title: Phosphatidylinositol 3-kinase-binding protein, PI3KAP/XB130, is required for cAMP-induced amplification of IGF mitogenic activity in FRTL-5 thyroid cells.
abstract: We previously demonstrated that long-term pretreatment of rat FRTL-5 thyroid cells with TSH or cAMP-generating reagents potentiated IGF-I-dependent DNA synthesis. Under these conditions, cAMP treatment increased tyrosine phosphorylation of a 125-kDa protein (p125) and its association with a p85 regulatory subunit of phosphatidylinositol 3-kinase (p85 PI3K), which were suggested to mediate potentiation of DNA synthesis. This study was undertaken to identify p125 and to elucidate its roles in potentiation of DNA synthesis induced by IGF-I. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis revealed p125 to be a rat ortholog of human XB130, which we named PI3K-associated protein (PI3KAP). cAMP treatment elevated PI3KAP/XB130 mRNA and protein levels as well as tyrosine phosphorylation and interaction with p85 PI3K leading to increased PI3K activities associated with PI3KAP/XB130, supporting the role of PI3KAP/XB130 in DNA synthesis potentiation. Importantly, PI3KAP/XB130 knockdown attenuated cAMP-dependent potentiation of IGF-I-induced DNA synthesis. Furthermore, c-Src was associated with PI3KAP/XB130 and was activated in response to cAMP. Addition of Src family kinase inhibitors, PP1 or PP2, during cAMP treatment abolished tyrosine phosphorylation of PI3KAP/XB130 and its interaction with p85 PI3K. Finally, introduction of PI3KAP/XB130 into NIH3T3 fibroblasts lacking endogenous PI3KAP/XB130 enhanced IGF-I-induced DNA synthesis; however, a mutant Y72F incapable of binding to p85 PI3K did not show this response. Together, these data indicate that cAMP-dependent induction of PI3KAP/XB130, which is associated with PI3K, is required for enhancement of IGF mitogenic activities.
authors.affiliation: Department of Animal Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Yamanaka
authors.forename: Takeshi
authors.initials: T
authors.lastname: Akama
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Fukushima
authors.forename: Taku
authors.initials: T
authors.lastname: Nedachi
authors.forename: Chie
authors.initials: C
authors.lastname: Kawasaki
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Chida
authors.forename: Shiro
authors.initials: S
authors.lastname: Minami
authors.forename: Koichi
authors.initials: K
authors.lastname: Suzuki
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Hakuno
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Takahashi
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D049109:Cell Proliferation; D000242:Cyclic AMP / Q000502:physiology*; D004261:DNA Replication; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000502:physiology*; D008297:Male; D051379:Mice; D008934:Mitogens / Q000502:physiology*; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D017422:Sequence Analysis, DNA; D013961:Thyroid Gland / Q000166:cytology*; D013972:Thyrotropin / Q000502:physiology; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D008934:Mitogens; C574981:PI3KAP protein, rat; D007334:Insulin-Like Growth Factor I; D013972:Thyrotropin; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D019061:src-Family Kinases