A Novel Protein Kinase B (PKB)/AKT-binding Protein Enhances PKB Kinase Activity and Regulates DNA Synthesis

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 twohybrid 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 APEinduced 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.The serine/threonine protein kinase PKB 1 (also called Akt) is thought to be a key mediator of signal transduction. Upon growth factor stimulation, a family of lipid kinases known as class 1 phosphoinositide 3-kinases (PI 3-kinases) is recruited to the plasma membrane. PI 3-kinases phosphorylate phosphatidylinositol 4,5-bisphosphate at the D-3 position of the inositol ring, converting it to phosphatidylinositol 3,4,5-trisphosphate. Following the activation of PI 3-kinase, PKBs are recruited to the plasma membrane through direct contact of the pleckstrin homology (PH) domain with phosphatidylinositol 3,4,5-trisphosphate and are phosphorylated at Thr 308 by PDK1 and at Ser 473 by PDK2, a kinase of which the molecular structure has not yet been identified (1, 2). AGC kinases other than PKB are also known to be regulated by PI 3-kinase, and PKB acts downstream from PI 3-kinase to regulate numerous biological processes, such as proliferation, antiapoptosis, cell growth, and glucose metabolism (1, 2).PKB has a wide range of substrates, including GSK-3, FKHR (FoxO1), FKHR-L1 (FoxO3), AFX (FoxO4), and eNOS, all of which have the consensus motif RXRXX(S/T) (3, 4). Protein kinases do not generally form stable complexes with their substrates, although PKB has been shown to exist in a stable complex with several of its substrates including MDM2, p21 Cip1 /WAF1, an...

Sap1 Promotes the Association of the Replication Fork Protection Complex With Chromatin and Is Involved in the Replication Checkpoint in Schizosaccharomyces pombe

“…Moreover, we have consistently found putative Sap1 homologs in a variety of other organisms that do not employ mating-type switching. For example, Sap1 shows significant similarity to Girdin (also known as APE, GIV, or Hook-related protein) in humans and mice (Anai et al 2005;Enomoto et al 2005;Le-Niculescu et al 2005;Simpson et al 2005) and also to the Xenopus LOC431973 protein (supplemental Figure S1A at http://www.genetics.org/ supplemental/), indicating its conservation throughout evolution. However, we could not identify a Sap1-like protein in budding yeast.…”

Functional characterization of the guanine nucleotide exchange factor (GEF) motif of GIV protein reveals a threshold effect in signaling

“…GIV has been previously reported by us (8,14) and others (11,12) to be an enhancer of PI3K-Akt signaling. We previously found that activation of Gαi3 by GIV is required for the efficient activation of Akt upon stimulation of receptor tyrosine kinases (RTKs) (8,10,14,16,19) as well as G-protein-coupled-receptors (GPCRs) (8,19).…”

“…Gα-interacting, vesicleassociated protein, Girdin) is a nonreceptor GEF that activates G proteins via an evolutionarily conserved, well-defined motif (8). A major function of GIV's GEF motif is to enhance the activation of the phosphoinositide 3 kinase (PI3K)-Akt pathway in response to different stimuli (8,(10)(11)(12)(13). GIV has been shown to be essential for cell migration during a variety of physiologic and pathologic processes, i.e., wound healing (14), macrophage chemotaxis (14), tumor cell migration (8,10), neuronal development (12), and endothelial cell migration (15), as well as for inhibiting autophagy (16).…”

The GAPs, GEFs, GDIs and…now, GEMs: New kids on the heterotrimeric G protein signaling block

“…Three other groups co-discovered GIV nearly simultaneously. Anai et al 18 described it as a binding partner and activator of Akt kinase and named it APE (Akt-Phosphorylation Enhancer). Simpson et al 19 reported it as a Hook-domain containing microtubule-interacting protein which binds the large GTPase dynamin and modulates endocytic trafficking; they named it HkRP1 (Hook-Related Protein 1).…”