Abstract
Protein kinase B (PKB)/Akt reportedly plays a role in the survival and/or proliferation of cells. We identified a novel protein, which binds to PKB, using a yeast two-hybrid screening system. This association was demonstrated not only in vivo by overexpressing both proteins or by coimmunoprecipitation of the endogenous proteins, but also in vitro using glutathione S-transferase fusion proteins. Importantly, this protein specifically associates with the C terminus of PKB but not with other AGC kinases and enhances PKB phosphorylation and kinase activation without growth factor stimulation. Thus, we termed this Akt-specific binding protein APE (Akt-phosphorylation enhancer). Since APE-induced phosphorylation of PKB did not occur in cells treated with wortmannin or LY294002, APE itself is not a kinase but seems to enhance or prolong the phosphoinositide 3-kinase-dependent phosphorylation of PKB. In cells in which APE was suppressed by small interfering RNA, DNA synthesis was significantly reduced with suppression of PKB phosphorylation, suggesting a synergistic role of APE in PKB-induced proliferation. On the other hand, in cells overexpressing both PKB and APE, despite markedly increased basal phosphorylation of PKB, both DNA rereplication and subsequent Chk2 phosphorylation and apoptosis were seen, suggesting the involvement of APE in the regulation of cell cycling replication licensing. Taking these observations together, APE appears to be a novel regulator of PKB phosphorylation. Furthermore, the interaction between APE and PKB, possibly dependent on the expression levels of both proteins, may be a novel molecular mechanism leading to proliferation and/or apoptosis.
Highlights
The serine/threonine protein kinase PKB1 is thought to be a key mediator of signal transduction
Our mouse cDNA is very likely to be full-length, and this protein was subsequently shown to enhance the phosphorylation of Protein kinase B (PKB), such that we designated the clone APE (Akt-phosphorylation enhancer)
APE overexpression increased PKB phosphorylation, in a titer-dependent manner, and the maximal phosphorylation of PKB obtained by APE overexpression was comparable with that achieved by long term pervanadate stimulation (Fig. 5B). These results suggest that APE overexpression can induce essentially maximal phosphorylation of PKB on Thr308 and Ser473, which indicates that APE is an enhancer of PKB in vivo
Summary
The serine/threonine protein kinase PKB1 ( called Akt) is thought to be a key mediator of signal transduction. TRB3 was identified as a negative modulator of the PKB type [12], a contradictory report was very recently published [13]. These results indicate that understanding PKB modulation is important for elucidating the mechanism of PKB activation and its regulation of cellular functions. Overexpression of this protein markedly enhances phosphorylation of Thr308 and Ser473 in PKB, leading to its kinase activation and phosphorylation of its downstream substrates such as GSK-3 and FKHR. Suppression of APE using RNA interference significantly reduces PKB phosphorylation and PKB kinase activity We termed this protein APE (Akt-phosphorylation enhancer) and demonstrate the possible role of APE in DNA synthesis and apoptosis in cooperation with PKB
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