Measuring intracellular metabolism has increasingly led to important insights in biomedical research. 13C tracer analysis, although less information-rich than quantitative 13C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting 13C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments.
Phosphatidylinositol 3-kinase (PI3-K) phosphorylates the 3-position of phosphatidylinositol to give rise to three signaling phospholipids. Binding of the pleckstrin homology (PH) domain of Akt to membrane PI(3)P's causes the translocation of Akt to the plasma membrane bringing it into contact with membrane-bound Akt kinase (PDK1 and 2), which phosphorylates and activates Akt. Akt inhibits apoptosis by phosphorylating Bad, thus promoting its binding to and blockade of the activity of the cell survival factor Bcl-x. Herein we present the synthesis and biological activity of several novel phosphatidylinositol analogues and demonstrate the ability of the carbonate group to function as a surrogate for the phosphate moiety. Due to a combination of their PI3-K and Akt inhibitory activities, the PI analogues 2, 3, and 5 proved to be good inhibitors of the growth of various cancer cell lines with IC(50) values in the 1-10 microM range. The enhanced Akt inhibitory activity of the axial hydroxymethyl-bearing analogue 5 compared to its equatorial counterpart 6 is rationalized based upon postulated differences in the H-bonding patterns of these compounds in complex with a homology modeling generated structure of the PH domain of Akt. This work represents the first attempt to examine the effects of 3-modified PI analogues on these two crucial cell signaling proteins, PI3-K and Akt, in an effort to better understand their cell growth inhibitory properties.
The formation of G-quadruplex structures within the nuclease hypersensitive element (NHE) III 1 region of the c-myc promoter and the ability of these structures to repress c-myc transcription have been well established. However, just how these extremely stable DNA secondary structures are transformed to activate c-myc transcription is still unknown. NM23-H2/nucleoside diphosphate kinase B has been recognized as an activator of c-myc transcription via interactions with the NHE III 1 region of the c-myc gene promoter. Through the use of RNA interference, we confirmed the transcriptional regulatory role of NM23-H2. In addition, we find that further purification of NM23-H2 results in loss of the previously identified DNA strand cleavage activity, but retention of its DNA binding activity. NM23-H2 binds to both single-stranded guanine-and cytosine-rich strands of the c-myc NHE III 1 and, to a lesser extent, to a random single-stranded DNA template. However, it does not bind to or cleave the NHE III 1 in duplex form. Significantly, potassium ions and compounds that stabilize the G-quadruplex and i-motif structures have an inhibitory effect on NM23-H2 DNA-binding activity. Mutation of Arg 88 to Ala 88 (R88A) reduced both DNA and nucleotide binding but had minimal effect on the NM23-H2 crystal structure. On the basis of these data and molecular modeling studies, we have proposed a stepwise trapping-out of the NHE III 1 region in a single-stranded form, thus allowing single-stranded transcription factors to bind and activate c-myc transcription. Furthermore, this model provides a rationale for how the stabilization of the G-quadruplex or i-motif structures formed within the c-myc gene promoter region can inhibit NM23-H2 from activating c-myc gene expression.
Ganglioside GM3 inhibits epidermal growth factor (EGF)-dependent cell proliferation in a variety of cell lines. Both in vitro and in vivo, this glycosphingolipid inhibits the kinase activity of the EGF receptor (EGFR). Furthermore, membrane preparations containing EGFR can bind to GM3-coated surfaces. These data suggest that GM3 may interact directly with the EGFR. In this study, the interaction of gangliosides with the extracellular domain (ECD) of the EGFR was investigated. The purified human recombinant ECD from insect cells bound directly to ganglioside GM3. The ganglioside interaction site appears to be distinct from the EGF-binding site. In agreement with previous reports on the effects of specific gangliosides on EGFR kinase activity, the ECD preferentially interacted with GM3. The order of relative binding of other gangliosides investigated was as follows: GM3 > > GM2, GD3, GM4 > GM1, GD1a, GD1b, GT1b, GD2, GQ1b > lactosylceramide. These data suggest that NeuAc-lactose is essential for binding and that any sugar substitution reduces binding. In agreement with the specificity of soluble ECD binding to gangliosides, GM3 specifically inhibited EGFR autophosphorylation. Identification of a ganglioside interaction site on the ECD of the EGFR is consistent with the hypothesis that endogenous GM3 may function as a direct modulator of EGFR activity.
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