A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs

Helen McNeill, Axel Knebel, J. Simon C. Arthur, Ana Cuenda, Philip Cohen

    Research output: Contribution to journalArticle

    51 Citations (Scopus)

    Abstract

    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 Ser200 in vitro by SAPK3/p38gamma, SAPK4/p38delta and JNK (c-Jun N-terminal kinase), but weakly by SAPK2a/p38alpha, SAPK2b/p38ß2 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/p38alpha and SAPK2b/p38ß2) and/or PD 184352 (which inhibits the activation of ERK1 and ERK2), and was similar in fibroblasts lacking both SAPK3/p38gamma and SAPK4/p38delta 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.

    Original languageEnglish
    Pages (from-to)391-400
    Number of pages10
    JournalBiochemical Journal
    Volume384
    Issue numberPt 2
    DOIs
    Publication statusPublished - 1 Dec 2004

    Keywords

    • Animals
    • Arsenites
    • Humans
    • Polyubiquitin
    • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
    • Fibroblasts
    • Mice, Knockout
    • Protein Subunits
    • Multienzyme Complexes
    • Molecular Sequence Data
    • Mitogen-Activated Protein Kinases
    • Adenosine Triphosphatases
    • Cell Extracts
    • Sodium Compounds
    • Cell Cycle Proteins
    • Osmotic Pressure
    • Immunoprecipitation
    • Amino Acid Sequence
    • Mice
    • Protein Binding
    • p38 Mitogen-Activated Protein Kinases
    • Leupeptins
    • Cells, Cultured
    • Kidney
    • Peptides
    • Substrate Specificity
    • Protein Structure, Tertiary
    • Cell Line

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