Onisha Patel

Doublecortin-like kinase 1 (DCLK1) is a proposed driver of gastric cancer (GC) that phosphorylates serine and threonine residues. Here, we showed that the kinase activity of DCLK1 orchestrated cancer cell–intrinsic and–extrinsic processes that led to pro-invasive and pro-metastatic reprogramming of GC cells. Inhibition of the kinase activity of DCLK1 reduced the growth of subcutaneous xenograft tumors formed from MKN1 human gastric carcinoma cells in mice and decreased the abundance of the stromal markers α-Sma, vimentin, and collagen. Similar effects were seen in mice with xenograft tumors formed from MKN1 cells expressing a kinase-inactive DCLK1 mutant (MKN1 ^D511N ). MKN1 ^D511N cells also had reduced in vitro migratory potential and stemness compared with control cells. Mice orthotopically grafted with MKN1 cells overexpressing DCLK1 (MKN1 ^DCLK1 ) showed increased invasiveness and had a greater incidence of lung metastases compared with those grafted with control MKN1 cells. Mechanistically, we showed that the chemokine CXCL12 acted downstream of DCLK1 in cultured MKN1 cells and in mice subcutaneously implanted with gastric tumors formed by MKN1 ^DCLK1 cells. Moreover, inhibition of the kinase activity of DCLK1 or the expression of DCLK1 ^D511N reversed the pro-tumorigenic and pro-metastatic phenotype. Together, this study establishes DCLK1 as a broadly acting and potentially targetable promoter of GC.

PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate the molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding and signaling via Grb2. Together, our studies provide a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer.

PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate new molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding. Together, our studies provide a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer.

The bacterial genus Mycobacterium includes important pathogens, most notably M. tuberculosis, which infects one-quarter of the entire human population, resulting in around 1.4 million deaths from tuberculosis each year. Mycobacteria, and the closely related corynebacteria, synthesize a class of abundant glycolipids, the phosphatidyl-myo-inositol mannosides (PIMs). PIMs serve as membrane anchors for hyperglycosylated species, lipomannan (LM) and lipoarabinomannan (LAM), which are surface-exposed and modulate the host immune response. Previously, in studies using the model species Corynebacterium glutamicum, NCgl2760 was identified as a novel membrane protein that is required for the synthesis of full-length LM and LAM. Here, the first crystal structure of its ortholog in Mycobacterium smegmatis, MSMEG_0317, is reported at 1.8 Å resolution. The structure revealed an elongated β-barrel fold enclosing two distinct cavities and one α-helix extending away from the β-barrel core, resembling a `cone with a flake' arrangement. Through xenon derivatization and structural comparison with AlphaFold2-derived predictions of the M. tuberculosis homolog Rv0227c, structural elements were identified that may undergo conformational changes to switch from `closed' to `open' conformations, allowing cavity access. An AlphaFold2-derived NCgl2760 model predicted a smaller β-barrel core with an enclosed central cavity, suggesting that all three proteins, which were collectively termed LmcA, may have a common mechanism of ligand binding through these cavities. These findings provide new structural insights into the biosynthetic pathway for a family of surface lipoglycans with important roles in mycobacterial pathogenesis.

The pseudokinase scaffolds PEAK1 and PEAK2 are implicated in cancer cell migration and metastasis. We characterized the regulation and role of the third family member PEAK3 in cell signaling. Similar to PEAK1 and PEAK2, PEAK3 formed both homotypic and heterotypic complexes. In addition, like PEAK1, it bound to the adaptors Grb2 and CrkII. However, unlike PEAK1 and PEAK2, homodimerized PEAK3 also interacted with the ARF GTPase-activating protein ASAP1, the E3 ubiquitin ligase Cbl, and the kinase PYK2. Dimerization and subsequent phosphorylation on Tyr ²⁴ , likely by a Src family kinase, were required for the binding of PEAK3 to Grb2 and ASAP1. Interactions with Grb2, CrkII, ASAP1, Cbl, and PYK2 exhibited contrasting dynamics upon cell stimulation with epidermal growth factor (EGF), in part due to PEAK3 dephosphorylation mediated by the phosphatase PTPN12. Overexpressing PEAK3 in mesenchymal-like MDA-MB-231 breast cancer cells enhanced cell elongation in a manner dependent on PEAK3 dimerization, and manipulation of PEAK3 expression demonstrated a positive role for this scaffold in regulating cell migration. Overexpressing PEAK3 in PEAK1/2 double-knockout MCF-10A breast epithelial cells enhanced acinar growth, impaired basement membrane integrity, and promoted invasion in three-dimensional cultures, with the latter two effects dependent on the binding of PEAK3 to Grb2 and ASAP1. PEAK1 and PEAK2 quantitatively and temporally influenced PEAK3 function. These findings characterize PEAK3 as an integral, signal-diversifying member of the PEAK family with scaffolding roles that promote cell proliferation, migration, and invasion.

