Mutational Analysis of the Mitochondrial Copper Metallochaperone Cox17

Heaton, Daren N.; Nittis, Thalia; Srinivasan, Chandra; Winge, Dennis R.

doi:10.1074/jbc.m006639200

Cited by 97 publications

(122 citation statements)

References 28 publications

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“…First, endogenous PKA was detected in the outer membrane and inner membrane space of highly purified, fractionated mitochondria. 4 Second, upon stimulation of HeLa cells with CPT-cAMP, an increase in phospho-ChChd3 Thr 10 is detected in intact mitochondria by confocal microscopy. 5 There are many different protein kinases in the cell.…”

Section: Discussionmentioning

confidence: 99%

“…Polyclonal antisera to full-length ChChd3 were generated in rabbits against the peptide sequence QAKKESEHQRRLKQARDLERER (Invitrogen). Affinity-purified polyclonal antibodies to phosphoChChd3 Thr 10 were generated in rabbits against an N-terminal peptide RRVT(PO 3 )FEADENENC (Pacific Immunology Corp, Ramona, CA).…”

Section: Two-dimensional Electrophoresis/maldi-tof-proteinsmentioning

confidence: 99%

“…Using 32 P-labeled N 6 (benzyl)-ATP and the purified engineered catalytic-subunit, we identified three novel PKA substrates in mouse mitochondria including ChChd3 and protein disulfide isomerase A5. ChChd3 is endogenously expressed in mitochondria and may play a role in metal binding similar to Cox17 and Cox19 (10).…”

mentioning

confidence: 99%

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Identification of ChChd3 as a Novel Substrate of the cAMP-dependent Protein Kinase (PKA) Using an Analog-sensitive Catalytic Subunit

Schauble

King

Darshi

et al. 2007

Journal of Biological Chemistry

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Due to the numerous kinases in the cell, many with overlapping substrates, it is difficult to find novel substrates for a specific kinase. To identify novel substrates of cAMP-dependent protein kinase (PKA), the PKA catalytic subunit was engineered to accept bulky N 6 -substituted ATP analogs, using a chemical genetics approach initially pioneered with v-Src (1). Methionine 120 was mutated to glycine in the ATP-binding pocket of the catalytic subunit. To express the stable mutant C-subunit in Escherichia coli required co-expression with PDK1. This mutant protein was active and fully phosphorylated on Thr 197 and Ser 338 . Based on its kinetic properties, the engineered C-subunit preferred N 6 (benzyl)-ATP and N 6 (phenethyl)-ATP over other ATP analogs, but still retained a 30 M K m for ATP. This mutant recombinant C-subunit was used to identify three novel PKA substrates. One protein, a novel mitochondrial ChChd protein, ChChd3, was identified, suggesting that PKA may regulate mitochondria proteins.The cAMP-dependent protein kinase (PKA) 3 is expressed in all mammalian cells and plays a major role in processes such as metabolic control, memory, growth, development, and apoptosis. Thus, it has many substrates, which usually are highly tissue specific. One such example is the bifunctional enzyme phosphofructokinase-2 (PFK2), which upon phosphorylation by PKA is converted to its phosphatase active mode in liver, but is stabilized in its kinase mode in heart, and is not a PKA substrate in skeletal muscle (1). Although PKA has been widely studied in most cells, the direct substrates of PKA are difficult to distinguish from downstream substrates that are phosphorylated as a result of the PKA activation. A possible way to determine whether identified substrates are modified by the kinase directly or indirectly via an intermediary kinase is to engineer the ATP-binding pocket of the kinase in question to accept a bulky ATP analog that cannot be used by the wild type kinase. This strategy was developed initially for a tyrosine kinase, v-Src, (2) and has subsequently been used for other protein kinases (3). Shah et al. (4) found that a single mutation conferred specificity for ATP analogs that have a large substituent on the nitrogen at position 6 (N 6 ) on the purine ring of ATP. Two residues that were found within 5 Å of the N 6 position of ATP were based initially on the structures of PKA and CDK2 (cyclindependent kinase 2) since the crystal structure of v-Src was not available at the time (2). In v-Src, these residues are Val 323 and Ile 338 , while the corresponding residues in PKA are Val 104 and Met 120 . Although the Val 323 mutation was found to be not important for conferring specificity in v-Src, mutation of Ile 338 to Ala did alter the specificity of v-Src and allowed the enzyme to use an orthogonal bulky ATP analog. Once the crystal structure of hematopoietic cell kinase (Hck), a Src family tyrosine kinase was available, molecular modeling was employed to explore more fully the binding pocket of Hck to find t...

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Section: Discussionmentioning

confidence: 99%

Section: Two-dimensional Electrophoresis/maldi-tof-proteinsmentioning

confidence: 99%

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Identification of ChChd3 as a Novel Substrate of the cAMP-dependent Protein Kinase (PKA) Using an Analog-sensitive Catalytic Subunit

Schauble

King

Darshi

et al. 2007

Journal of Biological Chemistry

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“…The roles of Cox17, Sco1, and Cox11 in copper metallation of CcO were substantiated by the observation that each protein is a Cu(I)-binding protein and that mutations that abrogate in vivo function attenuate Cu(I) binding (11,(13)(14)(15). Cox17 is a soluble protein that localizes within the IMS and also within the cytoplasm (8).…”

mentioning

confidence: 99%

Specific Copper Transfer from the Cox17 Metallochaperone to Both Sco1 and Cox11 in the Assembly of Yeast Cytochrome c Oxidase

Horng

Cobine

Maxfield³

et al. 2004

Journal of Biological Chemistry

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277

218

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“…Cox17 is found in both the cytosol and mitochondria. It was originally thought that Cox 17 binds copper in the cytosol, translocates into mitochondria and delivers copper to Cox1 and Cox2 [48][49][50]. However, further studies in yeast showed that in Cox 17 deficient cells, CcO activity can be fully restored by mitochondrial restricted expression of Cox17 [51].…”

Section: Copper Metabolism In Eukaryotic Cellsmentioning

confidence: 99%