In Situ Kinase Profiling Reveals Functionally Relevant Properties of Native Kinases

SummaryProtein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here, we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against >200 kinases in native cell proteomes and reveal biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected on live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading

Capillary electrophoresis with multiphoton-exited fluorescence : native fluorescence, enzymatic assays, and ultra-fast separations

textAnalysis of single neurons and/or single synaptic vesicles will uncover heterogeneity that is masked by the ensemble averaging required of many current techniques. In the Shear lab, we have developed instrumentation to perform capillary electrophoresis (CE) coupled to multiphoton excitation (MPE) as a means to improve detection limits for bioactive molecules in such volume limited samples. CE has provided significant advancements in this arena due to its ability to fractionate sample volumes of less than one picoliter and the relative ease with which highly sensitive detection strategies can be employed. Moreover, the use of extremely narrow separation channels (~600 nm) permits the collection of low volume samples without excessive dilution and provides the spatial and temporal resolution necessary to investigate heterogeneous microenvironments. Such low vii volume analyses necessitate the use of sensitive detection strategies due to the limited sample quantity within small inner diameter (i.d.) separation channels. Multiphoton-excited fluorescence is an excellent strategy for such low volume detection due to the inherently small excitation volumes, improved spectral isolation of the signal from the source, and the ability to probe diverse chromophores with a single, tunable source. Here we present the use of MPE with CE and demonstrate marked improvements in mass sensitivity for several neurotransmitters and neuropeptides (Chapter 2). Moreover, the versatility of this approach is demonstrated through the analysis of enzymatic reactions (Chapters 3 and 5) and photochemical derivatization (Chapter 4). We demonstrate a new platform for ultra-fast separations based on multiphoton-excited photochemistry in Chapter 4. Future efforts will demonstrate the capacity of CE-MPE to probe volume limited biological microenvironments such as single synaptic vesicles.Chemistry and Biochemistr

Multiphoton-Excited Serotonin Photochemistry

We report photochemical and photophysical studies of a multiphoton-excited reaction of serotonin that previously has been shown to generate a photoproduct capable of emitting broadly in the visible spectral region. The current studies demonstrate that absorption of near-infrared light by an intermediate state prepared via three-photon absorption enhances the photoproduct formation yield, with the largest action cross sections (∼10(−19) cm(2)) observed at the short-wavelength limit of the titanium:sapphire excitation source. The intermediate state is shown to persist for at least tens of nanoseconds and likely to be different from a previously reported oxygen-sensitive intermediate. In addition, the two-photon fluorescence action spectrum for the fluorescent photoproduct was determined and found to have a maximum at ∼780 nm (3.2 eV). A general mechanism for this photochemical process is proposed

Correction: Identification of a Tumor Specific, Active-Site Mutation in Casein Kinase 1α by Chemical Proteomics.

[This corrects the article DOI: 10.1371/journal.pone.0152934.]

Monitoring Native p38α:MK2/3 Complexes via Trans Delivery of an ATP Acyl Phosphate Probe

Here we describe a chemical proteomics strategy using ATP acyl phosphates to measure the formation of a protein:protein complex between p38α and mapkap kinases 2 and/or 3. Formation of the protein:protein complex results in a new probe labeling site on p38α that can be used to quantify the extent of interaction in cell lysates and the equilibrium binding constant for the interaction in vitro. We demonstrate through RNA interference that the labeling site is dependent on formation of the protein:protein complex in cells. Further, we identify that active-site-directed, small-molecule inhibitors of MK2/3 selectively inhibit the heterodimer-dependent probe labeling, whereas p38α inhibitors do not. These findings afford a new method to evaluate p38α and MK2/3 inhibitors within native biological systems and a new tool for improved understanding of p38α signaling pathways