Doublecortin-like kinase 1 (DCLK1) is an understudied bi-functional kinase with a proven role in tumour growth and development. However, the presence of tissue-specific spliced DCLK1 isoforms with distinct biological functions have challenged the development of effective strategies to understand the role of DCLK1 in oncogenesis. Recently, DCLK1-IN-1 was reported as a highly selective DCLK1 inhibitor, a powerful tool to dissect DCLK1 biological functions. Here, we report the crystal structures of DCLK1 kinase domain in complex with DCLK1-IN-1 and its precursors. Combined, our data rationalises the structure-activity relationship that informed the development of DCLK1-IN-1 and provides the basis for the high selectivity of DCLK1-IN-1, with DCLK1-IN-1 inducing a drastic conformational change of the ATP binding site. We demonstrate that DCLK1-IN-1 binds DCLK1 long isoforms but does not prevent DCLK1’s Microtubule-Associated Protein (MAP) function. Together, our work provides an invaluable structural platform to further the design of isoform-specific DCLK1 modulators for therapeutic intervention.

EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell–cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic activities are thought to initiate an intracellular signalling cascade, EphB6 and EphA10 are classified as catalytically dead, raising the question of how non-catalytic functions contribute to Eph receptor signalling homeostasis. In this study, we have characterised the biochemical properties and topology of the EphB6 and EphA10 intracellular regions comprising the juxtamembrane (JM) region, pseudokinase and SAM domains. Using small-angle X-ray scattering and cross-linking-mass spectrometry, we observed high flexibility within their intracellular regions in solution and a propensity for interaction between the component domains. We identified tyrosine residues in the JM region of EphB6 as EphB4 substrates, which can bind the SH2 domains of signalling effectors, including Abl, Src and Vav3, consistent with cellular roles in recruiting these proteins for downstream signalling. Furthermore, our finding that EphB6 and EphA10 can bind ATP and ATP-competitive small molecules raises the prospect that these pseudokinase domains could be pharmacologically targeted to counter oncogenic signalling.

The study of pseudokinases has uncovered that catalysis‐independent functions play a critical role in cell signalling regulation. However, how pseudokinases dynamically assemble and regulate oncogenic signalling pathways remains, in most cases, unclear due to a limited knowledge of the structural determinants that are critical for their functions. Here, we review the recent progress made to unravel the role of the PEAK family of pseudokinases, which comprises SgK269, SgK223 and the recently identified PEAK3, in assembling specific oncogenic signalling pathways that contribute to the progression of several aggressive cancers. We focus on recent structural advances revealing that SgK269 and SgK223 can homo‐ and heteroassociate via a unique dimerisation domain, comprising conserved regulatory helices directly surrounding the pseudokinase domain, which is also conserved in PEAK3. We also highlight a potential oligomerisation mechanism driven by the pseudokinase domain. While it is likely that homo‐ or heterodimerisation and oligomerisation mechanisms contribute to the assembly of complex signalling hubs and provide a means to spatially and temporally modulate and diversify signalling outputs, the exact role that these oncogenic scaffolds play in regulating cell migration, invasion and morphology remains unclear. Here, we attempt to link their structural characteristics to their cellular functions by providing a thorough analysis of the signalling transduction pathways they are known to modulate.

The mammalian pseudokinase SgK223, and its structurally related homologue SgK269, are oncogenic scaffolds that nucleate the assembly of specific signalling complexes and regulate tyrosine kinase signalling. Both SgK223 and SgK269 form homo- and hetero-oligomers, a mechanism that underpins a diversity of signalling outputs. However, mechanistic insights into SgK223 and SgK269 homo- and heterotypic association are lacking. Here we present the crystal structure of SgK223 pseudokinase domain and its adjacent N- and C-terminal helices. The structure reveals how the N- and C-regulatory helices engage in a novel fold to mediate the assembly of a high-affinity dimer. In addition, we identified regulatory interfaces on the pseudokinase domain required for the self-assembly of large open-ended oligomers. This study highlights the diversity in how the kinase fold mediates non-catalytic functions and provides mechanistic insights into how the assembly of these two oncogenic scaffolds is achieved in order to regulate signalling output.