Abstract: Unc-51-like kinase 4 (ULK4) is a pseudokinase conserved in most eukaryotes, yet ULK4 signaling mechanisms remain enigmatic. In this issue of Structure, Preuss and colleagues report a structure of the ATP-bound ULK4 pseudokinase domain, supported by proteomic analysis of the ULK4 interactome and in-depth evolutionary analysis of the intriguingULK4 pseudokinase domain. Unc-51-like kinase 4 (ULK4) is a pseudokinase conserved in most eukaryotes, yet ULK4 signaling mechanisms remain enigmatic. In this issue of Structure, Preuss and colleagues report a structure of the ATP-bound ULK4 pseudokinase domain, supported by proteomic analysis of the ULK4 interactome and in-depth evolutionary analysis of the intriguingULK4 pseudokinase domain. Pseudokinases are components of kinomes throughout the kingdoms of life (Kwon et al., 2019Kwon A. Scott S. Taujale R. Yeung W. Kochut K.J. Eyers P.A. Kannan N. Tracing the origin and evolution of pseudokinases across the tree of life.Sci. Signal. 2019; 12: eaav3810Crossref PubMed Scopus (30) Google Scholar). They function primarily as scaffold-based signaling hubs but can also allosterically modulate canonical kinases and, in several cases, perform cryptic enzymology involving an “atypical” mechanism of catalysis. Despite lacking several of the active site acid motifs that define “canonical” protein kinases (Figure 1), the human pseudokinase Unc-51-like kinase 4 (ULK4) possesses an affinity for ATP, although this interaction is metal independent (Murphy et al., 2014Murphy J.M. Zhang Q. Young S.N. Reese M.L. Bailey F.P. Eyers P.A. Ungureanu D. Hammaren H. Silvennoinen O. Varghese L.N. et al.A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties.Biochem. J. 2014; 457: 323-334Crossref PubMed Scopus (166) Google Scholar). Low-affinity small molecule kinase inhibitors of ULK4 have recently been described that, along with structural evidence, suggest that they target the nucleotide-binding site (Khamrui et al., 2020Khamrui S. Ung P.M.U. Secor C. Schlessinger A. Lazarus M.B. High-Resolution Structure and Inhibition of the Schizophrenia-Linked Pseudokinase ULK4.J. Am. Chem. Soc. 2020; 142: 33-37Crossref PubMed Scopus (8) Google Scholar). The pharmacological tractability of ULK4 is of potential importance because murine ULK4 controls the extent of the neural stem cell pool (Liu et al., 2016Liu M. Guan Z. Shen Q. Flinter F. Domínguez L. Ahn J.W. Collier D.A. O’Brien T. Shen S. Ulk4 Regulates Neural Stem Cell Pool.Stem Cells. 2016; 34: 2318-2331Crossref PubMed Scopus (13) Google Scholar), and single nucleotide polymorphisms (SNPs) in ULK4 have been linked to hypertension and schizophrenia. What is currently lacking is an understanding of how ULK4 performs its biological role(s), including detailed knowledge of specific subcellular partners and the extent of ULK4 regulatory nodes. In this issue of Structure, a study by Preuss and colleagues moves the ULK4 field forward several steps with the presentation of a high-resolution structure (PDB: 6TSZ) of the human ULK4-ATPγS complex (Preuss et al., 2020Preuss F. Chatterjee D. Mathea S. Shrestha S. St-Germain J. Saha M. Kannan N. Raught B. Rottapel R. Knapp S. Nucleotide Binding, Evolutionary Insights, and Interaction Partners of the Pseudokinase Unc-51-like Kinase 4.Structure. 2020; 28 (this issue): 1184-1196Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). This extends related work in which ULK4 was co-crystallized (Khamrui et al., 2020Khamrui S. Ung P.M.U. Secor C. Schlessinger A. Lazarus M.B. High-Resolution Structure and Inhibition of the Schizophrenia-Linked Pseudokinase ULK4.J. Am. Chem. Soc. 2020; 142: 33-37Crossref PubMed Scopus (8) Google Scholar) in the presence of an ATP-competitive inhibitor (PDB: 6U5L). The unusual ATP binding mode seen in the new study (Preuss et al., 2020Preuss F. Chatterjee D. Mathea S. Shrestha S. St-Germain J. Saha M. Kannan N. Raught B. Rottapel R. Knapp S. Nucleotide Binding, Evolutionary Insights, and Interaction Partners of the Pseudokinase Unc-51-like Kinase 4.Structure. 2020; 28 (this issue): 1184-1196Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar) leads to the proposal that ULK4 utilizes a conserved residue, Lys39, distinct from the canonical “VAIK” Lys (Figure 1). The study is consistent with previous pseudokinase nucleotide-screening experiments, which demonstrated metal-independent nucleotide binding to ULK4 through a variety of approaches (Eyers and Murphy, 2013Eyers P.A. Murphy J.M. Dawn of the dead: protein pseudokinases signal new adventures in cell biology.Biochem. Soc. Trans. 2013; 41: 969-974Crossref PubMed Scopus (61) Google Scholar; Khamrui et al., 2020Khamrui S. Ung P.M.U. Secor C. Schlessinger A. Lazarus M.B. High-Resolution Structure and Inhibition of the Schizophrenia-Linked Pseudokinase ULK4.J. Am. Chem. Soc. 2020; 142: 33-37Crossref PubMed Scopus (8) Google Scholar). The structural data are also supported by biophysical analysis of recombinant ULK4, which co-purifies with a host nucleotide when overexpressed in bacteria, is stabilized some 20°C by ATP (and destabilized by Mg2+ ions), and exhibits very stable ATP association during extended molecular dynamics simulations in silico. An important, and very welcome, additional element to this study is the first evolutionary analysis of ULK4 pseudokinase domain sequences across an exceptionally broad spread of eukaryotic organisms. An initial finding is that ULK4 is defined by two co-evolved sequences that distinguish it from closely related canonical kinases. These ULK4-specific features likely contribute to the fold of the pseudokinase domain and potentially could form docking interactions with partner proteins. The elegant analysis of 277 distinct ULK4 sequences now permits these motifs to be employed confidently to define ULK4 within any kinome. The analysis also concludes that although mammalian ULK4 homologs from a variety of species contain an intact (though still atypical) Gly-rich loop (Figure 1), the absence of canonical Asn and Asp residues explains why metal binding (and catalysis) is absent. Second, a broad comparison of ULK4 catalytic residues in various ancestral lineages reveals a number of vertebrate and non-vertebrate traits (Figure 1). Remarkably, although essentially all ULK4 proteins possess a rudimentary Gly-rich loop, suggesting the ability to accommodate ATP (and assuming neutralization of the negative charge of the phosphate groups), the degree of conservation of other canonical amino acids varies significantly, but in a phylogenetically consistent manner, across phyla. For example, in protist and plant ULK4 orthologs, a full “complement” of catalytic residue is nearly always present (Figure 1), which challenges the assignment as pseudokinases in a large number of species. However, Arabidopsis ULK4 (also known as RUNKEL/RUK) has previously been demonstrated to be devoid of phosphotransferase activity in vitro, and transgenic ULK4 lacking “canonical” amino acids does not rescue the lethality of Arabidopsis “ruk” mutants in vivo (Krupnova et al., 2009Krupnova T. Sasabe M. Ghebreghiorghis L. Gruber C.W. Hamada T. Dehmel V. Strompen G. Stierhof Y.D. Lukowitz W. Kemmerling B. et al.Microtubule-associated kinase-like protein RUNKEL needed [corrected] for cell plate expansion in Arabidopsis cytokinesis.Curr. Biol. 2009; 19: 518-523Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Another notable aspect of the new study are cellular interaction studies performed using BioID (FLAG-BirA∗ fused to ULK4), which marries mass spectrometry to human cell extract immunoprecipitation and reveals cellular interactors of both the ULK4 pseudokinase and C-terminal helical HEAT/armadillo repeat domains. Many of the identified ULK4 interaction partners are previously known centrosomal or tubulin-associated proteins, and the discovery of “canonical” protein kinase targets (including ROCK1/2 and STK36; Figure 1), as well as the protein phosphatase PTPN14, increases the likelihood that ULK4 cellular scaffolding performs phosphoregulatory roles. These will be important avenues for further investigation. Consistent with the finding that human ULK4 binds to the microtubule-binding augmin/HAUS complex and kinesin family motor proteins, Arabidopsis ULK4/RUNKEL has previously been shown to bind to microtubules (Krupnova et al., 2009Krupnova T. Sasabe M. Ghebreghiorghis L. Gruber C.W. Hamada T. Dehmel V. Strompen G. Stierhof Y.D. Lukowitz W. Kemmerling B. et al.Microtubule-associated kinase-like protein RUNKEL needed [corrected] for cell plate expansion in Arabidopsis cytokinesis.Curr. Biol. 2009; 19: 518-523Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and controls localization of the plant kinesin protein HINKEL to regulate the phragmoblast MAPK pathway (Krupnova et al., 2013Krupnova T. Stierhof Y.D. Hiller U. Strompen G. Müller S. The microtubule-associated kinase-like protein RUNKEL functions in somatic and syncytial cytokinesis.Plant J. 2013; 74: 781-791Crossref PubMed Scopus (12) Google Scholar). Intriguingly, ancient eukaryotic genomes, including the ciliate Tetrahymena and Placazoans, perhaps the simplest of all multicellular animals, also contain ULK4-like genes, which exhibit a range of shuffled catalytic motifs that might support catalytic phosphotransferase activity (Figure 1). However, various lines of evidence currently support the assertion that ULK4-dependent biology occurs independently of catalysis (but might require conserved nucleotide binding), despite rather marked differences in the evolved machinery between species, which could reflect a requirement to support an appropriate fold or scaffold disposition for species-specific roles. Similar conformational flexibility has been established multiple times for canonical protein kinases, which can operate in both catalytic and non-catalytic modes (Jacobsen and Murphy, 2017Jacobsen A.V. Murphy J.M. The secret life of kinases: insights into non-catalytic signalling functions from pseudokinases.Biochem. Soc. Trans. 