Andrew F. Wilks

Two novel protein-tyrosine kinases, each with a second phosphotransferase-related catalytic domain, define a new class of protein kinase.

The protein-tyrosine kinases (PTKs) are a burgeoning family of proteins, each of which bears a conserved domain of 250 to 300 amino acids capable of phosphorylating substrate proteins on tyrosine residues. We recently exploited the existence of two highly conserved sequence elements within the catalytic domain to generate PTK-specific degenerate oligonucleotide primers (A. F. Wilks, Proc. Natl. Acad. Sci. USA 86:1603-1607, 1989). By application of the polymerase chain reaction, portions of the catalytic domains of several novel PTKs were amplified. We describe here the primary sequence of one of these new PTKs, JAK1 (from Janus kinase), a member of a new class of PTK characterized by the presence of a second phosphotransferase-related domain immediately N terminal to the PTK domain. The second phosphotransferase domain bears all the hallmarks of a protein kinase, although its structure differs significantly from that of the PTK and threonine/serine kinase family members. A second member of this family (JAK2) has been partially characterized and exhibits a similar array of kinase-related domains. JAK1 is a large, widely expressed membrane-associated phosphoprotein of approximately 130,000 Da. The PTK activity of JAK1 has been located in the C-terminal PTK-like domain. The role of the second kinaselike domain is unknown.

Kinase-negative mutants of JAK1 can sustain interferon-gamma-inducible gene expression but not an antiviral state.

The receptor‐associated protein tyrosine kinases JAK1 and JAK2 are both required for the interferon (IFN)‐gamma response. The effects of expressing kinase‐negative JAK mutant proteins on signal transduction in response to IFN‐gamma in wild‐type cells and in mutant cells lacking either JAK1 or JAK2 have been analysed. In cells lacking endogenous JAK1 the expression of a transfected kinase‐negative JAK1 can sustain substantial IFN‐gamma‐inducible gene expression, consistent with a structural as well as an enzymic role for JAK1. Kinase‐negative JAK2, expressed in cells lacking endogenous JAK2, cannot sustain IFN‐gamma‐inducible gene expression, despite low level activation of STAT1 DNA binding activity. When expressed in wild‐type cells, kinase‐negative JAK2 acts as a dominant‐negative inhibitor of the IFN‐gamma response. Further analysis of the JAK/STAT pathway suggests a model for the IFN‐gamma response in which the initial phosphorylation of JAK1 and JAK2 is mediated by JAK2, whereas phosphorylation of the IFN‐gamma receptor is normally carried out by JAK1. The efficient phosphorylation of STAT 1 in the receptor‐JAK complex may again depend on JAK2. Interestingly, a JAK1‐dependent signal, in addition to STAT1 activation, appears to be required for the expression of the antiviral state.

Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death

Significance The four-helix bundle (4HB) domain of Mixed Lineage Kinase Domain-Like (MLKL) bears two clusters of residues that are required for cell death by necroptosis. Mutations within a cluster centered on the α4 helix of the 4HB domain of MLKL prevented its membrane translocation, oligomerization, and ability to induce necroptosis. This cluster is composed principally of acidic residues and therefore challenges the idea that the 4HB domain engages negatively charged phospholipid membranes via a conventional positively charged interaction surface. The importance of membrane translocation to MLKL-mediated death is supported by our identification of a small molecule that binds the MLKL pseudokinase domain and retards membrane translocation to inhibit necroptotic signaling. Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3–mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.

Ryk-deficient mice exhibit craniofacial defects associated with perturbed Eph receptor crosstalk

