Chandana Kondapalli discovers the substrate for the Parkinson's disease associated kinase, PINK1
Mutations in the protein kinase PINK1 were discovered in 2004 in patients with early-onset Parkinson's disease. PINK1 is unique among all protein kinases since it contains an N-terminal mitochondrial targeting motif and its catalytic domain is also unusual as it possesses three unique insertions between the beta strands that make up the typical fold of the N-lobe of protein kinases. Despite intensive research the substrate for PINK1 remained unknown.
Chandana Kondapalli, a PhD student co-supervised by Miratul Muqit and Dario Alessi, set out to discover the substrate of PINK1. When Chandana embarked on her studies, she found that recombinant PINK1 when expressed in mammalian cells was inactive which limited the ability to use traditional biochemical approaches to identify substrates. In a separate project, another PhD student in the lab, Helen Woodroof had discovered that insect orthologues of PINK1 were constitutively active as judged by their ability to phosphorylate generic substrates. Chandana decided to test whether insect PINK1 could phosphorylate 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. Excitingly she found that PINK1 could only phosphorylate one of these proteins namely the RING E3 ligase Parkin, which is commonly mutated in early-onset Parkinson's disease. Chandana next mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Serine 65 (Ser65).
A major question was how phosphorylation at Ser65 by PINK1 affected Parkin function. A recent study by Helen Walden's group in London had suggested that Parkin was inactive when expressed in vitro suggesting that it required to be activated. Another PhD student in the lab, Agne Kazlauskaite, sought to test the hypothesis that PINK1 phosphorylation at Ser65 may activate Parkin. Before Agne could perform her experiments, Axel Knebel and Clare Johnson, based in the SCILLS Protein Production Unit, first generated milligram amounts of highly purified Parkin using a method adapted from the Walden lab. Agne then developed an assay whereby she could test Parkin activity in vitro following phosphorylation by PINK1. Remarkably Agne found that Parkin was active only when it was phosphorylated by wild-type but not a kinase-inactive version of PINK1. Furthermore, a Ser65Ala mutant of Parkin could not be activated by PINK1 suggesting that Ser65 phosphorylation was indeed required for Parkin activation.
In further experiments, Chandana was able to show that human PINK1 is normally inactive in cells but that it can become active following mitochondrial depolarisation. Under these conditions she could confirm that human PINK1 can phosphorylate Parkin at Ser65 using mass spectrometry and a phosphospecific antibody against this site. She also mapped a PINK1 autophosphorylation site by mass spectrometry at residue Threonine 257 (Thr257) and confirmed this using a phosphospecific antibody against Thr257. A major hurdle in studying PINK1 in cells has been the detection of endogenous levels of PINK1. However, Ning Zhang, a post-doc in the lab, was able to detect endogenous levels of PINK1 following mitochondrial depolarisation and importantly showed that endogenous PINK1 could phosphorylate Parkin at Ser65.
Chandana's findings suggest that Parkin is a bona fide PINK1 substrate and indicate that monitoring phosphorylation of Parkin at Ser65 and/or PINK1 at Thr257 represent the first biomarkers for examining the activity of the PINK1-Parkin signalling pathway in vivo. The next major challenge in the field is to identify physiological substrates of Parkin but this research also suggests that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for Parkinson's disease.
To read a copy of Chandana's paper published in Open Biology, click here.
Chandana Kondapalli, a PhD student co-supervised by Miratul Muqit and Dario Alessi, set out to discover the substrate of PINK1. When Chandana embarked on her studies, she found that recombinant PINK1 when expressed in mammalian cells was inactive which limited the ability to use traditional biochemical approaches to identify substrates. In a separate project, another PhD student in the lab, Helen Woodroof had discovered that insect orthologues of PINK1 were constitutively active as judged by their ability to phosphorylate generic substrates. Chandana decided to test whether insect PINK1 could phosphorylate 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. Excitingly she found that PINK1 could only phosphorylate one of these proteins namely the RING E3 ligase Parkin, which is commonly mutated in early-onset Parkinson's disease. Chandana next mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Serine 65 (Ser65).
A major question was how phosphorylation at Ser65 by PINK1 affected Parkin function. A recent study by Helen Walden's group in London had suggested that Parkin was inactive when expressed in vitro suggesting that it required to be activated. Another PhD student in the lab, Agne Kazlauskaite, sought to test the hypothesis that PINK1 phosphorylation at Ser65 may activate Parkin. Before Agne could perform her experiments, Axel Knebel and Clare Johnson, based in the SCILLS Protein Production Unit, first generated milligram amounts of highly purified Parkin using a method adapted from the Walden lab. Agne then developed an assay whereby she could test Parkin activity in vitro following phosphorylation by PINK1. Remarkably Agne found that Parkin was active only when it was phosphorylated by wild-type but not a kinase-inactive version of PINK1. Furthermore, a Ser65Ala mutant of Parkin could not be activated by PINK1 suggesting that Ser65 phosphorylation was indeed required for Parkin activation.
In further experiments, Chandana was able to show that human PINK1 is normally inactive in cells but that it can become active following mitochondrial depolarisation. Under these conditions she could confirm that human PINK1 can phosphorylate Parkin at Ser65 using mass spectrometry and a phosphospecific antibody against this site. She also mapped a PINK1 autophosphorylation site by mass spectrometry at residue Threonine 257 (Thr257) and confirmed this using a phosphospecific antibody against Thr257. A major hurdle in studying PINK1 in cells has been the detection of endogenous levels of PINK1. However, Ning Zhang, a post-doc in the lab, was able to detect endogenous levels of PINK1 following mitochondrial depolarisation and importantly showed that endogenous PINK1 could phosphorylate Parkin at Ser65.
Chandana's findings suggest that Parkin is a bona fide PINK1 substrate and indicate that monitoring phosphorylation of Parkin at Ser65 and/or PINK1 at Thr257 represent the first biomarkers for examining the activity of the PINK1-Parkin signalling pathway in vivo. The next major challenge in the field is to identify physiological substrates of Parkin but this research also suggests that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for Parkinson's disease.
To read a copy of Chandana's paper published in Open Biology, click here.