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.
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.
Seki
Takahiro
T
Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
Irie
Naoki
N
Nakamura
Kyoko
K
Sakaue
Hiroshi
H
Ogawa
Wataru
W
Kasuga
Masato
M
Yamamoto
Hideyuki
H
Ohmori
Shiho
S
Saito
Naoaki
N
Sakai
Norio
N
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D016466:CHO Cells; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D018698:Glutamic Acid / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D010769:Phosphothreonine; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; C087857:protein kinase C gamma; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578564:Pdpk1 protein, mouse; C578579:Pdpk1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D051745:Protein Kinase C-delta