Abstract
The serine/threonine kinase, Akt (protein kinase B) plays a central role in the regulation of intracellular cell survival. Recently, we demonstrated that the proto-oncogene TCL1, overexpressed in human T-cell prolymphocytic leukemia, is an Akt kinase co-activator. Tightly restricted TCL1 gene expression in early developmental cells suggested that the TCL1 gene is regulated at a transcriptional level. To characterize how TCL1 gene expression is regulated, we cloned the 5'-promoter of the TCL1 gene located at human chromosome 14q32. The 5'-TCL1 promoter region contains a TATA box with cis-regulatory elements for Nur77/NGFI-B (nerve growth factor-responsive element (NBRE), CCAAGGTCA), NFkappaB, and fork head transcription factor. Nur77/NGFI-B, an orphan receptor superfamily transcription factor implicated in T-cell apoptosis, is a substrate for Akt. We hypothesized that TCL1 transactivity is regulated through Akt-induced phosphorylation of Nur77/NGFI-B in vivo. In an electrophoretic mobility shift assay with chromosomal immunoprecipitation assays, wild-type Nur77, but not S350A mutant Nur77, could specifically bind to TCL1-NBRE. A luciferase assay demonstrated that TCL1-NBRE is required for inhibition of TCL1 transactivity upon nerve growth factor/platelet-derived growth factor stimulation, which activates Akt and phosphorylates Nur77. Using a chromosomal immunoprecipitation assay with reverse transcription-PCR, nerve growth factor stimulation inhibited binding of endogenous Nur77 to TCL1-NBRE, in turn, suppressing TCL1 gene expression. The results together establish that TCL1-NBRE is a novel negative regulatory element of Nur77 (NGFI-B). To the best of our knowledge, TCL1-NBRE is the first direct target of Nur77 involving the regulation of intracellular cell death survival. This Akt-induced inhibitory mechanism of TCL1 should play an important role in immunological and/or neuronal development in vivo.
Highlights
Ptosis [1,2,3]
In this regard it was striking that a putative FKHRL consensus binding site (CAAAATAA, located from Ϫ857 to 850) as well as a Nur77/NGFI-B binding site (NBRE, CCAAGGTCA, located from Ϫ340 to Ϫ332) were present within 1000 of the 5Ј-TCL1 proximal promoter region (Fig. 1B)
Nur77 is an orphan receptor superfamily transcription factor that is implicated in neuronal development and apoptosis [20]
Summary
Ptosis [1,2,3]. Recently, we demonstrated that the proto-oncogene TCL1, whose function was previously unknown, is an Akt kinase co-activator that physically binds and, activates Akt [4, 5]. By EMSA and luciferase reporter assays, we demonstrated that Nur77 (NGFI-B family) bound to the NBRE site of the TCL1 promoter (TCL1-NBRE, AAGGTCA), and negatively regulated TCL1 gene expression by Akt stimulation.
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