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Fused protein of δPKC activation loop and PDK1‐interacting fragment (δAL‐PIF) functions as a pseudosubstrate and an inhibitory molecule for PDK1 when expressed in cells
Author(s) -
Seki Takahiro,
Irie Naoki,
Nakamura Kyoko,
Sakaue Hiroshi,
Ogawa Wataru,
Kasuga Masato,
Yamamoto Hideyuki,
Ohmori Shiho,
Saito Naoaki,
Sakai Norio
Publication year - 2006
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/j.1365-2443.2006.01003.x
Subject(s) - protein kinase c , biology , microbiology and biotechnology , phosphorylation , protein kinase a , kinase , protein kinase b , apoptosis , biochemistry
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 δAL‐PIF, and characterized its properties in cultured cells. δAL‐PIF consists of two fused proteins of the protein kinase Cδ (δPKC) activation loop (δAL) and PDK1‐interacting fragment (PIF). The phosphorylation of δAL‐PIF was detected with anti‐δPKC phospho‐Thr 505 ‐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 δAL‐PIF, basal phosphorylation level at the activation loop of PKBα, δPKC and γPKC was reduced, compared with that in control cells, suggesting that δAL‐PIF functions as an inhibitory molecule for PDK1. δAL‐PIF affected the stability, translocation and endogenous activity of PKCs. These effects of δAL‐PIF on γPKC properties were confirmed by investigation using conditioned PDK1 knockout cells. Furthermore, apoptosis frequently occurred in cells expressing δAL‐PIF for 3 days. These findings revealed that δAL‐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.