No abstract

SUMMARY Melanoma and other cancers harbor oncogenic mutations in the protein kinase B-Raf, which leads to constitutive activation and dysregulation of MAP kinase signaling. In order to elucidate molecular determinants responsible for B-Raf control of cancer phenotypes, we present a method for phosphoprotein profiling, using negative ionization mass spectrometry to detect phosphopeptides based on their fragment ion signature caused by release of PO3−. The method provides an alternative strategy for phosphoproteomics, circumventing affinity enrichment of phosphopeptides and isotopic labeling of samples. Ninety phosphorylation events were regulated by oncogenic B-Raf signaling, based on their responses to treating melanoma cells with MKK1/2 inhibitor. Regulated phosphoproteins included known signaling effectors and cytoskeletal regulators. We investigated MINERVA/FAM129B, a target belonging to a protein family with unknown category and function, and established the importance of this protein and its MAP kinase-dependent phosphorylation in controlling melanoma cell invasion into 3-dimensional collagen matrix.

Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) Analysis of the expression of fusion genes, in which the coding sequence of a reporter gene is placed under the control of putative regulatory elements of a test gene, has resulted in the identification of various cis-acting elements of eucaryotic and viral genes which interact with trans-acting factors to control transcription in mammalian cells (8,28,30). The two classes of cis-acting elements which have been identified are termed promoter and enhancer elements. Promoter elements are characterized by their dependence on position and orientation with respect to the transcription start site (8,28,30). These are typified by the CAAT, SP1, and TATA elements. Enhancer elements are characterized by their ability to exert effects relatively independently of distance from and orientation to the transcription start site (41). Enhancers may be constitutively active, as in the cases of the simian virus 40 enhancer (34) and the basal-level enhancers of the human metallothionein Ila gene (16, 40), or they may be regulatable, as exemplified by the metalregulatory elements of the human metallothionein IIa gene (22,23) and the steroid hormone response elements (22,23,37). Typically, deletion of a regulatable enhancer sequence affects regulated expression but has little or no effect on basal expression of the gene.The gene encoding phosphoenolpyruvate carboxykinase (PEPCK) is regulated at the transcriptional level by a number of hormones (3,10,24,39

Significant differences in biochemical parameters between normal and tumor tissues offer an opportunity to chemically design drug carriers which respond to these changes and deliver the drugs at the desired site. For example, overexpression of the matrix metalloproteinase-9 (MMP-9) enzyme in the extracellular matrix of tumor tissues can act as a trigger to chemically modulate the drug delivery from the carriers. In this study, we have synthesized an MMP-9-cleavable, collagen mimetic lipopeptide which forms nanosized vesicles with the POPC, POPE-SS-PEG, and cholesteryl-hemisuccinate lipids. The lipopeptide retains the triple-helical conformation when incorporated into these nanovesicles. The PEG groups shield the substrate lipopeptides from hydrolysis by MMP-9. However, in the presence of elevated glutathione levels, the PEG groups are reductively removed, exposing the lipopeptides to MMP-9. The resultant peptide-bond cleavage disturbs the vesicles’ lipid bilayer, leading to the release of encapsulated contents. These PEGylated nanovesicles are capable of encapsulating the anticancer drug gemcitabine with 50% efficiency. They were stable in physiological conditions and in human serum. Effective drug release was demonstrated using the pancreatic ductal carcinoma cells (PANC-1 and MIAPaCa-2) in two-dimensional and three-dimensional “tumor-like” spheroid cultures. A reduction in tumor growth was observed after intravenous administration of the gemcitabine-encapsulated nanovesicles in the xenograft model of athymic, female nude mice.

Mammalian cGMP- and cAMP-dependent protein kinase show considerable similarity in amino acid sequence, although they specifically bind different cyclic nucleotides. Results of cGMP analogue binding experiments, combined with modeling of the cGMP binding sites by analogy to the structure of the homologous catabolite gene activator protein, suggest that a threonine residue forms a hydrogen bond with the 2-NH2 of cGMP. This threonine is invariant in all cGMP binding domains, but the corresponding residue in 23 out of 24 cAMP binding sites of protein kinases is alanine, which cannot form the same hydrogen bond. This alanine/threonine difference has the potential for discriminating between cAMP and cGMP and may be important in the evolutionary divergence of cyclic nucleotide binding sites.