Selenocysteine-Specific Mass Spectrometry Reveals Tissue-Distinct Selenoproteomes and Candidate Selenoproteins.

Selenoproteins, defined by the presence of selenocysteines (Sec), play important roles in a wide range of biological processes. All known selenoproteins are marked by the presence of Sec insertion sequence (SECIS) at their mRNA. The lack of an effective analytical method has hindered our ability to explore the selenoproteome and new selenoproteins beyond SECIS. Here, we develop a Sec-specific mass spectrometry-based technique, termed "SecMS," which allows the systematic profiling of selenoproteomes by selective alkylation of Sec. Using SecMS, we quantitatively characterized the age- and stress-regulated selenoproteomes for nine tissues from mice of different ages and mammalian cells, demonstrating tissue-specific selenoproteomes and an age-dependent decline in specific selenoproteins in brains and hearts. We established an integrated platform using SecMS and SECIS-independent selenoprotein (SIS) database and further identified five candidate selenoproteins. The application of this integrated platform provides an effective strategy to explore the selenoproteome independent of SECIS.

Protein expression profiles of C3H 10T1/2 murine fibroblasts and of isogenic cells transformed by the H1047R mutant of phosphoinositide 3-kinase (PI3K).

We have used stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with tandem mass spectrometry to characterize the proteomes of two isogenic cell lines that differ in the expression of a single oncoprotein,p110α of PI3K, carrying the H1047R mutation. 51,510 peptides were identified and assigned to 4,201 proteins. Most notable among the proteins that show increased expression in the oncogenically transformed cells are several involved in the interferon response including Isg15, Ifit1, Igtp and Oas2 (interferon stimulated gene 15, interferon-induced protein with tetratricopeptide repeats 1, interferon gamma-inducible GTP-binding protein, 2'-5'-oligoadenylate synthetase 2). Prominent among the downregulated proteins are several involved in cell adhesion as well as proteins that are affected by the negative feedback from PI3K signaling. The differential expressions documented in this analysis suggest novel links between oncogenic PI3K and several signaling pathways. These links will be explored in future studies.

PI3K and STAT3: a new alliance.

UNLABELLED: Recent proteomic data have uncovered an interdependence of PI3K and STAT3. In PI3K-tranformed murine cells, STAT3 is phosphorylated on Y705 and activated in a PI3K-dependent manner. Dominant negative STAT3 interferes with PI3K-induced oncogenic transformation. Phosphorylation of STAT3 in PI3K-transformed murine cells is mediated by the TEC kinase BMX. Observations on glioblastoma stem cells reveal similar critical roles for STAT3 and BMX. The new data document an important role of STAT3 in PI3K-driven oncogenic transformation and mark BMX as a promising therapeutic target that could enhance the effectiveness of PI3K inhibitors. SIGNIFICANCE: The PI3K­TOR and STAT3 signaling pathways represent two distinct regulatory networks. The discovery of a functional link between these pathways is significant for our understanding of PI3K- and STAT3-driven oncogenic mechanisms and identifies the TEC kinase BMX as a new cancer target.

Phosphatidylinositol 4,5-bisphosphate-specific AKT1 is oncogenic.

The protein kinase AKT1 (v-akt murine thymoma viral oncogene homolog 1), also referred to as protein kinase B (PKB), is an essential mediator of the phosphatidylinositol 3-kinase signaling pathway. Elevated activity of AKT1 is common in human cancer. Localization at the plasma membrane, leading to enhanced phosphorylation and activation of AKT1, is an important factor determining the oncogenicity of this kinase. Although the phosphatidylinositol 3-kinase signaling pathway is frequently upregulated in cancer, cancer-specific mutations in AKT1 are not common. Recently, such a mutation has been identified in breast, colon and ovarian cancers. The mutation is located in the pleckstrin homology (PH) domain of AKT1 and results in a glutamic acid to lysine substitution at residue 17. The resultant change in the conformation of the PH domain facilitates membrane binding of the mutant protein. Here we show that exchange of the PH domain leading to preferential binding of phosphatidylinositol 4,5-bisphosphate (PIP(2)) over phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) constitutively activates AKT1. AKT1 with this altered PIP affinity induces oncogenic transformation in cultures of chicken embryo fibroblasts and causes neoplastic growth and angiogenesis in the chorioallantoic membrane of the chicken embryo. Gain-of-function mutants of AKT1 may not be affected by PI3K inhibitors that are currently in development. Therefore, AKT1 remains a distinct and important cancer target.