Putative link between Polo-like kinases (PLKs) and Toll-like receptor (TLR) signaling in transformed and primary human immune cells.

Toll-like receptors (TLRs) are important sentinels of bacterial and viral infection and thus fulfil a critical sensory role in innate immunity. Polo-like kinases (PLKs), a five membered family of Ser/Thr protein kinases, have long been studied for their role in mitosis and thus represent attractive therapeutic targets in cancer therapy. Recently, PLKs were implicated in TLR signaling in mice but the role of PLKs in TLR signaling in untransformed primary immune cells has not been addressed, even though PLK inhibitors are in clinical trials. We here identified several phospho-serine and phospho-threonine residues in the known TLR pathway kinases, Interleukin-1 receptor-associated kinase (IRAK) 2 and IRAK4. These sites lie in canonical polo-box motifs (PBM), sequence motifs known to direct recruitment of PLKs to client proteins. Interestingly, PLK1 was phosphorylated and PLK 2 and 3 mRNA induced upon TLR stimulation in primary immune cells, respectively. In whole blood, PLK inhibition disparately affected TLR mediated cytokine responses in a donor- and inhibitor-dependent fashion. Collectively, PLKs may thus potentially interface with TLR signaling in humans. We propose that temporary PLK inhibitor-mediated blockade of TLR-signaling in certain patients receiving such inhibitors during cancer treatment may cause adverse effects such as an increased risk of infections due to a then compromised ability of the TLR recognition system to sense and initiate cytokine responses to invading microbes.

The N-terminal loop of IRAK-4 death domain regulates ordered assembly of the Myddosome signalling scaffold.

Activation of Toll-like receptors induces dimerization and the recruitment of the death domain (DD) adaptor protein MyD88 into an oligomeric post receptor complex termed the Myddosome. The Myddosome is a hub for inflammatory and oncogenic signaling and has a hierarchical arrangement with 6-8 MyD88 molecules assembling with exactly 4 of IRAK-4 and 4 of IRAK-2. Here we show that a conserved motif in IRAK-4 (Ser8-X-X-X-Arg12) is autophosphorylated and that the phosphorylated DD is unable to form Myddosomes. Furthermore a mutant DD with the phospho-mimetic residue Asp at this position is impaired in both signalling and Myddosome assembly. IRAK-4 Arg12 is also essential for Myddosome assembly and signalling and we propose that phosphorylated Ser8 induces the N-terminal loop to fold into an α-helix. This conformer is stabilised by an electrostatic interaction between phospho-Ser8 and Arg12 and would destabilise a critical interface between IRAK-4 and MyD88. Interestingly IRAK-2 does not conserve this motif and has an alternative interface in the Myddosome that requires Arg67, a residue conserved in paralogues, IRAK-1 and 3(M).

Two human MYD88 variants, S34Y and R98C, interfere with MyD88-IRAK4-myddosome assembly.

Innate immune receptors detect microbial pathogens and subsequently activate adaptive immune responses to combat pathogen invasion. MyD88 is a key adaptor molecule in both Toll-like receptor (TLR) and IL-1 receptor superfamily signaling pathways. This is illustrated by the fact that human individuals carrying rare, naturally occurring MYD88 point mutations suffer from reoccurring life-threatening infections. Here we analyzed the functional properties of six reported non-synonymous single nucleotide polymorphisms of MYD88 in an in vitro cellular system. Two variants found in the MyD88 death domain, S34Y and R98C, showed severely reduced NF-κB activation due to reduced homo-oligomerization and IRAK4 interaction. Structural modeling highlights Ser-34 and Arg-98 as residues important for the assembly of the Myddosome, a death domain (DD) post-receptor complex involving the DD of MyD88, IRAK4, and IRAK2 or IRAK1. Using S34Y and R98C as functional probes, our data show that MyD88 homo-oligomerization and IRAK4 interaction is modulated by the MyD88 TIR and IRAK4 kinase domain, demonstrating the functional importance of non-DD regions not observed in a recent Myddosome crystal structure. The differential interference of S34Y and R98C with some (IL-1 receptor, TLR2, TLR4, TLR5, and TLR7) but not all (TLR9) MyD88-dependent signaling pathways also suggests that receptor specificities exist at the level of the Myddosome. Given their detrimental effect on signaling, it is not surprising that our epidemiological analysis in several case-control studies confirms that S34Y and R98C are rare variants that may drastically contribute to susceptibility to infection in only few individuals.

An oligomeric signaling platform formed by the Toll-like receptor signal transducers MyD88 and IRAK-4.

Toll-like receptors (TLRs) mediate responses to pathogen-associated molecules as part of the vertebrate innate immune response to infection. Receptor dimerization is coupled to downstream signal transduction by the recruitment of a post-receptor complex containing the adaptor protein MyD88 and the IRAK protein kinases. In this work, we show that the death domains of human MyD88 and IRAK-4 assemble into closed complexes having unusual stoichiometries of 7:4 and 8:4, the Myddosome. Formation of the Myddosome is likely to be a key event for TLR4 signaling in vivo as we show here that pathway activation requires that the receptors cluster into lipid rafts. Taken together, these findings indicate that TLR activation causes the formation of a highly oligomeric signaling platform analogous to the death-inducing signaling complex of the Fas receptor pathway.

LEA (late embryonic abundant)-like protein Hsp 12 (heat-shock protein 12) is present in the cell wall and enhances the barotolerance of the yeast Saccharomyces cerevisiae.

Yeast cells Saccharomyces cerevisiae, late embryogenic abundant-like stress response protein Hsp 12 (heat-shock protein 12) were found by immunocytochemistry to be located both in the cytoplasm and in the cell wall, from where they could be extracted with dilute NaOH solutions. Yeast cells with the Hsp 12 gene disrupted were unable to grow in the presence of either 12 mM caffeine or 0.43 mM Congo Red, molecules known to affect cell-wall integrity. The volume of yeast cells were less affected by rapid changes in the osmolality of the growth medium when compared with the wild-type yeast cells, suggesting a role for Hsp 12 in the flexibility of the cell wall. This was also suggested by subjecting the yeast cells to rapid changes in barometric pressure where it was found that wild-type yeast cells were more resistant to cellular breakage.

Significant quantities of the glycolytic enzyme phosphoglycerate mutase are present in the cell wall of yeast Saccharomyces cerevisiae.

NaOH was used to extract proteins from the cell walls of the yeast Saccharomyces cerevisiae. This treatment was shown not to disrupt yeast cells, as NaOH-extracted cells displayed a normal morphology upon electron microscopy. Moreover, extracted and untreated cells had qualitatively similar protein contents upon disruption. When yeast was grown in the presence of 1 M mannitol, two proteins were found to be present at an elevated concentration in the cell wall. These were found to be the late-embryogenic-abundant-like protein heat-shock protein 12 and the glycolytic enzyme phosphoglycerate mutase. The presence of phosphoglycerate mutase in the cell wall was confirmed by immunocytochemical analysis. Not only was the phosphoglycerate mutase in the yeast cell wall found to be active, but whole yeast cells were also able to convert 3-phosphoglycerate in the medium into ethanol, provided that the necessary cofactors were present.