BACE1, a membrane-bound aspartyl protease that is implicated in Alzheimer’s disease, is the first protease to cut the amyloid precursor protein resulting in the generation of amyloid-β and its aggregation to form senile plaques, a hallmark feature of the disease. Few other native BACE1 substrates have been identified despite its relatively loose substrate specificity. We report a bioinformatics approach identifying several putative BACE1 substrates. Using our algorithm, we successfully predicted the cleavage sites for 70% of known BACE1 substrates and further validated our algorithm output against substrates identified in a recent BACE1 proteomics study that also showed a 70% success rate. Having validated our approach with known substrates, we report putative cleavage recognition sequences within 962 proteins, which can be explored using in vivo methods. Approximately 900 of these proteins have not been identified or implicated as BACE1 substrates. Gene ontology cluster analysis of the putative substrates identified enrichment in proteins involved in immune system processes and in cell surface protein-protein interactions.
Tandem mass spectrometry (MS/MS) has enabled researchers to analyze complex biological samples since the original concept inception. It facilitates the identification and quantification of modifications within tens of thousands of proteins in a single large-scale proteomic experiment. Phosphorylation analysis, as one of the most common and important post-translational modifications, has particularly benefited from such progress in the field. Here we showcase the technique through in-depth analyses of B cell signaling based on quantitative phosphoproteomics. As a complement to the previously described PolyMAC-Ti (polymer-based metal ion affinity capture using titanium) reagent, we introduce here PolyMAC-Fe, which utilizes a different metal ion, Fe(III). An extensive comparison using the different available MS/MS fragmentations techniques was made between PolyMAC-Fe, PolyMAC-Ti and IMAC (immobilized metal ion affinity chromatography) reagents in terms of specificity, reproducibility and type of phosphopeptides being enriched. PolyMAC-Fe based chelation demonstrated good selectivity and unique specificity toward phosphopeptides, making it useful in specialized applications. We have combined PolyMAC-Ti and PolyMAC-Fe, along with SILAC-based quantitation and large-scale fractionation, for quantitative B cell phosphoproteomic analyses. The complementary approach allowed us to identify a larger percentage of multiply phosphorylated peptides than with PolyMAC-Ti alone. Overall, out of 13,794 unique phosphorylation sites identified, close to 20% were dependent on BCR signaling. These sites were further mapped to a variety of major signaling networks, offering more detailed information about the biochemistry of B cell receptor engagement.
Background: Syk is a tyrosine kinase with both tumor promoting and tumor suppressing activities in cancer cells. Results: Protein kinase A is phosphorylated on a C-terminal tyrosine by Syk. Conclusion: The phosphorylation of PKA inhibits its activity and its ability to activate CREB. Significance: The phosphorylation by Syk of PKA inhibits its participation in downstream signaling pathways.
Engagement
of the B cell receptor for antigen (BCR) leads to immune
responses through a cascade of intracellular signaling events. Most
studies to date have focused on the BCR and protein tyrosine phosphorylation.
Because spleen tyrosine kinase, Syk, is an upstream kinase in multiple
BCR-regulated signaling pathways, it also affects many downstream
events that are modulated through the phosphorylation of proteins
on serine and threonine residues. Here, we report a novel phosphopeptide
enrichment strategy and its application to a comprehensive quantitative
phosphoproteomics analysis of Syk-dependent downstream signaling events
in B cells, focusing on serine and threonine phosphorylation. Using
a combination of the Syk inhibitor piceatannol, SILAC quantification,
peptide fractionation, and complementary PolyMAC-Ti and PolyMAC-Zr
enrichment techniques, we analyzed changes in BCR-stimulated protein
phosphorylation that were dependent on the activity of Syk. We identified
and quantified over 13 000 unique phosphopeptides, with a large
percentage dependent on Syk activity in BCR-stimulated B cells. Our
results not only confirmed many known functions of Syk, but more importantly,
suggested many novel roles, including in the ubiquitin proteasome
pathway, that warrant further exploration.
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