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.
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.
Misra
Uma K
UK
Department of Pathology, Duke University Medical Center, Durham, NC 27710, United States.
Kaczowka
Steven J
SJ
Pizzo
Salvatore V
SV
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013873:Thionucleotides / Q000494:pharmacology*; D014162:Transfection
C482601:Epac protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D011518:Proto-Oncogene Proteins; D012330:RNA, Double-Stranded; C490393:Tcl1 protein, mouse; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D051057:Proto-Oncogene Proteins c-akt