Abstract
Molybdopterin (MPT) synthase is an essential enzyme involved in the synthesis of the molybdenum cofactor precursor molybdopterin. The molybdenum cofactor biosynthetic pathway is conserved from prokaryotes to Metazoa. CG10238 is the Drosophila homolog of the MoaE protein, a subunit of MPT synthase, and is found in a fusion with the mitogen-activated protein kinase (MAPK)-upstream protein kinase-binding inhibitory protein (MBIP). This fused protein inhibits the activation of c-Jun N-terminal kinase (JNK). dMoaE (CG10238) carries out this function as a subunit of the ATAC histone acetyltransferase complex. In this study, we demonstrate that Drosophila MoaE (CG10238) also interacts with Drosophila MoaD and with itself to form a complex with stoichiometry identical to the MPT synthase holoenzyme in addition to its function in ATAC. We also show that sequence determinants that regulate MAPK signaling are located within the MoaE region of dMoaE (CG10238). Analysis of other metazoan MBIPs reveals that MBIP protein sequences have an N-terminal region that appears to have been derived from the MoaE protein, although it has lost residues responsible for catalytic activity. Thus, intact and modified copies of the MoaE protein may have been conscripted to play a new, noncatalytic role in MAPK signaling in Metazoa as part of the ATAC complex.
Highlights
From the ‡Stowers Institute for Medical Research, Kansas City, Missouri 64110, the ¶Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas 66160, and the Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas 66160
We found that the ATAC subunit dMoaE (CG10238, NP_651340.1) associates with the protein components of the Jun N-terminal kinase (JNK) signaling pathway and inhibited JNK activation in response to osmotic stress [5]. dMoaE (CG10238) is annotated in Flybase as encoding the Drosophila homolog of the molybdopterin synthase large subunit
In Drosophila, MoaE is translationally fused to MBIP, a protein that is implicated in the regulation of mitogen-activated protein kinase (MAPK) signaling in mammals [8]
Summary
Ion Exchange (Mono Q) Chromatography—The S2 cell nuclear extracts were loaded on the Mono Q ion exchange column (Amersham Biosciences) running a gradient from buffer (A) containing 100 mM NaCl, 50 mM Tris-HCl, 10% glycerol, and 0.1% Tween 20 to buffer (B) containing 1 M NaCl, 50 mM Tris-HCl, 10% glycerol, and 0.1% Tween 20 to buffer. The individually purified recombinant proteins were coimmunoprecipitated using anti-FLAG M2 affinity gel (Sigma) or monoclonal anti-HA-agarose (Sigma), and bound proteins were analyzed by Western blotting as previously described [5]. To estimate relative protein levels, spectral counts were normalized for each nonredundant protein k detected in a particular MudPIT analysis, and distributed normalized spectral abundance factors (dNSAFs) were calculated as follows [22], dNSAFi ϭ dSAFiN dSAFi iϭ. There are additional columns for each run: sS, spectral counts for peptides shared between protein isoforms; uS, spectral counts for peptides uniquely matching the protein; dS, spectral counts for peptides shared between proteins are counted only once and distributed according to the spectral count contribution of peptides unique to each isoform (as a way to estimate the relative proportion between isoforms); and dNSAF, NSAF are calculated based on distributed spectral counts (with shared spectral counts distributed among isoforms)
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