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Genes to Cells (2006) 11, 1051-1070. doi:10.1111/j.1365-2443.2006.01003.x
© 2006 Blackwell Publishing or its licensors
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Fused protein of {delta}PKC activation loop and PDK1-interacting fragment ({delta}AL-PIF) functions as a pseudosubstrate and an inhibitory molecule for PDK1 when expressed in cells

Takahiro Seki1, Naoki Irie2,a, Kyoko Nakamura3, Hiroshi Sakaue3, Wataru Ogawa3, Masato Kasuga3, Hideyuki Yamamoto4, Shiho Ohmori2, Naoaki Saito2 and Norio Sakai1,*

1 Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734–8551, Japan
2 Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan
3 Division of Diabetes, Digestive and Kidney Disease, Department of Clinical Molecular Medicine, Kobe University Graduated School of Medicine, Kobe 650-0017, Japan
4 Department of Molecular Pharmacology, Graduate School of Medical Science, Kumamoto University, Kumamoto 860-8556, Japan

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 {delta}AL-PIF, and characterized its properties in cultured cells. {delta}AL-PIF consists of two fused proteins of the protein kinase C{delta} ({delta}PKC) activation loop ({delta}AL) and PDK1-interacting fragment (PIF). The phosphorylation of {delta}AL-PIF was detected with anti-{delta}PKC 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 {delta}AL-PIF, basal phosphorylation level at the activation loop of PKB{alpha}, {delta}PKC and {gamma}PKC was reduced, compared with that in control cells, suggesting that {delta}AL-PIF functions as an inhibitory molecule for PDK1. {delta}AL-PIF affected the stability, translocation and endogenous activity of PKCs. These effects of {delta}AL-PIF on {gamma}PKC properties were confirmed by investigation using conditioned PDK1 knockout cells. Furthermore, apoptosis frequently occurred in cells expressing {delta}AL-PIF for 3 days. These findings revealed that {delta}AL-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.

Communicated by: Kozo Kaibuchi

aPresent address: Department of Growth Regulation, Institute of Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606–8507, Japan

* Correspondence: E-mail: nsakai{at}hiroshima-u.ac.jp




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[Abstract] [Full Text] [PDF]


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