A novel protein kinase B (PKB)/AKT-binding protein enhances PKB kinase activity and regulates DNA synthesis.
Protein kinase B (PKB)/Akt reportedly plays a role in the survival and/or proliferation of cells. We identified a novel protein, which binds to PKB, using a yeast two-hybrid screening system. This association was demonstrated not only in vivo by overexpressing both proteins or by coimmunoprecipitation of the endogenous proteins, but also in vitro using glutathione S-transferase fusion proteins. Importantly, this protein specifically associates with the C terminus of PKB but not with other AGC kinases and enhances PKB phosphorylation and kinase activation without growth factor stimulation. Thus, we termed this Akt-specific binding protein APE (Akt-phosphorylation enhancer). Since APE-induced phosphorylation of PKB did not occur in cells treated with wortmannin or LY294002, APE itself is not a kinase but seems to enhance or prolong the phosphoinositide 3-kinase-dependent phosphorylation of PKB. In cells in which APE was suppressed by small interfering RNA, DNA synthesis was significantly reduced with suppression of PKB phosphorylation, suggesting a synergistic role of APE in PKB-induced proliferation. On the other hand, in cells overexpressing both PKB and APE, despite markedly increased basal phosphorylation of PKB, both DNA rereplication and subsequent Chk2 phosphorylation and apoptosis were seen, suggesting the involvement of APE in the regulation of cell cycling replication licensing. Taking these observations together, APE appears to be a novel regulator of PKB phosphorylation. Furthermore, the interaction between APE and PKB, possibly dependent on the expression levels of both proteins, may be a novel molecular mechanism leading to proliferation and/or apoptosis.
Anai
Motonobu
M
Department of Internal Medicine, Institute for Adult Diseases, Asahi Life Foundation, 1-6-1, Marunouchi, Chiyoda-ku, Tokyo 100-0005, Japan.
Shojima
Nobuhiro
N
Katagiri
Hideki
H
Ogihara
Takehide
T
Sakoda
Hideyuki
H
Onishi
Yukiko
Y
Ono
Hiraku
H
Fujishiro
Midori
M
Fukushima
Yasushi
Y
Horike
Nanao
N
Viana
Amelia
A
Kikuchi
Masatoshi
M
Noguchi
Noriko
N
Takahashi
Shinichiro
S
Takata
Kuniaki
K
Oka
Yoshitomo
Y
Uchijima
Yasunobu
Y
Kurihara
Hiroki
H
Asano
Tomoichiro
T
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism* / Q000502:physiology; D002522:Chlorocebus aethiops; D004247:DNA / Q000096:biosynthesis*; D004789:Enzyme Activation / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D004247:DNA; D008840:Microfilament Proteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; C494918:AKT1 protein, human; C527550:Ccdc88a protein, mouse; D033921:Vesicular Transport Proteins