Pseudokinases are members of the protein kinase superfamily but signal primarily through noncatalytic mechanisms. Many pseudokinases contribute to the pathologies of human diseases, yet they remain largely unexplored as drug targets owing to challenges associated with modulation of their biological functions. Our understanding of the structure and physiological roles of pseudokinases has improved substantially over the past decade, revealing intriguing similarities between pseudokinases and their catalytically active counterparts. Pseudokinases often adopt conformations that are analogous to those seen in catalytically active kinases and, in some cases, can also bind metal cations and/or nucleotides. Several clinically approved kinase inhibitors have been shown to influence the noncatalytic functions of active kinases, providing hope that similar properties in pseudokinases could be pharmacologically regulated. In this Review, we discuss known roles of pseudokinases in disease, their unique structural features and the progress that has been made towards developing pseudokinase-directed therapeutics.

Prospects for pharmacological targeting of pseudokinases

Protein phosphorylation by eukaryotic protein kinases (ePKs) is a fundamental mechanism of cell signaling in all organisms. In model vertebrates, ~10% of ePKs are classified as pseudokinases, which have amino acid changes within the catalytic machinery of the kinase domain that distinguish them from their canonical kinase counterparts. However, pseudokinases still regulate various signaling pathways, usually doing so in the absence of their own catalytic output. To investigate the prevalence, evolutionary relationships, and biological diversity of these pseudoenzymes, we performed a comprehensive analysis of putative pseudokinase sequences in available eukaryotic, bacterial, and archaeal proteomes. We found that pseudokinases are present across all domains of life, and we classified nearly 30,000 eukaryotic, 1500 bacterial, and 20 archaeal pseudokinase sequences into 86 pseudokinase families, including ~30 families that were previously unknown. We uncovered a rich variety of pseudokinases with notable expansions not only in animals but also in plants, fungi, and bacteria, where pseudokinases have previously received cursory attention. These expansions are accompanied by domain shuffling, which suggests roles for pseudokinases in plant innate immunity, plant-fungal interactions, and bacterial signaling. Mechanistically, the ancestral kinase fold has diverged in many distinct ways through the enrichment of unique sequence motifs to generate new families of pseudokinases in which the kinase domain is repurposed for noncanonical nucleotide binding or to stabilize unique, inactive kinase conformations. We further provide a collection of annotated pseudokinase sequences in the Protein Kinase Ontology (ProKinO) as a new mineable resource for the signaling community.

Tracing the origin and evolution of pseudokinases across the tree of life

The 21st century is witnessing an explosive surge in our understanding of pseudoenzyme-driven regulatory mechanisms in biology. Pseudoenzymes are proteins that have sequence homology with enzyme families but that are proven or predicted to lack enzyme activity due to mutations in otherwise conserved catalytic amino acids. The best-studied pseudoenzymes are pseudokinases, although examples from other families are emerging at a rapid rate as experimental approaches catch up with an avalanche of freely available informatics data. Kingdom-wide analysis in prokaryotes, archaea and eukaryotes reveals that between 5 and 10% of proteins that make up enzyme families are pseudoenzymes, with notable expansions and contractions seemingly associated with specific signaling niches. Pseudoenzymes can allosterically activate canonical enzymes, act as scaffolds to control assembly of signaling complexes and their localization, serve as molecular switches, or regulate signaling networks through substrate or enzyme sequestration. Molecular analysis of pseudoenzymes is rapidly advancing knowledge of how they perform noncatalytic functions and is enabling the discovery of unexpected, and previously unappreciated, functions of their intensively studied enzyme counterparts. Notably, upon further examination, some pseudoenzymes have previously unknown enzymatic activities that could not have been predicted a priori. Pseudoenzymes can be targeted and manipulated by small molecules and therefore represent new therapeutic targets (or anti-targets, where intervention should be avoided) in various diseases. In this review, which brings together broad bioinformatics and cell signaling approaches in the field, we highlight a selection of findings relevant to a contemporary understanding of pseudoenzyme-based biology.

Emerging concepts in pseudoenzyme classification, evolution, and signaling

There's more to death than life: Noncatalytic functions in kinase and pseudokinase signaling.