Secondary palate formation is a complex process that is frequently disturbed in mammals, resulting in the birth defect cleft palate. Gene targeting has identified components of cytokine/growth factor signalling systems such as Tgf-α/Egfr, Eph receptors B2 and B3 (Ephb2 and Ephb3, respectively), Tgf-β2, Tgf-β3 and activin-βA (ref. 3) as regulators of secondary palate development. Here we demonstrate that the mouse orphan receptor ‘related to tyrosine kinases’ (Ryk) is essential for normal development and morphogenesis of craniofacial structures including the secondary palate. Ryk belongs to a subclass of catalytically inactive, but otherwise distantly related, receptor protein tyrosine kinases (RTKs). Mice homozygous for a null allele of Ryk have a distinctive craniofacial appearance, shortened limbs and postnatal mortality due to feeding and respiratory complications associated with a complete cleft of the secondary palate. Consistent with cleft palate phenocopy in Ephb2/Ephb3-deficient mice and the role of a Drosophila melanogaster Ryk orthologue, Derailed, in the transduction of repulsive axon pathfinding cues, our biochemical data implicate Ryk in signalling mediated by Eph receptors and the cell-junction–associated Af-6 (also known as Afadin). Our findings highlight the importance of signal crosstalk between members of different RTK subfamilies.

JAK protein tyrosine kinases: their role in cytokine signalling

Protein tyrosine kinases (PTKs) are integral components of the cellular machinery that mediates the transduction and/or processing of many extra- and intracellular signals. Members of the JAK family of intracellular PTKs (JAK1, JAK2 and TYK2) are characterized by the possession of a PTK-related domain and five additional homology domains, in addition to a classical PTK domain. An important breakthrough in the understanding of JAK kinases function(s) has come from the recent observations that many cytokine receptors compensate for their lack of a PTK domain by utilizing members of the JAK family for signal transduction.

Zebrafish stat3 is expressed in restricted tissues during embryogenesis and stat1 rescues cytokine signaling in a STAT1-deficient human cell line.

Transcription factors of the STAT family are required for cellular responses to multiple signaling molecules. After ligand binding‐induced activation of cognate receptors, STAT proteins are phosphorylated, hetero‐ or homodimerize, and translocate to the nucleus. Subsequent STAT binding to specific DNA elements in the promoters of signal‐responsive genes alters the transcriptional activity of these loci. STAT function has been implicated in the transduction of signals for growth, reproduction, viral defense, and immune regulation. We have isolated and characterized two STAT homologs from the zebrafish Danio rerio. The stat3 gene is expressed in a tissue‐restricted manner during embryogenesis, and larval development with highest levels of transcript are detected in the anterior hypoblast, eyes, cranial sensory ganglia, gut, pharyngeal arches, cranial motor nuclei, and lateral line system. In contrast, the stat1 gene is not expressed during early development. The stat3 gene maps to a chromosomal position syntenic with the mouse and human STAT3 homologs, whereas the stat1 gene does not. Despite a higher rate of evolutionary change in stat1 relative to stat3, the stat1 protein rescues interferon‐signaling functions in a STAT1‐deficient human cell line, indicating that cytokine‐signaling mechanisms are likely to be conserved between fish and tetrapods. Dev Dyn 1999;215:352–370.

The application of the polymerase chain reaction to cloning members of the protein tyrosine kinase family

Degenerate oligodeoxyribonucleotide (oligo) primers derived from amino acid (aa) sequence motifs held in common between all members of the protein tyrosine kinase (PTK) family were used to prime the amplification of PTK-related sequences from a variety of murine cDNA sources, including the haemopoietic cell lines, FDC-P1 and WEHI-3B D+, peritoneal macrophages and whole brain. Several parameters, such as the length (short, i.e., less than 20 nucleotides (nt) vs. long, i.e., greater than 30 nt) and degeneracy (i.e., moderately degenerate vs. highly degenerate) of the oligo primers and the temperature of the extension phase of the reaction, were examined. The data from these analyses suggest that the most effective type of primer in this application of the polymerase chain reaction is a short, moderately degenerate oligo such as that which might be derived from the small patches of aa sequence homology that are frequently found to be held in common among members of protein families. In addition to a number of previously described PTK sequences, a novel mammalian PTK-related sequence was uncovered.

A CSF-1 receptor kinase inhibitor targets effector functions and inhibits pro-inflammatory cytokine production from murine macrophage populations

CSF‐1 regulates macrophage differentiation, survival, and function, and is an attractive therapeutic target for chronic inflammation and malignant diseases. Here we describe the effects of a potent and selective inhibitor of CSF‐1R—CYC10268—on CSF‐1R‐dependent signaling. In in vitro kinase assays, CYC10268 was active in the low nanomolar range and showed selectivity over other kinases such as Abl and Kit. CYC10268 blocked survival mediated by CSF‐1R in primary murine bone marrow‐derived macrophages (BMM) and in the factor‐dependent cell line Ba/F3, in which the CSF‐1R was ectopically expressed. CYC10268 also inhibited CSF‐1 regulated signaling (Akt, ERK‐1/2), gene expression (urokinase plasminogen activator, toll‐like receptor 9, and apolipoprotein E), and priming of LPS‐inducible cytokine production in BMM. In thioglycollate‐elicited peritoneal macrophages (TEPM), which survive in the absence of exogenous CSF‐1, CYC10268 impaired LPS‐induced cytokine production and regulated expression of known CSF‐1 target genes. These observations support the conclusion that TEPM are CSF‐1 autocrine and that CSF‐1 plays a central role in macrophage effector functions during inflammation. CSF‐1R inhibitors such as CYC10268 provide a powerful tool to dissect the role of the CSF‐1/CSF‐1R signaling system in a range of biological systems and have potential for a number of therapeutic applications.—Irvine, K. M., Burns, C. J., Wilks, A. F., Su, S., Hume, D. A., Sweet, M. J. A CSF‐1 receptor kinase inhibitor targets effector functions and inhibits pro‐inflammatory cytokine production from murine macrophage populations. FASEB J. 20, 1315–1326 (2006)

Placenta Growth Factor and Vascular Endothelial Growth Factor are Co-Expressed During Early Embryonic Development

We have used the polymerase chain reaction to identify mouse proteins similar in primary structure to the endothelial cell mitogen Vascular Endothelial Growth Factor (VEGF). One amplified product encoded mouse Placenta Growth Factor (PIGF). The pattern of PIGF gene expression in mouse embryos was studied by in situ hybridization. Transcripts encoding mouse PIGF were abundant in trophoblastic giant cells associated with the parietal yolk sac at early stages of embryogenesis. VEGF transcripts were also detected in trophoblastic giant cells raising the possibility that these cells may secrete heterodimers consisting of one PIGF subunit and one VEGF subunit. The secretion of PIGF and VEGF by trophoblastic giant cells is likely to be the signal which initiates and co-ordinates vascularization in the deciduum and placenta during early embryogenesis.

Colony-stimulating factor-1 (CSF-1) delivers a proatherogenic signal to human macrophages

M‐CSF/CSF‐1 supports the proliferation and differentiation of monocytes and macrophages. In mice, CSF‐1 also promotes proinflammatory responses in vivo by regulating mature macrophage functions, but little is known about the acute effects of this growth factor on mature human macrophages. Here, we show that in contrast to its effects on mouse bone marrow‐derived macrophages, CSF‐1 did not induce expression of urokinase plasminogen activator mRNA, repress expression of apolipoprotein E mRNA, or prime LPS‐induced TNF and IL‐6 secretion in human monocyte‐derived macrophages (HMDM) from several independent donors. Instead, we show by expression profiling that CSF‐1 modulates the HMDM transcriptome to favor a proatherogenic environment. CSF‐1 induced expression of the proatherogenic chemokines CXCL10/IFN‐inducible protein 10, CCL2, and CCL7 but repressed expression of the antiatherogenic chemokine receptor CXCR4. CSF‐1 also up‐regulated genes encoding enzymes of the cholesterol biosynthetic pathway (HMGCR, MVD, IDI1, FDPS, SQLE, CYP51A1, EBP, NSDHL, DHCR7, and DHCR24), and expression of ABCG1, encoding a cholesterol efflux transporter, was repressed. Consistent with these effects, CSF‐1 increased levels of free cholesterol in HMDM, and the selective CSF‐1R kinase inhibitor GW2580 ablated this response. These data demonstrate that CSF‐1 represents a further link between inflammation and cardiovascular disease and suggest two distinct mechanisms by which CSF‐1, which is known to be present in atherosclerotic lesions, may contribute to plaque progression.