RRC ID 42738
著者 Seki T, Irie N, Nakamura K, Sakaue H, Ogawa W, Kasuga M, Yamamoto H, Ohmori S, Saito N, Sakai N.
タイトル Fused protein of deltaPKC activation loop and PDK1-interacting fragment (deltaAL-PIF) functions as a pseudosubstrate and an inhibitory molecule for PDK1 when expressed in cells.
ジャーナル Genes Cells
Abstract To elucidate the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in cellular signaling, we constructed and expressed a pseudosubstrate of PDK1, designated as deltaAL-PIF, and characterized its properties in cultured cells. deltaAL-PIF consists of two fused proteins of the protein kinase Cdelta (deltaPKC) activation loop (deltaAL) and PDK1-interacting fragment (PIF). The phosphorylation of deltaAL-PIF was detected with anti-deltaPKC phospho-Thr505-specific antibody and was increased in proportion to the expression level of co-expressed GST-PDK1, indicating that it acts as a pseudosubstrate of PDK1. In cells expressing deltaAL-PIF, basal phosphorylation level at the activation loop of PKBalpha, deltaPKC and gammaPKC was reduced, compared with that in control cells, suggesting that deltaAL-PIF functions as an inhibitory molecule for PDK1. deltaAL-PIF affected the stability, translocation and endogenous activity of PKCs. These effects of deltaAL-PIF on gammaPKC properties were confirmed by investigation using conditioned PDK1 knockout cells. Furthermore, apoptosis frequently occurred in cells expressing deltaAL-PIF for 3 days. These findings revealed that deltaAL-PIF served as an effective pseudosubstrate and an inhibitory molecule for PDK1, suggesting that this molecule can be used as a tool for investigating PDK-mediated cellular functions as well as being applicable for anti-cancer therapy.
巻・号 11(9)
ページ 1051-70
公開日 2006-9-1
DOI 10.1111/j.1365-2443.2006.01003.x
PII GTC1003
PMID 16923125
MeSH 3-Phosphoinositide-Dependent Protein Kinases Adenosine Triphosphate / pharmacology Animals Apoptosis / drug effects CHO Cells COS Cells Cells, Cultured Chlorocebus aethiops Cricetinae Cricetulus Enzyme Activation / drug effects Gene Expression* / drug effects Glutamic Acid / pharmacology Mice Mice, Knockout Phosphorylation / drug effects Phosphothreonine / metabolism Protein Binding / drug effects Protein Kinase C / metabolism Protein Kinase C-delta / metabolism* Protein Serine-Threonine Kinases / metabolism* Protein Transport / drug effects Proto-Oncogene Proteins c-akt / metabolism Rats Recombinant Fusion Proteins / chemistry Recombinant Fusion Proteins / metabolism*
IF 1.655
引用数 5
WOS 分野 GENETICS & HEREDITY CELL BIOLOGY
リソース情報
ヒト・動物細胞