A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs
A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs
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DOI：
10.1042/bj20041498
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发表时间：
2004-12-01
影响因子：
4.1
通讯作者：
Cohen, P
中科院分区：
文献类型：
--
作者：
McNeill, H;Knebel, A;Cohen, P
一种广泛表达的蛋白质含有乌巴（泛素相关）和UBX（泛素样）结构域，被鉴定为SAPKs（应激激活蛋白激酶）的底物。被称为SAKS 1（SAPK底物-1），其在体外被SAPK 3/p38 γ、SAPK 4/p38 δ和JNK（c-Jun N-末端激酶）在Ser（200）处有效磷酸化，但被SAPK 2a/p38 α、SAPK 2b/p38 β 2或ERK（细胞外信号调节激酶）2微弱磷酸化。丝氨酸（200），位于UBX结构域的N-末端，在HEK-293（人胚肾）细胞中响应于应激物而磷酸化。SB 203580（SAPK 2a/p38 a和SAPK 2b/p38 β 2的抑制剂）和/或PD 184352（抑制ERK 1和ERK 2的活化）不能阻止磷酸化，并且在缺乏SAPK 3/p38 γ和SAPK 4/p38 δ或JNK 1和JNK 2的成纤维细胞中相似。SAKS 1在体外分别通过乌巴和UBX结构域结合泛素四聚体和VCP。通过提高多聚泛素化蛋白质的水平来增强与固定化GST（谷胱甘肽S-转移酶）-SAKS 1结合的细胞提取物中VCP的量，而提取物中的SAKS 1和VCP与针对S5 a的抗体共免疫沉淀，S5 a是结合多聚泛素化蛋白质的19 S蛋白酶体亚基的组分。PNGase（肽N-聚糖酶）与VCP形成1：1复合物，因此也与固定的GST-SAKS 1结合。我们认为，SAKS 1可能是一个适配器，指导VCP的多聚泛素化蛋白，和PNGase错误折叠的糖蛋白，促进其破坏的蛋白酶体。
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 Ser(200) 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. Ser(200), 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/p38a 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) ill 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 coimmumoprecipitated 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.