A widely expressed protein containing UBA (ubiquitin-associated) and UBX (ubiquitin-like) domains was identified as a substrate of SAPKs (stress-activated protein kinases). Termed SAKS1 (SAPK substrate-1), it was phosphorylated efficiently at Ser200in vitro by SAPK3/p38γ, SAPK4/p38δ and JNK (c-Jun N-terminal kinase), but weakly by SAPK2a/p38α, SAPK2b/p38β2 or ERK (extracellular-signal-regulated kinase) 2. Ser200, situated immediately N-terminal to the UBX domain, became phosphorylated in HEK-293 (human embryonic kidney) cells in response to stressors. Phosphorylation was not prevented by SB 203580 (an inhibitor of SAPK2a/p38α and SAPK2b/p38β2) and/or PD 184352 (which inhibits the activation of ERK1 and ERK2), and was similar in fibroblasts lacking both SAPK3/p38γ and SAPK4/p38δ or JNK1 and JNK2. SAKS1 bound ubiquitin tetramers and VCP (valosin-containing protein) in vitro via the UBA and UBX domains respectively. The amount of VCP in cell extracts that bound to immobilized GST (glutathione S-transferase)–SAKS1 was enhanced by elevating the level of polyubiquitinated proteins, while SAKS1 and VCP in extracts were coimmunoprecipitated with an antibody raised against S5a, a component of the 19 S proteasomal subunit that binds polyubiquitinated proteins. PNGase (peptide N-glycanase) formed a 1:1 complex with VCP and, for this reason, also bound to immobilized GST–SAKS1. We suggest that SAKS1 may be an adaptor that directs VCP to polyubiquitinated proteins, and PNGase to misfolded glycoproteins, facilitating their destruction by the proteasome.
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December 2004
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Research Article|
November 23 2004
A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs
Helen McNEILL;
Helen McNEILL
1MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Axel KNEBEL;
Axel KNEBEL
1MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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J. Simon C. ARTHUR;
J. Simon C. ARTHUR
1MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Ana CUENDA;
Ana CUENDA
1MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Philip COHEN
Philip COHEN
1
1MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
1To whom correspondence should be addressed (email p.cohen@dundee.ac.uk).
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Publisher: Portland Press Ltd
Received:
September 03 2004
Accepted:
September 13 2004
Accepted Manuscript online:
September 13 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2004
Biochem J (2004) 384 (2): 391–400.
Article history
Received:
September 03 2004
Accepted:
September 13 2004
Accepted Manuscript online:
September 13 2004
Citation
Helen McNEILL, Axel KNEBEL, J. Simon C. ARTHUR, Ana CUENDA, Philip COHEN; A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs. Biochem J 1 December 2004; 384 (2): 391–400. doi: https://doi.org/10.1042/BJ20041498
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