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Nijsje Dorman, Ph.D., Patrick C.H. Lo, Ph.D., and Kristie Nybo, Ph.D.
BioTechniques, Vol. 49, No. 3, September 2010, pp. 615–617
Full Text (PDF)

Disruptive Technology

A powerful way to dissect signal transduction pathways is to observe the effects of turning a kinase off or on. Although there are many strategies for regulating kinases, such as sequestration, degradation, or chemically induced activation, each suffers from troubling limitations. For instance, methods that confine or degrade the kinase could also impinge on its non-enzymatic functions, while more precise interventions may need to be redesigned for each kinase studied. An allosteric activation approach described by Karginov et al. in Nature Biotechnology addresses these shortcomings with a method that relies on the much-exploited interaction between the FK506-binding protein (FKBP12), the FKBP12-rapamycin-binding (FRB) domain, and rapamycin. A truncated form of FKBP12, insertable FKBP12 (iFKBP), was designed to be positioned adjacent to the glycine (G) loop, a conserved structural feature that interacts with the ATP necessary for kinase function. Disruption of this region with iFKBP is expected to destabilize the G loop, compromising phosphate transfer. However, rapamycin and FRB stabilize iFKBP, restoring the functionality of the G loop and the catalytic activity of the kinase. As a first test of the approach, the authors worked with focal adhesion kinase (FAK), a protein that has both scaffold and phosphorylation functions. RapR-FAK, the iFKBP-containing, rapamycin-regulatable version of FAK, was essentially catalytically inactive under normal conditions. In the presence of expressed FRB, however, RapR-FAK was restored to near–wild-type activity just 2 min after the addition of 50 nM rapamycin. Importantly, the noncatalytic behavior of FAK was otherwise unaffected by this engineered allosteric regulatory module. As with wild-type protein, RapR-FAK was sensitive to autoinhibition, and the protein's interactions with known binding partners and its intracellular localization were preserved. Because rapamycin has immunosuppressive effects that may interfere with pathway characterization, the authors confirmed that two nonimmunosuppressive analogs of rapamycin also provided regulated activation, a finding that suggests the method could be used in vivo. The broader applicability of the approach was demonstrated by introducing iFKBP into Src and p38, making them rapamycin-inducible and supporting the authors’ contention that their method should be applicable to any tyrosine or serine/threonine kinase.

Karginov et al. 2010. Engineered allosteric activation of kinases in living cells. Nat. Biotechnol. 28:743-747.

Growing Apart

Profiling the differences between two distinct cell types that differentiate from a single progenitor has been facilit in vivo studies by recent methods such as the isolation of transcripts from ribosomes containing cell-specifically tagged ribosomal or poly(A) proteins-binding However, such techniques do not also amine epigenetic factors involve differentiation, such as DNA methylation posttranslational modifications of his tones, and nucleosome remodeling to remedy this omission, Deal and Henikoff writing in Developmental Cell, describe a method for studying both the nuclear RNA—which studies have shown to be comparable to total cellular mRNA— and chromatin of a specific cell type by isolating its transgenically tagged nuclei. Developed for use in Arabidopsis, this method involves cotransformation of a constitutively expressed biotin ligase gene with a construct consisting of a cell type–specific promoter driving a fusion of a subdomain of the outer nuclear envelope–targeted Arabidopsis Ran GTPaseactivating protein 1 (RanGAP1), GFP, and biotin ligase recognition peptide. Coexpression of the biotin ligase and the fusion protein produces biotin-studdenuclei that can be purified by streptavidinconjugated magnetic beads. As a test system, Deal and Henikoff studied the hair and non-hair cells of Arabidopsis root epidermis, which derive from a shared progenitor. Nuclei from each cell type were purified from 3 g root tissue, with recovery of 1–3 × 105 nuclei per preparation. Total nuclear RNA was extracted and gene expression levels were measured by microarray. Nuclei preparations were also used for chromatin profiling via chromatin immunoprecipitation with antibodies directed against methylated histone H3, and assay of immunoprecipitated chromatin fragments on tiling arrays. By combining the chromatin profiling and gene expression data, the authors showed that transcription-associated histone methylation enhanced and polycomb silencing associated histone methylation was diminished in the cell type where a particular gene was more highly expressed. Although similar analyses would be possible using isolation by flow cytometry, the authors report that in their hands, cell sorting resulted 50% purity and 10% recovery compared to >90% purity and 70% recovey with the nuclei-tagging method. The new technique should be transferableto any organism amenable to transgenic tagging, although an alternative to the RanGAP1 domain would be needed for nonplant systems. Based on the method's simplicity (unlike laser-capture microdissection or cell sorting, no specialized equipment is required) and applicability to both gene and chromatin profiling, it should become a key tool for parsing developmental pathways.

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