Interaction between TCL1 and Epac1 in the activation of Akt kinases in plasma membranes and nuclei of 8-CPT-2-O-Me-cAMP-stimulated macrophages

Abstract

Epac1 is a cAMP-stimulated guanine exchange factor that activates Rap1. The protein product of the T cell leukemia 1 (TCL1) proto-oncogene binds to Akt enhancing its kinase activity. TCL1 and Epac promote cellular proliferation because of their activating effects on Akt. Employing macrophages, we have studied the mechanisms whereby these proteins function in the regulation of Akt kinase activity. Cells were treated with 8-CPT-2-O-Me-cAMP, a cAMP analog which acts selectively and specifically via Epac1. Epac1 co-immunoprecipitated with TCL1 in plasma membrane and nuclear fractions of 8-CPT-2-O-Me-cAMP-stimulated macrophages. Interaction of TCL1 and Epac1 was also observed in a [125I]GST-Epac1 pulldown assay. A two-threefold increase in Akt Thr-308 and Akt Ser-473 protein kinase activities and their phosphoprotein levels was observed in TCL1 immunoprecipitates of plasma membranes and nuclei of the treated cells. Elevated Akt Thr-308 protein kinase activity and its phosphoprotein levels were significantly reduced in TCL1 immunoprecipitates of plasma membranes of 8-CPT-2-O-Me-cAMP-treated cells where Epac1 gene expression was silenced. In contrast, Akt Ser-473 protein kinase activity and its phosphoprotein levels were reduced only in plasma membranes. Our studies suggest that a ternary complex of TCL1, Epac1, and Akt forms in activated macrophages both promoting Akt activation and regulating intracellular distribution of Akt.

Figure 1

Co-immunoprecipitation of Epac1 with TCL1 in TCL1 immunoprecipitates of plasma membrane and nuclear fractions of cells treated with: (1) buffer and (2) 8-CPT-2-O-Me-cAMP (200 μM/30 min). Panel A. A representative immunoblot of three to four experiments of Epac1 and TCL1 in plasma membrane (□) and nuclear fraction (■) obtained by precipitation with an anti-TCL1 antibody. The bar diagrams above respective immunoblots show changes in protein levels of Epac1 and TCL1 in arbitrary units ± SE from three to four individual experiments. Panel B. An immunoblot of Epac1 and TCL1 in cell lysates of cells treated with: (1) buffer; and (2) 8-CPT-2-O-Me-cAMP. Panel C. Specificity of Epac1 and TCL1 antibodies. Immunoblots of non-immune IgG immunoprecipitates of plasma membrane and nuclear fractions of cells treated with (1) buffer; and (2) 8-CPT-2-O-Me-cAMP probes with Epac1 and TCL1 antibodies. This study demonstrates the specifity of the method.

Figure 2

[¹²⁵I]GST-Epac1 pulldown assay showing association of TCL1 with Epac1. [¹²⁵I]GST-Epac1 -glutathione-Sepharose 4B beads were incubated with: (1) lysates of buffer-treated cells; (2) lysates of 8-CPT-2-O-Me-cAMP-treated cells; and (3) purified TCL1 protein, respectively. Panel A. Autoradiograph of [¹²⁵I]GST-Epac1 in Epac1 and TCL1 immunoblots, respectively. S in Panel A is the [¹²⁵I]GST-Epac1 protein standard. Panel B. Immunoblots of Epac1 and TCL1, respectively of protein adsorbed on [¹²⁵I]GST-Epac1-glutathione-Sepharose beads. S in Panel B is the TCL1 and [¹²⁵I]GST-Epac1 protein standards, respectively. Panel C. [¹²⁵I]GST-Epac1 in autoradiographs (Panel A) and TCL1 and Epac1 immunoblot (Panel B), respectively expressed in arbitrary units (× 10³) as the mean ± SE from two experiments performed in duplicate are shown below the respective panel. In the left panel, TCL1, the bars represent: [¹²⁵I]GST-Epac1 by autoradiograph (□) and TCL1 protein by immunoblot (■). In the right Panel, the bars are: [¹²⁵I]GST-Epac1 by autoradiograph (□)and Epac1 protein by immunoblot (■).

Figure 3

Upregulation of Akt^Thr-308 and Akt^Ser-473 protein kinase activities in the TCL1 immunoprecipitates of plasma membrane and nuclear fractions. Panel A. The bars are: (1) Akt^Thr-308 kinase activity in plasma membranes (pm) of buffer (□) and 8-CPT-2-O-Me-cAMP-stimulated cells (■); 2) Akt^Ser-473 kinase activities plasma membranes of buffer (□) and 8-CPT-8-O-Me-cAMP-treated cells (■); (3) Akt^Thr-308 kinase activities in nuclei (nuc) of cells treated with buffer ( )or 8-CPT-2-O-Me-cAMP ( ); and (4) Akt^Ser-473 kinase activities in nuclei of cells treated with buffer ( ) and 8-CPT-2-O-Me-cAMP ( ). Akt1 kinase activities are expressed as [³³P] γATP incorporated (pmol/mg protein) and are mean ± SE from three experiments. Immunoblots representative of three experiments of p-Akt^Thr-308 and p-Akt^Ser-473 of plasma membranes and nuclei are shown below the bar graph where (1) indicates buffer-treated and (2) 8-CPT-2-O-Me-cAMP-treated. Panel B. Specificity of antibodies employed in these studies. Immunoblots of non-immune IgG immunoprecipitates of plasma membrane and nuclear fractions of cells treated with: (1) buffer or (2) 8-CPT-2-O-Me-cAMP probed for p-Akt^Thr-308 and p-Akt^Ser-473.

Figure 4

Immunoblots showing the changes in TCL1, Epac1, p-Akt^Thr-308 and p-Akt^Ser-473 in TCL1 immunoprecipitates (Panel A) and Epac1 immunoprecipitates (Panel B) of plasma membranes (pm) and nuclei (nuc) of 8-CPT-2-O-Me-cAMP-treated cells after silencing Epac1 gene expression. The lanes are (1) lipofectamine + buffer; (2) lipofectamine + 8-CPT-2-O-Me-cAMP (200 μm/30 min); (3) Epac1 dsRNA + 8-CPT-2-O-Me-cAMP; and (4) scrambled dsRNA + 8-CPT-2-O-Me-cAMP. The protein loading control actin is also shown. Immunoblots shown are representative of two experiments performed in duplicate. The changes in levels of Epac1, TCL1, p-Akt^Thr-308 and p-Akt^Ser-473 in TCL1 and Epac1 immunoprecipitates, respectively are shown above the respective immunoblots and are expressed in arbitrary units ± SE from two experiments performed in duplicate. The bars in the respective panels are: p-Akt^Thr-308 (□) and p-Akt^Ser-473 (■).

Figure 5

Nuclear/perinuclear region localization of Epac1 and TCL1 in cells treated with 8-CPT-2-O-Me-cAMP, buffer, or anti-IgG by confocal microscopy.