NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database revealed high sequence identity of the product of this gene to serinethreonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identity was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity.The short arm of human chromosome 3 harbors a number of tumor suppressor genes. Two of these genes were cloned and characterized: the von Hippel-Lindau disease gene at 3p25 (33) and the hereditary nonpolyposis colorectal cancer gene at 3p21-p22 (6, 42). At least three other tumor suppressor loci are considered to be located on 3p: 3p25, which is frequently deleted in breast cancers (8); the 3p21 region, which is involved in the origin and/or development of small cell lung cancer (SCLC) and non-SCLC (4), testicular (37), ovarian (56), and renal cell (29, 63) carcinomas; and the 3p12-p13 region, which shows loss of heterozygosity or homozygous deletions of markers in several cases of breast cancer (8), SCLC (34,44), and renal cell carcinoma (38). In the case of SCLC, heterozygous deletions of large segments of 3p are frequently observed, complicating the definition of the tumor suppressor locus. Recently homozygous deletions have been found in several SCLC-derived cell lines (13, 28), defining the putative SCLC gene locus to 3p21.3. At the same time, a 2-Mb DNA fragment from the 3p21-22 region, overlapping with the SCLC cell line NCI-H740 deletion, was shown to suppress tumorigenicity in nude mice when transfected into a mouse fibrosarcoma cell line (26). In the present study, NotI linking clones, which are markers of human genes (1), were used to identify transcribed sequences within the large NCI-H740 deletion. One of the genes identified within this region codes for a sequence which has extensive homology to the previously identified gene for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2).MAPKAP kinase 2 was originally isolated from rabbit skeletal muscle (50). In vitro studies have shown that the mitogenactivated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) isoforms are the only detectable MAPKAP kinase 2-reactivating enzymes in sk...
A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule–induced protein down-regulation through drug “off-targets” might be relevant for other inhibitors that serendipitously target pseudokinases.
Highlights d Structure of the ULK4 ATP complex reveals a unique ATP binding mode d Disease-associated mutations modulate ATP binding and ULK4 stability d Loss of canonical motifs co-occurred in evolution with a specific activation loop d BioID suggests a role of ULK4 regulating centrosomal and cytoskeletal functions
Pseudoenzymes are present within many, but not all, known enzyme families and lack one or more conserved canonical amino acids that help define their catalytically active counterparts. Recent findings in the pseudokinase field confirm that evolutionary repurposing of the structurally defined bilobal protein kinase fold permits distinct biological functions to emerge, many of which rely on conformational switching, as opposed to canonical catalysis. In this analysis, we evaluate progress in evaluating several members of the 'dark' pseudokinome that are pertinent to help drive this expanding field. Initially, we discuss how adaptions in erythropoietin-producing hepatocellular carcinoma (Eph) receptor tyrosine kinase domains resulted in two vertebrate pseudokinases, EphA10 and EphB6, in which co-evolving sequences generate new motifs that are likely to be important for both nucleotide binding and catalysis-independent signalling. Secondly, we discuss how conformationally flexible Tribbles pseudokinases, which have radiated in the complex vertebrates, control fundamental aspects of cell signalling that may be targetable with covalent small molecules. Finally, we show how species-level adaptions in the duplicated canonical kinase protein serine kinase histone (PSKH)1 sequence have led to the appearance of the pseudokinase PSKH2, whose physiological role remains mysterious. In conclusion, we show how the patterns we discover are selectively conserved within specific pseudokinases, and that when they are modelled alongside closely related canonical kinases, many are found to be located in functionally important regions of the conserved kinase fold. Interrogation of these patterns will be useful for future evaluation of these, and other, members of the unstudied human kinome.
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