title: A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs.
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/p38beta2 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/p38beta2) 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.
authors.affiliation: MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: McNeill
authors.forename: Axel
authors.initials: A
authors.lastname: Knebel
authors.forename: J Simon C
authors.initials: JS
authors.lastname: Arthur
authors.forename: Ana
authors.initials: A
authors.lastname: Cuenda
authors.forename: Philip
authors.initials: P
authors.lastname: Cohen
mesh_terms: D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D000818:Animals; D018053:Arsenites / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000276:immunology / Q000378:metabolism*; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000201:enzymology; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D020928:Mitogen-Activated Protein Kinases / Q000172:deficiency / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry; D009997:Osmotic Pressure; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D025821:Polyubiquitin / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000276:immunology / Q000378:metabolism; D017670:Sodium Compounds / Q000494:pharmacology; D013379:Substrate Specificity; D000074405:Valosin Containing Protein; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D018053:Arsenites; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D007976:Leupeptins; D009097:Multienzyme Complexes; D010455:Peptides; D021122:Protein Subunits; D017670:Sodium Compounds; D025821:Polyubiquitin; C017947:sodium arsenite; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein; C000614179:Vcp protein, mouse; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde