ABSTRACT
Here we report the discovery that bifunctional thiol- and amine-reactive electrophiles serve as mechanism-based covalent cross-linkers for HECT E3 ubiquitin ligase-substrate pairs. We demonstrate that these chemical cross-linkers covalently cross-link the catalytic Cys residue of the yeast HECT E3 ubiquitin ligase Rsp5 with the Lys of the ubiquitination site in the model substrate Sic60-GFP. This work represents the first example of a mechanism-based covalent cross-link of HECT E3-substrate pairs that converts transiently interacting HECT E3-substrate pairs into stable, covalently cross-linked protein complexes, thereby facilitating their subsequent isolation, identification, and study.
Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitin-Protein Ligases/metabolism , Binding Sites , Cross-Linking Reagents/metabolism , Cyclin-Dependent Kinase Inhibitor Proteins/metabolism , Cysteine/chemistry , Endosomal Sorting Complexes Required for Transport/chemistry , Endosomal Sorting Complexes Required for Transport/genetics , Lysine/chemistry , Peptide Fragments/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Ubiquitin-Protein Ligase Complexes/chemistry , Ubiquitin-Protein Ligase Complexes/genetics , Ubiquitin-Protein Ligases/chemistry , UbiquitinationABSTRACT
The upstream protein kinases responsible for thousands of phosphorylation events in the phosphoproteome remain to be discovered. We developed a three-component chemical reaction which converts the transient noncovalent substrate-kinase complex into a covalently cross-linked product by utilizing a dialdehyde-based cross-linker, 1. Unfortunately, the reaction of 1 with a lysine in the kinase active site and an engineered cysteine on the substrate to form an isoindole cross-linked product could not be performed in the presence of competing cellular proteins due to nonspecific side reactions. In order to more selectively target the cross-linker to protein kinases in cell lysates, we replaced the weak, kinase-binding adenosine moiety of 1 with a potent protein kinase inhibitor scaffold. In addition, we replaced the o-phthaldialdehyde moiety in 1 with a less-reactive thiophene-2,3-dicarboxaldehyde moiety. The combination of these two structural modifications provides for cross-linking of a cysteine-containing substrate to its corresponding kinase in the presence of competing cellular proteins.
Subject(s)
Phosphotransferases/chemistry , Adenosine/chemistry , Cross-Linking Reagents/chemistry , Cysteine/chemistry , Fluorescent Dyes/chemistry , HeLa Cells , Humans , Lysine/chemistry , Models, Chemical , Molecular Conformation , Peptides/chemistry , Phosphorylation , Phosphotransferases/metabolism , Protein Structure, Tertiary , Proteins/chemistry , Thiophenes/chemistryABSTRACT
Protein phosphorylation represents the most dominant and evolutionary conserved posttranslational modification for information transfer in cells and organisms. The human genome encodes >500 protein kinases, and thousands of phosphorylation sites are present in mammalian proteome. To develop a global view of phosphorylation network, there is a need to map the connectivity between kinases and phosphoproteome. We developed a chemical kinase-substrate cross-linker 1 that converts transient kinase-substrate interactions into a covalently linked kinase-substrate complex in vitro and in the presence of cell lysates. The method can be applied to identify unknown upstream kinases responsible for phosphorylation events in cell lysates.
Subject(s)
Enzyme Assays/methods , Peptides/metabolism , Phosphotransferases/metabolism , Cell Line, Tumor , Cross-Linking Reagents/chemical synthesis , Cross-Linking Reagents/metabolism , Cysteine/metabolism , Fluorescent Dyes/metabolism , HeLa Cells , Humans , Peptides/chemical synthesis , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Staining and Labeling , Substrate SpecificityABSTRACT
Bistramide A (1) is a marine natural product with broad, potent antiproliferative effects. Bistramide A has been reported to selectively activate protein kinase C (PKC) delta, leading to the view that PKCdelta is the principal mediator of antiproliferative activity of this natural product. Contrary to this observation, we established that bistramide A binds PKCdelta with low affinity, does not activate this kinase in vitro and does not translocate GFP-PKCdelta. Furthermore, we identified actin as the cellular receptor of bistramide A. We report that bistramide A disrupts the actin cytoskeleton, inhibits actin polymerization, depolymerizes filamentous F-actin in vitro and binds directly to monomeric G-actin in a 1:1 ratio with a Kd of 7 nM. We also constructed a fully synthetic9 bistramide A-based affinity matrix and isolated actin as a specific bistramide A-binding protein. This activity provides a molecular explanation for the potent antiproliferative effects of bistramide A, identifying it as a new biochemical tool for studies of the actin cytoskeleton and as a potential lead for development of a new class of antitumor agents.
Subject(s)
Acetamides/pharmacology , Actins/drug effects , Pyrans/pharmacology , Acetamides/chemical synthesis , Acetamides/chemistry , Actins/metabolism , Animals , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Humans , In Vitro Techniques , Molecular Conformation , Protein Kinase C-delta/drug effects , Protein Kinase C-delta/metabolism , Pyrans/chemical synthesis , Pyrans/chemistry , Rats , Spiro Compounds/chemical synthesis , Spiro Compounds/chemistry , Spiro Compounds/pharmacology , Time FactorsABSTRACT
We have developed an efficient and highly stereocontrolled synthesis of bistramide A, a selective activator of protein kinase C isotype delta. Our synthetic strategy featured a novel bidirectional approach for spiroketal construction based on the ring-opening/cross-metathesis sequence employing a highly strained cyclopropenone acetal. The synthesis afforded the final target with the longest linear sequence of 15 steps and provided unambiguous structural determination of bistramide A, including assignment of the previously unknown C(37) stereochemistry.