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Methods for Identifying Substrates of Atypical Mitochondrial Kinases

Methods for Identifying Substrates of Atypical Mitochondrial Kinases. Grant Barber. Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB. Reversible phosphorylation is central to mitochondrial biology.

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Methods for Identifying Substrates of Atypical Mitochondrial Kinases

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  1. Methods for Identifying Substrates of Atypical Mitochondrial Kinases Grant Barber Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB

  2. Reversible phosphorylation is central to mitochondrial biology Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB

  3. Reversible phosphorylation is central to mitochondrial biology Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB

  4. Mutations in ADCK3, an atypical kinase, cause ARCA2 Patients with autosomal recessive cerebellar ataxia 2 (ARCA2) have cerebellar atrophy. • Patients with ARCA 2 have severe CoQ10 deficiency and subsequent mitochondrial dysfunction. Lagier-Tourenneet al. 2008 Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB

  5. Reasons to study ADCK family of atypical kinases • Role of kinases in mitochondrial biology • Essential for Coenzyme Q biosynthesis • Pathophysiology of ARCA2 Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB

  6. Atypical kinases may phosphorylate either a lipid or protein Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB

  7. Identification of Atypical Kinase Substrates using Radiolabeling Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB

  8. Techniques developed for Kinase-Substrate screening • Liposomes: required to solubilize individual lipids in kinase assays • TLC: allows separation of lipids • Radiolabeling • ADP-Glo: high-throughput, luminescent kinase assay to test specific kinase-substrate interactions Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB

  9. Liposomes for testing lipid substrates PIP2/PS liposomes Intensity Intensity Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB

  10. TLC to detect phosphorylated lipids Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB

  11. Radiolabeling: Mitoliposomes for presentation of lipid substrates Mitochondrial lysate Mitoliposomes Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB

  12. Radiolabeling of Mitochondrial lysates Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB

  13. ADP-Glo Assay is a high-throughput, luminescent kinase assay Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB

  14. Summary • Here, I developed a broad method to establish kinase-substrate interactions, including a rigorous, high-throughput assay to validate interactions. • Mitoliposomes, a novel creation, present the mitochondrial lipidome without endogenous kinases. • Next Step: Use radiolabeling to detect substrates of ADCK family of atypical mitochondrial kinases. Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis Grant Barber--ASBMB

  15. Acknowledgments Dave Pagliarini Jon Stefely Andrew Reidenbach Adam Jochem

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