
Journal of Biological Chemistry, 2013Co-Authors: Masahiro Iguchi, Kei Okatsu, Fumika Koyano, Hidetaka Kosako, Mayumi Kimura, Keiji Tanaka, Yuki Kujuro, Norihiro Suzuki, Shinichiro Uchiyama, Noriyuki MatsudaAbstract:Abstract PINK1 and Parkin are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation.
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mitochondrial hexokinase hki is a novel substrate of the Parkin ubiquitin ligase
Biochemical and Biophysical Research Communications, 2012Co-Authors: Kei Okatsu, Fumika Koyano, Etsu Go, Mayumi Kimura, Keiji Tanaka, Shunichiro Iemura, Tohru Natsume, Noriyuki MatsudaAbstract:Abstract Dysfunction of Parkin, a RING-IBR-RING motif containing protein, causes autosomal recessive familial Parkinsonism. Biochemically, Parkin is a ubiquitin-ligating enzyme (E3) that catalyzes ubiquitin transfer from ubiquitin-activating and -conjugating enzymes (E1/E2) to a substrate. Recent studies have revealed that Parkin localizes in the cytoplasm and its E3 activity is repressed under steady-state conditions. In contrast, Parkin moves to mitochondria with low membrane potential, thereby activating the latent enzymatic activity of the protein, which in turn triggers Parkin-mediated ubiquitylation of numerous mitochondrial substrates. However, the mechanism of how Parkin-catalyzed ubiquitylation maintains mitochondrial integrity has yet to be determined. To begin to address this, we screened for novel Parkin substrate(s) and identified mitochondrial hexokinase I (HKI) as a candidate. Following a decrease in membrane potential, Parkin ubiquitylation of HKI leads to its proteasomal degradation. Moreover, most disease-relevant mutations of Parkin hinder this event and endogenous HKI is ubiquitylated upon dissipation of mitochondrial membrane potential in genuine-Parkin expressing cells, suggesting its physiological importance.
Richard J Youle - One of the best experts on this subject based on the ideXlab platform.
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pink1 phosphorylates ubiquitin to activate Parkin e3 ubiquitin ligase activity
Journal of Cell Biology, 2014Co-Authors: Lesley A Kane, Adam I Fogel, Soojay Banerjee, Koji Yamano, Shireen A Sarraf, Michael Lazarou, Yan Li, Richard J YouleAbstract:PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin’s ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.
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pink1 drives Parkin self association and hect like e3 activity upstream of mitochondrial binding
Journal of Cell Biology, 2013Co-Authors: Michael Lazarou, Derek P Narendra, Soojay Banerjee, Ephrem Tekle, Richard J YouleAbstract:Genetic studies indicate that the mitochondrial kinase PINK1 and the RING-between-RING E3 ubiquitin ligase Parkin function in the same pathway. In concurrence, mechanistic studies show that PINK1 can recruit Parkin from the cytosol to the mitochondria, increase the ubiquitination activity of Parkin, and induce Parkin-mediated mitophagy. Here, we used a cell-free assay to recapitulate PINK1-dependent activation of Parkin ubiquitination of a validated mitochondrial substrate, mitofusin 1. We show that PINK1 activated the formation of a Parkin–ubiquitin thioester intermediate, a hallmark of HECT E3 ligases, both in vitro and in vivo. Parkin HECT-like ubiquitin ligase activity was essential for PINK1-mediated Parkin translocation to mitochondria and mitophagy. Using an inactive Parkin mutant, we found that PINK1 stimulated Parkin self-association and complex formation upstream of mitochondrial translocation. Self-association occurred independent of ubiquitination activity through the RING-between-RING domain, providing mechanistic insight into how PINK1 activates Parkin.
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pink1 is selectively stabilized on impaired mitochondria to activate Parkin
PLOS Biology, 2010Co-Authors: Derek P Narendra, Clement A. Gautier, Derfen Suen, Mark R. Cookson, Jie Shen, Atsushi Tanaka, Richard J YouleAbstract:Loss-of-function mutations in PINK1 and Parkin cause Parkinsonism in humans and mitochondrial dysfunction in model organisms. Parkin is selectively recruited from the cytosol to damaged mitochondria to trigger their autophagy. How Parkin recognizes damaged mitochondria, however, is unknown. Here, we show that expression of PINK1 on individual mitochondria is regulated by voltage-dependent proteolysis to maintain low levels of PINK1 on healthy, polarized mitochondria, while facilitating the rapid accumulation of PINK1 on mitochondria that sustain damage. PINK1 accumulation on mitochondria is both necessary and sufficient for Parkin recruitment to mitochondria, and disease-causing mutations in PINK1 and Parkin disrupt Parkin recruitment and Parkin-induced mitophagy at distinct steps. These findings provide a biochemical explanation for the genetic epistasis between PINK1 and Parkin in Drosophila melanogaster. In addition, they support a novel model for the negative selection of damaged mitochondria, in which PINK1 signals mitochondrial dysfunction to Parkin, and Parkin promotes their elimination.
