The E3 ligase TTC3 facilitates ubiquitination and degradation of phosphorylated Akt.

The serine threonine kinase Akt is a core survival factor that underlies a variety of human diseases. Although regulatory phosphorylation and dephosphorylation have been well documented, the other posttranslational mechanisms that modulate Akt activity remain unclear. We show here that tetratricopeptide repeat domain 3 (TTC3) is an E3 ligase that interacts with Akt. TTC3 contains a canonical RING finger motif, a pair of tetratricopeptide motifs, a putative Akt phosphorylation site, and nuclear localization signals, and is encoded by a gene within the Down syndrome (DS) critical region on chromosome 21. TTC3 is an Akt-specific E3 ligase that binds to phosphorylated Akt and facilitates its ubiquitination and degradation within the nucleus. Moreover, DS cells exhibit elevated TTC3 expression, reduced phosphorylated Akt, and accumulation in the G(2)M phase, which can be reversed by TTC3 siRNA or Myr-Akt. Thus, interaction between TTC3 and Akt may contribute to the clinical symptoms of DS.

Identification of nerve growth factor-responsive element of the TCL1 promoter as a novel negative regulatory element.

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.