2017; 45: 665-681Crossref PubMed Scopus (42) Google Scholar). However, it should be noted that this model does not formally preclude auto- or transphosphorylation for some orthologs, notably plants and protist ULK4 (Figure 1), and expanded biochemical studies with purified ULK4 proteins and a panel of potential substrates, including some of the interactome identified by Preuss and colleagues, might well throw up interesting findings. What is clear from the convincing analysis presented is that species-specific preservation/degradation of active site motifs in ULK4 (notably the loss of the β3-Lys residue; Figure 1) occurs concurrently with the appearance of a unique helical (and highly extended) activation loop. Moreover, in ULK4, this potentially drives interaction and stabilization of co-conserved residues in the key regulatory αC helix (Preuss et al., 2020Preuss F. Chatterjee D. Mathea S. Shrestha S. St-Germain J. Saha M. Kannan N. Raught B. Rottapel R. Knapp S. Nucleotide Binding, Evolutionary Insights, and Interaction Partners of the Pseudokinase Unc-51-like Kinase 4.Structure. 2020; 28 (this issue): 1184-1196Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). The elegant structural dynamics and evolutionary analysis (Preuss et al., 2020Preuss F. Chatterjee D. Mathea S. Shrestha S. St-Germain J. Saha M. Kannan N. Raught B. Rottapel R. Knapp S. Nucleotide Binding, Evolutionary Insights, and Interaction Partners of the Pseudokinase Unc-51-like Kinase 4.Structure. 2020; 28 (this issue): 1184-1196Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar) also extends earlier pseudokinase studies by revealing an unexpected diversity of the ULK4 catalytic machinery present within, for example, animal and plant lineages. Similar structural informatics approaches have also been used to probe pseudokinase-specific adaptions between species, (Eyers et al., 2017Eyers P.A. Keeshan K. Kannan N. Tribbles in the 21st century: the evolving roles of tribbles pseudokinases in biology and disease.Trends Cell Biol. 2017; 27: 284-298Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), and as demonstrated for the pseudokinase MLKL (Garnish et al., 2020Garnish S.E. Yeung W. Coursier D. Birkinshaw R.W. Sandow J.J. Lehmann W.I.L. Liang L.Y. Lucet I.S. Chalmers J.D. Patrick W.M. et al.Distinct pseudokinase domain conformations underlie divergent activation mechanisms among vertebrate MLKL orthologues.Nat. Commun. 2020; 11: 3060Crossref PubMed Scopus (19) Google Scholar) and in evolutionary transitions between pseudokinase and “canonical” kinase sequences that have evolved in the “dark” pseudokinase PSKH2 (Shrestha et al., 2020Shrestha S. Byrne D.P. Harris J.A. Kannan N. Eyers P.A. Cataloguing the dead: breathing new life into pseudokinase research.FEBS J. 2020; 287: 4150-4169Crossref PubMed Scopus (14) Google Scholar). Naturally, many questions remain. For example, to what extent does the N-terminally localized ULK4 pseudokinase domain communicate with the C-terminal HEAT/Armadillo-like repeats found in abundance in ULK4 homologs (e.g., 5 in human, 12 in Arabidopsis)? Moreover, are these extended helical regions involved in microtubule binding across species, as previously demonstrated in Arabidopsis ULK4? How, and why, do “pseudosubstrates” interact with the ULK4 pseudokinase domain, and is ULK4 an allosteric regulator of canonical kinases, as has been demonstrated for several other pseudokinases? In conclusion, it is clear that the multidisciplinary analysis of pseudokinases continues to enrich the study of cell signaling. Moreover, “The Incredible Hulk” axiom that “you wouldn’t like me when I’m angry” can also be applied here in the context of the ULK4 pseudokinase; although, in most living systems, “you wouldn’t like me if I were catalytically active” appears to be a more appropriate epitaph. Thanks to James Murphy and Sam Eyers for their help with the assembly of Figure 1. Research in the author's lab is supported by funding from North West Cancer Research ( CR1097 and CR1208 ), the NIH (A data analytics framework for mining the dark kinome, U01CA239106 ) and UKRI/BBSRC ( BB/N021703/1 and BB/S018514/1 ). Nucleotide Binding, Evolutionary Insights, and Interaction Partners of the Pseudokinase Unc-51-like Kinase 4Preuss et al.StructureAugust 18, 2020In BriefA high-resolution structure of the understudied pseudokinase ULK4 binding ATPγS in an unusual manner is presented by Preuss et al. Evolutionarily, the loss of canonical motifs was compensated by the co-emerging of additional structural elements. Cellular interaction studies imply a centrosomal and tubulin-associated role of ULK4. Full-Text PDF