Aberrant expression of receptor tyrosine kinase AXL is linked to metastasis. AXL can be activated by its ligand GAS6 or by other kinases, but the signaling pathways conferring its metastatic activity are unknown. Here, we define the AXL-regulated phosphoproteome in breast cancer cells. We reveal that AXL stimulates the phosphorylation of a network of focal adhesion (FA) proteins, culminating in faster FA disassembly. Mechanistically, AXL phosphorylates NEDD9, leading to its binding to CRKII which in turn associates with and orchestrates the phosphorylation of the pseudo-kinase PEAK1. We find that PEAK1 is in complex with the tyrosine kinase CSK to mediate the phosphorylation of PAXILLIN. Uncoupling of PEAK1 from AXL signaling decreases metastasis in vivo, but not tumor growth. Our results uncover a contribution of AXL signaling to FA dynamics, reveal a long sought-after mechanism underlying AXL metastatic activity, and identify PEAK1 as a therapeutic target in AXL positive tumors. AXL receptor tyrosine kinase has a role in metastasis but the mechanism is unclear. In this study, the authors show that AXL activation can control focal adhesion dynamics via PEAK1 and that AXL-mediated PEAK1 phosphorylation is required for metastasis of triple negative breast cancer cells in vivo.

/pdf/axl-confers-cell-migration-and-invasion-by-hijacking-a-peak1-1uaf90o1oo.pdf

AXL confers cell migration and invasion by hijacking a PEAK1-regulated focal adhesion protein network

The pseudo-kinase and signaling protein Pragmin has been linked to cancer by regulating protein tyrosine phosphorylation via unknown mechanisms. Here we present the crystal structure of the Pragmin 906-1368 amino acids C-terminus, which encompasses its kinase domain. We show that Pragmin contains a classical protein kinase fold devoid of catalytic activity. A particular inhibitory triad, conserved in a Pragmin/SgK269/PEAK1/C19orf35 superfamily, tightly holds the catalytic lysine (K997) to prevent ATP binding. By proteomics, we discovered that this pseudo-kinase uses the tyrosine kinase CSK to induce protein tyrosine phosphorylation in human cells. Interestingly, the protein kinase domain is bordered by N- and C-terminal extensions forming an original dimerization domain that regulates Pragmin self-association and stimulates CSK activity. A1329E mutation in the C-terminal extension destabilizes Pragmin dimerization and reduces CSK activation. Thus, our results reveal a new dimerization mechanism by which a pseudo-kinase can induce protein tyrosine phosphorylation.

https://www.biorxiv.org/content/early/2017/11/27/218669.full.pdf

Dimerization of the Pragmin pseudo-kinase regulates protein tyrosine phosphorylation

The pseudo-kinase and signaling protein Pragmin has been linked to cancer by regulating protein tyrosine phosphorylation via unknown mechanisms. Here we present the crystal structure of the Pragmin 906- 1368 amino acids C-terminus, which encompasses its kinase domain. We show that Pragmin contains a classical protein kinase fold devoid of catalytic activity. A particular inhibitory triad, conserved in a Pragmin/SgK269/PEAK1/C19orf35 superfamily, tightly holds the catalytic lysine (K997) to prevent ATP binding. By proteomics, we discovered that this pseudo-kinase uses the tyrosine kinase CSK to induce protein tyrosine phosphorylation in human cells. Interestingly, the protein kinase domain is bordered by N- and C-terminal extensions forming an original dimerization domain that regulates Pragmin self-association and stimulates CSK activity. A1329E mutation in the C-terminal extension destabilizes Pragmin dimerization and reduces CSK activation. Thus, our results reveal a new dimerization mechanism by which a pseudo-kinase can induce protein tyrosine phosphorylation.

Dimerization of the Pragmin Pseudo-Kinase Regulates Protein Tyrosine Phosphorylation

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Dimerization of the Pragmin pseudo-kinase regulates protein tyrosine phosphorylation

Dimerization of the Pragmin Pseudo-Kinase Regulates Protein Tyrosine Phosphorylation

Dimerization of the Pragmin Pseudo-Kinase Regulates Protein Tyrosine Phosphorylation