Large-scale Proteomics Analysis of the Human Kinome

Abstract

Members of the human protein kinase superfamily are the major regulatory enzymes involved in the activity control of eukaryotic signal transduction pathways. As protein kinases reside at the nodes of phosphorylation-based signal transmission, comprehensive analysis of their cellular expression and site-specific phosphorylation can provide important insights into the architecture and functionality of signaling networks. However, in global proteome studies, low cellular abundance of protein kinases often results in rather minor peptide species that are occluded by a vast excess of peptides from other cellular proteins. These analytical limitations create a rationale for kinomewide enrichment of protein kinases prior to mass spectrometry analysis. Here, we employed stable isotope labeling by amino acids in cell culture (SILAC) to compare the binding characteristics of three kinase-selective affinity resins by quantitative mass spectrometry. The evaluated pre-fractionation tools possessed pyrido[2,3-d]pyrimidine-based kinase inhibitors as immobilized capture ligands and retained considerable subsets of the human kinome. Based on these results, an affinity resin displaying the broadly selective kinase ligand VI16832 was employed to quantify the relative expression of more than 170 protein kinases across three different, SILAC-encoded cancer cell lines. These experiments demonstrated the feasibility of comparative kinome profiling in a compact experimental format. Interestingly, we found high levels of cytoplasmic and low levels of receptor tyrosine kinases in MV4 -11 leukemia cells compared with the adherent cancer lines HCT116 and MDA-MB-435S. The VI16832 resin was further exploited to pre-fractionate kinases for targeted phosphoproteomics analysis, which revealed about 1200 distinct phosphorylation sites on more than 200 protein kinases. This hitherto largest

A Small Molecule Inhibits Akt through Direct Binding to Akt and Preventing Akt Membrane Translocation

“…Membrane Translocation-To explore the mechanism by which API-1 inhibits Akt, we performed a protein kinase-compound binding assay because API-1 contains an amino group that has been shown to bind to NHS-activated Sepharose (22)(23)(24). After immobilization, compound-bound Sepharose beads were incubated with recombinant Akt protein.…”

“…The assay for API-1 binding to Akt was performed essentially as previously described for other kinase inhibitors that contain an amino group (22)(23)(24). API-1 was immobilized on Sepharose beads (GE Healthcare) through covalent linkage using its amino group (Fig.…”

Pyk2 cytonuclear localization: mechanisms and regulation by serine dephosphorylation

“…S778 is located in a canonical PKA phosphorylation site and mutation of either S778 or the two basic residues involved in kinase interaction resulted in GFPPyk2 700-841 nuclear relocalization. Large-scale analysis of protein phosphorylation using mass spectrometry has found that S778 is one of the phosphorylated residues of Pyk2 in vivo [47,48]. However, until now, nothing was known about the specific function of S778 and this is the first time a role of Pyk2 S/T phosphorylation has been identified.…”

Cdk1-mediated phosphorylation of Cdc7 suppresses DNA re-replication

“…This may be due to Cdc7 autophosphorylation 14 or phosphorylation by other kinases such as polo-like kinase (Plk), as large scale proteomics studies indicated that Plk phosphorylates Cdc7 at Serine 27 and 277 in prometaphase. 26,28,[30][31][32][33] In any event, our data demonstrate that Cdc7 is phosphorylated at multiple sites in a mitotic Cdkdependent manner around prometaphase.…”