13 research outputs found
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
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
Sequence analysis of cloned PCR products from the 815zp tumor sample.
<p>A) A representative clone showing the mutated nucleotide (*). B) Alignment of all 41 sequences. Differences between pseudogene and CSNK1A1 are indicated by a plus symbol.</p
Immunoblot of recombinant CSNK1A1 samples.
<p>HEK293 cells were transfected with either no DNA (Mock), <b>CSNK1A1</b> wild type or D136N plasmids (CK1-WT and CK1-M, respectively).</p
The structure of an ATP acyl phosphate probe containing a desthiobiotin affinity tag and a schematic mechanism for covalent labeling of conserved lysines in the kinase active site.
<p>The structure of an ATP acyl phosphate probe containing a desthiobiotin affinity tag and a schematic mechanism for covalent labeling of conserved lysines in the kinase active site.</p