PIM-Related Kinases Selectively Regulate Olfactory Sensations in Caenorhabditis elegans

Mammalian PIM Kinases and C. elegans PRKs are true orthologs

The C. elegans prk-1 and prk-2 genes that encode PRKs had previously been mapped to chromosome III and sequenced (Ronald Plasterk, The Netherlands; wormbase.org). An amino acid sequence alignment between the two C. elegans PRKs and the three human or mouse PIM kinases revealed that they are well conserved with ∼40% amino acid similarity (Fig. 1A). Expectedly, the highest identities were observed within their kinase domains, especially at the ATP-binding site and the catalytically active site. By contrast, the C-terminal tails of C. elegans PRKs are much longer than those of their mammalian orthologs, and are not conserved even between PRK-1 and PRK-2.

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Figure 1.

Mammalian PIM kinases and C. elegans PRKs are true orthologs. A, Amino acid sequences of C. elegans PRK-1 (NM_001276777.1) and PRK-2 (CAA84323.2) were aligned with Homo sapiens PIM-1 (NP_001230115.1), PIM-2 (NP_006866.2), and PIM-3 (NP_001001852.2), and Mus musculus PIM-1 (NP_032868.2), PIM-2 (NP_613072.1), and PIM-3 (NP_663453.1), using the Clustal X 2.0 software (Thompson et al., 1997). Identical or similar amino acids present in all or only in some of them are marked with black or gray backgrounds, respectively. Positions of the conserved ATP-binding and catalytically active sites are highlighted with red and blue lines, respectively. B, The amino acid sequences of the C. elegans (PRK-1 and PRK-2), Danio rerio (PIM-1, PIM-2, and PIM-3), Xenopus laevis (PIM-1 and PIM-3), Mus musculus (PIM-1, PIM-2, and PIM-3) and Homo sapiens (PIM-1, PIM-2, and PIM-3) were used to draw a phylogenetic tree of PIM orthologs and paralogs in distinct species (http://www.phylogeny.fr). The scale of 0.4 refers to 40% difference between sequences.

A phylogenetic tree (Fig. 1B) shows the evolutionary distance between PIM orthologs and paralogs in several species, including also the zebrafish Danio rerio and the African clawed frog Xenopus laevis. In mammalian species and in frogs, there is more homology between each PIM ortholog than between the distinct family members, PIM-1, PIM-2, and PIM-3. By contrast, the C. elegans and D. rerio orthologs are all significantly separated from the other species, with C. elegans proteins however being more homologous to mammalian ones than those from D. rerio. Interestingly and maybe also surprisingly, no obvious PIM orthologs have been found in the fruit fly Drosophila melanogaster (Manning, 2005; Peter Gallant, University of Würzburg, Germany, personal communication).

To demonstrate that the C. elegans PRKs are functional orthologs for mammalian PIM kinases, we took human PIM-1 and Ce PRK-2 proteins as representatives of each family and analyzed for their activities by radioactive in vitro kinase assays. As expected and shown in Figure 2A, both PIM-1 and PRK-2 were able to autophosphorylate themselves. More interestingly, also PRK-2 was able to phosphorylate known PIM substrates, such as NFATc1, suggesting that PIMs and PRKs are true orthologs sharing similar preferences for their target sites.

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Figure 2.

PIM and PRK kinases function similarly in vitro. A, Radioactive kinase assays were performed to analyze the in vitro ability of human PIM-1 and C. elegans PRK-2 to phosphorylate themselves or the PIM substrate NFATC1 (amino acids 1–418) in the absence (−) or presence (+) of 10 μm DHPCC-9. The intensities of phosphorylated proteins are shown at the top and the total amounts of proteins at the bottom. Shown are also the relative levels of phosphorylation of kinases and their substrates in control versus drug-treated samples in this dataset representing three independent experiments. B, PRK-1 autophosphorylation was analyzed in the absence (−) or presence (+) of 10 μm DHPCC-9, AZD-1208, or SGI-1776. The phosphorylated protein was visualized by immunoblotting with the phospho-RXXS*/T* antibody, which has several potential target sites in PRK-1.

