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.

Fig. 1.

Identification of the 125-kDa tyrosine-phosphorylated protein (p125). A, Quiescent FRTL-5 cells were treated with 1 mm Bt₂cAMP for 24 h. Cell lysates were prepared and subjected to immunoprecipitation with anti-phosphotyrosine (αphospho-Tyr) antibody, as described in Materials and Methods. Samples were analyzed by immunoblotting with anti-phosphotyrosine antibody or silver staining. B, The band corresponding to p125 in the cAMP-treated sample (indicated as pp125) was subjected to MALDI-TOF MS analysis. The gel region with a similar electrophoretic mobility in the untreated sample was used as a negative control. MS peaks detected only in the Bt₂cAMP-treated sample (arrowheads) were analyzed by peptide mass finger printing. C, Schematic diagram of mouse PI3KAP/XB130. Y represents putative tyrosine phosphorylation sites for Src family kinases, PH is the PH domain, and coiled-coil is the coiled-coil domain. Gray line represents the SH3 domain-binding motif. Amino acid numbers of putative tyrosine phosphorylation sites, and ends of each domain/motif are indicated. D, PI3KAP/XB130 mRNA levels in rat tissues or FRTL-5 cells treated with 1 mm Bt₂cAMP for 24 h were analyzed by RT-PCR. RPS-29 was analyzed as a loading control.

Fig. 2.

TSH or cAMP treatment increases gene expression and tyrosine phosphorylation of PI3KAP/XB130, leading to the increased formation of PI3KAP/XB130-PI3K complex. A–C, The 8-wk-old male Wistar rats were fed 0.03% MMI in drinking water for 4 wk; A, plasma TSH levels were evaluated by ELISA; B, thyroid glands were photographed before removal of any thyroid tissue, with the enlarged thyroid glands in MMI-treated rats and control thyroid glands indicated and representative photographs shown; C, relative mRNA levels of PI3KAP/XB130 in the thyroid glands were analyzed by real-time PCR. The means ± sem from seven rats are shown. D and E, Quiescent FRTL-5 cells were treated with 1 mm Bt₂cAMP for the indicated hours or with indicated concentrations of TSH for 24 h. PI3KAP/XB130 protein levels, tyrosine phosphorylation, and its interaction with p85 PI3K were examined by immunoprecipitation (IP) and immunoblotting (IB) analysis. PI3KAP/XB130 mRNA levels were analyzed by Northern blotting (NB). The results of ethidium bromide staining of 18S rRNA are shown for a loading control. The means ± sem of the densitometric analysis results of three independent experiments are shown in the lower graphs. F, Quiescent FRTL-5 cells were treated with 1 nm TSH or 1 mm Bt₂cAMP for 24 h. The cell lysates were subjected to immunoprecipitation followed by immunoblotting with indicated antibodies (inset), or PI3K activity was measured in the immunoprecipitates. The means ± sem of three replicate dishes are shown. Similar results were obtained in two independent experiments. *, P < 0.05.

Fig. 3.

Knockdown of PI3KAP/XB130 suppresses cAMP-dependent potentiation of IGF-I-induced DNA synthesis in FRTL-5 cells. FRTL-5 cells were transfected with control siRNA or siRNA against PI3KAP/XB130. After serum starvation, cells were pretreated with or without 1 mm Bt₂cAMP for 24 h. A, The knockdown efficiency was evaluated by immunoblotting (IB). The means ± sem of the densitometric analysis results of three independent experiments are shown in the lower graphs. B, PI3KAP/XB130 tyrosine phosphorylation and its interaction with p85 PI3K were examined by immunoprecipitation (IP) and immunoblotting analysis. C, PI3K activity was measured in the immunoprecipitates with anti-PI3KAP/XB130 antibody. The means ± sem of three replicate dishes are shown. Similar results were obtained in two independent experiments. D–F, After washing to remove reagents, cells were treated with 100 ng/ml IGF-I for 24 h (D) or 3–6 h (E and F). [Methyl-³H]thymidine incorporation into DNA was measured during the last 4 h (D). The means ± sem of three replicate wells are shown. Similar results were obtained in three independent experiments. Cyclin D1 protein levels were analyzed by immunoblotting (E), and the cyclin D1 mRNA levels were analyzed by RT-PCR (F). RPS29 was a loading control. The means ± sem of the densitometric analysis results of three independent experiments are shown in the right graphs (E and F). *, P < 0.05. n.s., Not significant.

Fig. 4.

PI3KAP/XB130 is phosphorylated by Src family tyrosine kinases and forms a complex with PI3K. Quiescent FRTL-5 cells were treated with or without 1 mm Bt₂cAMP for 24 h. A and B, During cAMP treatment, the indicated concentrations of PP1 or PP2 were added. PI3KAP/XB130 protein levels, tyrosine phosphorylation, and its interaction with p85 PI3K were examined by immunoprecipitation (IP) and immunoblotting (IB) analysis. The means ± sem of the densitometric analysis results of three independent experiments are shown in the lower graphs (A). PI3K activity was measured in the immunoprecipitates with anti-PI3KAP/XB130 antibody. The means ± sem of three independent experiments are shown (B). C and D, Before serum starvation and cAMP treatment, FRTL-5 cells were transfected with control siRNA or siRNA against Src. PI3KAP/XB130 protein levels, tyrosine phosphorylation, and its interaction with p85 PI3K were examined by immunoprecipitation and immunoblotting analysis (C). PI3K activity was measured in the immunoprecipitates with anti-PI3KAP/XB130 antibody. The means ± sem of three replicate dishes are shown (D). E, The interaction of PI3KAP/XB130 with Src was analyzed by immunoprecipitation followed by immunoblotting with the indicated antibodies. Nonimmune IgG was used as a negative control. Similar results were obtained in two independent experiments. F, Src protein and the Y416 phosphorylation levels were analyzed by immunoblotting. The means ± sem of the densitometric analysis results of three independent experiments are shown as pSrc to total Src ratio in the graphs. *, P < 0.05.

Fig. 5.

Interaction of PI3KAP/XB130 with PI3K is required for potentiation of DNA synthesis induced by IGF-I. NIH3T3 cells were infected with control retrovirus pMXs-neo (mock) or retrovirus encoding FLAG-PI3KAP/XB130 or FLAG-Y72F. A, The cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG antibody. The immunoprecipitates and the total cell lysates were subjected to immunoblotting (IB) with indicated antibodies. B, The retrovirus-infected cells were serum starved for 24 h and then treated with 100 ng/ml IGF-I for 16 h. [Methyl-³H]thymidine incorporation into DNA was measured during the last 4 h. The means ± sem of three replicate wells are shown. There are significant differences between values with different letters (P < 0.05). Similar results were obtained in three independent experiments.