Measurement of kinase activation in single mammalian cells

Meredith, Gavin; Sims, Christopher E.; Soughayer, Joseph S.; Allbritton, Nancy L.

doi:10.1038/73760

Cited by 126 publications

(146 citation statements)

References 36 publications

Supporting

Mentioning

144

Contrasting

Order By: Relevance

“…This CE-based method provides a quantitative measure of the fluorescent species obtained from single cells as previously described (37,43,45,46). After trypsin and TCEP exposure, the electrophoretic traces obtained from these cells (n = 12) demonstrated predominately a single peak that comigrated with the nonphosphorylated standard for the ABL peptide domain used in these experiments (Fig.…”

Section: A Peptide Cargo Is Efficiently Loaded Into Lymphocyte-derivementioning

confidence: 98%

Myristoyl-Based Transport of Peptides into Living Cells

et al. 2007

Self Cite

View full text Add to dashboard Cite

Translocation of membrane impermeant molecules to the interior of living cells is a necessity for many biochemical investigations. Myristoylation was studied as a means to introduce peptides into living cells. Uptake of a myristoylated, fluorescent peptide was efficient in the B lymphocyte cell line BA/F3. In contrast, this cell line was resistant to peptide uptake using a cell penetrating peptide derived from the TAT protein. In BA/F3 cells, membrane association was shown to be rapid reaching a maximum within 30 minutes. Cellular uptake of the peptide lagged the membrane association, but occurred within a similar time frame. Experiments performed at 37°C vs. 4°C demonstrated profound temperature dependence in the cellular uptake of myristoylated cargo. Myristoylated peptides with either positive or negative charge were shown to load efficiently. In contrast to TAT-conjugated cargo, pyrenebutyrate did not enhance cellular uptake of the myristoylated peptide. The myristoylated peptide did not adversely affect cell viability at concentrations up to 100 μM. This assessment of myristoyl-based transport provides fundamental data needed in understanding the intracellular delivery of myristoylated peptide cargoes for cellbased biochemical studies.

show abstract

Section: A Peptide Cargo Is Efficiently Loaded Into Lymphocyte-derivementioning

confidence: 98%

Myristoyl-Based Transport of Peptides into Living Cells

et al. 2007

Self Cite

View full text Add to dashboard Cite

show abstract

“…These probes have high value in measurements on primary cells since they are not genetically encoded. Similarly short peptide substrates have been used by Allbritton and colleagues to report intracellular kinase activity and this method would also benefit from the development of efficient peptide-based substrates for use as reporters of cellular enzyme activity (40).…”

Section: Introductionmentioning

confidence: 99%

Use of Docking Peptides to Design Modular Substrates with High Efficiency for Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase

et al. 2007

View full text Add to dashboard Cite

The mitogen-activated protein (MAP) kinase ERK plays a key role in the regulation of cellular proliferation. Mutations in the ERK cascade occur in 30% of malignant tumors. Thus understanding how the kinase identifies its cognate substrates as well as monitoring the activity of ERK is central to cancer research and therapeutic development. ERK binds to its protein targets, both downstream substrates and upstream activators, via a binding site distinct from the catalytic site of ERK. The substrate sequences that bind, or dock, to these sites on ERK influence the efficiency of phosphorylation. For this reason, simple peptide substrates containing only phosphorylation sequences typically possess low efficiencies for ERK. Appending short docking peptides derived from full-length protein substrates and activators of ERK to a phosphorylation sequence increased the affinity of ERK for the phosphorylation sequence by as much as 200-fold, while only slightly diminishing the maximal velocity of the reaction. The efficiency of the phosphorylation reaction was increased by up to 150 fold, while the specificity of the substrate for ERK was preserved. Simple, modular peptide substrates which can be easily tailored to possess high phosphorylation efficiencies will enhance our understanding of the regulation of ERK and provide a tool for the development of new kinase assays.

show abstract

“…Researchers must investigate the reactivity of a protein with its physiological partners to understand its role in cellular pathways; concentration measurements alone could be misleading, because the local environment of a protein can drastically alter its activity. Emerging capillary micro-/nanofabrication technologies allow the chemical reactivity of the target species to be investigated (3,(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Allbritton and coworkers (11,12) embarked on the development of this technology and used capillary electrophoresis to analyze intracellular kinase activity.…”

mentioning

confidence: 99%

“…Emerging capillary micro-/nanofabrication technologies allow the chemical reactivity of the target species to be investigated (3,(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Allbritton and coworkers (11,12) embarked on the development of this technology and used capillary electrophoresis to analyze intracellular kinase activity. The combination of these advances in characterizing the reactivity and concentrations of cellular compounds with even smaller, more intricate devices will certainly advance our knowledge of cellular heterogeneity and signaling cascades.…”

mentioning

confidence: 99%

Nanokit for single-cell electrochemical analyses

Pan

Jiang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

113

View full text Add to dashboard Cite

The development of more intricate devices for the analysis of small molecules and protein activity in single cells would advance our knowledge of cellular heterogeneity and signaling cascades. Therefore, in this study, a nanokit was produced by filling a nanometersized capillary with a ring electrode at the tip with components from traditional kits, which could be egressed outside the capillary by electrochemical pumping. At the tip, femtoliter amounts of the kit components were reacted with the analyte to generate hydrogen peroxide for the electrochemical measurement by the ring electrode. Taking advantage of the nanotip and small volume injection, the nanokit was easily inserted into a single cell to determine the intracellular glucose levels and sphingomyelinase (SMase) activity, which had rarely been achieved. High cellular heterogeneities of these two molecules were observed, showing the significance of the nanokit. Compared with the current methods that use a complicated structural design or surface functionalization for the recognition of the analytes, the nanokit has adapted features of the well-established kits and integrated the kit components and detector in one nanometer-sized capillary, which provides a specific device to characterize the reactivity and concentrations of cellular compounds in single cells.

show abstract

Measurement of kinase activation in single mammalian cells

Cited by 126 publications

References 36 publications

Myristoyl-Based Transport of Peptides into Living Cells

Myristoyl-Based Transport of Peptides into Living Cells

Use of Docking Peptides to Design Modular Substrates with High Efficiency for Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase

Nanokit for single-cell electrochemical analyses

Contact Info

Product

Resources

About