Akt1 contains a functional leucine-rich nuclear export sequence.

Nuclear Akt1 expression and Akt activation are common in cancer invasion. However, the mechanisms for this association and its causal role in invasion are uncertain. In an effort to identify potential mechanisms for regulating Akt subcellular localization, we analyzed the Akt gene sequences and identified a highly conserved leucine-rich potential nuclear export sequence (NES). Initial experiments demonstrated that leptomycin B induced nuclear Akt1 localization. Transient expression experiments demonstrated that, in comparison to wild-type Akt1, NES-mutated (AKT/NES) Akt1 has reduced interactions with CRM-1 and persistent nuclear localization. Subsequent stable transfection experiments in Akt1-/- fibroblasts confirmed that expression of AKT/NES resulted in persistent nuclear localization and activation1. Finally, stable expression of AKT/NES in Akt1-/- fibroblasts was sufficient to enhance cell migration in vitro. Thus, Akt1 contains a functional NES and mutation of the NES results in nuclear-predominant Akt1 activation that is sufficient to induce migration.

Akt: a potential target for thyroid cancer therapy.

Thyroid cancer is a heterogeneous disorder characterized by gene mutations that activate signaling pathways, and also by abnormalities in tumor suppressor genes and cell cycle proteins. Activation of the Akt/PKB signaling pathway appears to be an important event in thyroid tumorigenesis and, perhaps, in tumor progression too. Akt is activated in Cowden's syndrome through inactivation of PTEN, a negative regulator of Akt. Cowden's syndrome is an autosomal dominant multiorgan hamartoma syndrome characterized by benign and malignant thyroid tumors, breast cancers, and colon cancers. In addition, the Akt pathway appears to be activated in a significant proportion of sporadic thyroid cancers through activation of growth factor pathways by thyroid oncogenes and/or receptor overexpression. Disruption of PI3-kinase activity pharmacologically or disruption of Akt signaling using dominant negative cDNA expression have demonstrated salutary effects on several cancer models in vitro. Therefore, Akt represents an attractive target for pharmaceutical development for a variety of malignancies, including thyroid cancer.

Four naturally occurring mutations in the human GnRH receptor affect ligand binding and receptor function.

In the present study, we performed functional analyses of four mutations in the human GnRH receptor (GnRHR) gene, identified in patients with idiopathic hypogonadotropic hypogonadism. These mutations result in amino acid substitutions in the extracellular N-terminal domain (Thr32Ile), second extracellular loop (Cys200Tyr), third intracellular loop (Leu266Arg) and sixth transmembrane helix (Cys279Tyr). Immunocytochemical analysis of cells transfected with HA-tagged GnRHR constructs revealed that all four mutant receptors were present on the cell surface. However, all four mutant receptors failed to exhibit measurable specific GnRH binding and, except for Thr32Ile, any significant inositol phosphate accumulation after GnRH stimulation. In addition, Leu266Arg and Cys279Tyr receptors were unable to stimulate gonadotropin subunit or GnRHR gene promoter activity in response to GnRH. Interestingly, the Cys200Tyr mutant was able to stimulate gonadotropin subunit and GnRHR promoter activity, albeit with a higher EC(50) and a markedly reduced maximal response compared to wild type receptor. The Thr32Ile mutant was also able to stimulate gonadotropin subunit and GnRHR promoters, but with a further significant increase in EC(50). Similarly, this mutant partially retained the ability to activate extracellular signal-regulated kinase 1 and stimulate CRE-luciferase activity with an identical shift in EC(50). Taken together, the studies suggest that the Thr32Ile mutation reduces hGnRHR function primarily by reducing ligand binding affinity, and the Cys200Tyr mutation reduces cell surface receptor expression. All four amino acid substitutions interfered with ligand binding, and affected signal transduction and stimulation of gonadotropin and GnRHR gene expression in response to GnRH.