6 research outputs found
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
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
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
Analysis of a CSNK1A1 active site peptide from wild-type and mutant CSNK1A1 expressed in HEK293 cells.
<p>A) Amino acid sequence of the mutant peptide. B) LC-MS/MS spectra for wild-type and mutant protein. The mutant sample is off-set by 50 units for clarity and the region of interest is highlighted. C) Region of interest in the LC-MS/MS spectra for colon tumor and matched control samples. Each spectrum is normalized to the highest intensity ion. D) Extracted ion chromatograms from HEK293 lysates transfected with either wild-type or mutant CSNK1A1. The wild-type sample is normalized to the peak eluting at 63.0 minutes and the mutant sample is normalized to the peak eluting at 59.8 minutes.</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
Genomic DNA sequencing of CSNK1A1.
<p>A) PCR sequencing results for control using intronic primer. B) PCR sequencing results for patient 815zp using an intronic primer. In both cases, nucleotide of interest is highlighted with an asterisk.</p