ABSTRACT
Interleukin-1 receptor-associated kinase 3 (IRAK3) is a pseudokinase mediator in the human inflammatory pathway, and ablation of its function is associated with enhanced antitumor immunity. Traditionally, pseudokinases have eluded "druggability" and have not been considered tractable targets in the pharmaceutical industry. Herein we disclose a CRISPR/Cas9-mediated knockout of IRAK3 in monocyte-derived dendritic cells that results in an increase in IL-12 production upon lipopolysaccharide (LPS) stimulation. Furthermore, we disclose and characterize Degradomer D-1, which displays selective proteasomal degradation of IRAK3 and reproduces the 1L-12p40 increases observed in the CRISPR/Cas9 knockout.
Subject(s)
Cytokines , Interleukin-1 Receptor-Associated Kinases , Cytokines/metabolism , Humans , Interleukin-1 Receptor-Associated Kinases/metabolism , Interleukin-12/metabolism , Lipopolysaccharides/metabolism , Lipopolysaccharides/pharmacology , Monocytes/metabolismABSTRACT
Taspase1 is an Ntn-hydrolase overexpressed in primary human cancers, coordinating cancer cell proliferation, invasion, and metastasis. Loss of Taspase1 activity disrupts proliferation of human cancer cells in vitro and in mouse models of glioblastoma. Taspase1 is synthesized as an inactive proenzyme, becoming active upon intramolecular cleavage. The activation process changes the conformation of a long fragment at the C-terminus of the α subunit, for which no full-length structural information exists and whose function is poorly understood. We present a cloning strategy to generate a circularly permuted form of Taspase1 to determine the crystallographic structure of active Taspase1. We discovered that this region forms a long helix and is indispensable for the catalytic activity of Taspase1. Our study highlights the importance of this element for the enzymatic activity of Ntn-hydrolases, suggesting that it could be a potential target for the design of inhibitors with potential to be developed into anticancer therapeutics.
Subject(s)
Endopeptidases/chemistry , Endopeptidases/metabolism , Cloning, Molecular , Crystallography, X-Ray , Dynamic Light Scattering , Endopeptidases/genetics , Enzyme Activation , Humans , Models, Molecular , Protein Domains , Protein Structure, SecondaryABSTRACT
The recognition that Rho guanosine triphosphatases (GTPases) (Rho, Rac, and Cdc42) play important regulatory roles in many areas of cell biology has made the ability to measure their activity in cells an important biological tool. Because Rho GTPases become activated by conversion from guanosine diphosphate-bound states to guanosine triphosphate (GTP)-bound forms, affinity-based methods to detect the formation of GTP-Rho GTPases have been developed and are widely used for the purpose of assessing Rho GTPase activities in biological studies.
Subject(s)
Biological Assay/methods , rho GTP-Binding Proteins/metabolism , Apoptosis Regulatory Proteins , Enzyme Activation , GTP-Binding Proteins , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , p21-Activated Kinases , rho GTP-Binding Proteins/geneticsABSTRACT
Cytoskeletal remodeling is critical for cell adhesion, spreading, and motility. p21-activated kinase (PAK), an effector molecule of the Rho GTPases Rac and Cdc42, has been implicated in cytoskeletal remodeling and cell motility. PAK kinase activity and subcellular distribution are tightly regulated by rapid and transient localized Rac and Cdc42 activation, and by interactions mediated by adapter proteins. Here, we show that endogenous PAK is constitutively activated in certain breast cancer cell lines and that this active PAK is mislocalized to atypical focal adhesions in the absence of high levels of activated Rho GTPases. PAK localization to focal adhesions in these cells is independent of PAK kinase activity, NCK binding, or GTPase binding, but requires the association of PAK with PIX. Disruption of the PAK-PIX interaction with competitive peptides displaces PAK from focal adhesions and results in a substantial reduction in PAK hyperactivity. Moreover, disruption of the PAK-PIX interaction is associated with a dramatic decrease of PIX and paxillin in focal adhesions, indicating that PAK localization to these structures via PIX is required for the maintenance of paxillin- and PIX-containing focal adhesions. Abnormal regulation of PAK localization and activity may contribute to the tumorigenic properties of certain breast cancer cells.