Part of the phosphorylation samples had been pretreated with the pyrrolocarbazole carbaldehyde compound DHPCC-9, which has been shown to selectively inhibit catalytic activities of all three mammalian PIM kinases in vitro (Akué-Gédu et al., 2009), in cell-based assays (Santio et al., 2010) and under in vivo conditions in mice (Santio et al., 2015) or chicken eggs (Santio et al., 2016). At the 10 µm concentration used, DHPCC-9 was able to inhibit autophosphorylation of not only PIM-1, but also PRK-2 (Fig. 2A), indicating that the ATP-binding pockets of PIMs and PRKs are conserved enough to allow binding of the ATP-competitive drug. In addition, the abilities of both PIM-1 and PRK-2 to phosphorylate NFATC1 were compromised by DHPCC-9.

To determine whether the kinase activity of also PRK-1 can be reduced by DHPCC-9 and whether two other structurally unrelated PIM inhibitors, the imidazopyridazine SGI-1776 (Mumenthaler et al., 2009) and the thiazolidinedione AZD-1208 (Keeton et al., 2014) have similar effects in C. elegans, additional non-radioactive in vitro kinase assays were performed. There we analyzed the autophosphorylation activity of PRK-1 by immunoblotting with the phospho-RXXS*/T*-specific antibody, for which four potential target sites were identified from the PRK-1 amino acid sequence. Although PRK-1 was prominently phosphorylated in the presence of DMSO, its autophosphorylation was significantly reduced by 10 µm pretreatment with either DHPCC-9, AZD-1208 or SGI-1776 (Fig. 2C). Thus, although all PIM inhibitors reduced activities of PRKs less efficiently than that of PIM-1, their effects were strong enough to prompt us to use them as tools to investigate the physiological functions of PRKs in C. elegans.

PIM inhibitors specifically suppress attractive olfactory responses via AWC^ON neurons

We previously reported that mammalian PIM kinases are expressed in sensory organs, such as the olfactory epithelium (Eichmann et al., 2000). Because PRK-1, and possibly also PRK-2, are expressed in C. elegans head neurons, we wanted to investigate whether PRKs are involved in regulation of olfaction. Before carrying out behavioral assays, we experimentally determined that the maximal tolerated dosage of the PIM inhibitor DHPCC-9 in C. elegans is ∼ 200 µm. Then we synchronized wild-type animals, exposed populations of Day 1 adults to 10-200 µm concentrations of DHPCC-9 for 120 min, and conducted chemotaxis assays to AWC-sensed attractive odorants for another 120 min. At tested dilutions of odorants, control animals vigorously responded to butanone, benzaldehyde and isoamyl alcohol (IAA) with chemotaxis indices (CIs) of ∼ 0.9, whereas 2,3-pentanedione was less attractive with CI of 0.6 (Fig. 3A). More interestingly, animals pre-exposed to increasing concentrations of DHPCC-9 consistently displayed a significant dose-dependent decrease in their chemotaxis to butanone. The inhibitory effect of DHPCC-9 on the chemotactic behavior became evident already at the lowest 10 µm concentration. However, it was most prominent at the maximal dose of 200 µm, where animals were moving randomly to all directions, as if they did not sense the presence of the odorant. There was a statistically significant reduction (∼20%) in chemotaxis to benzaldehyde in DHPCC-9-treated animals compared with controls, whereas hardly any effects were observed by DHPCC-9 on chemotaxis to IAA or 2,3-pentanedione. It is known that butanone is sensed by AWC^ON neurons, 2,3-pentanedione by AWC^OFF neurons and benzaldehyde and IAA by both (Troemel et al., 1999). Thus, our data suggest that inhibition of PRK activity by DHPCC-9 specifically and asymmetrically suppresses olfactory sensing via AWC^ON neurons.

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Figure 3.

PIM inhibitors specifically suppress olfactory sensing via AWC^ON neurons. Synchronized young adult animals were exposed to indicated concentrations of PIM inhibitors DHPCC-9 (A, B), AZD-1208 (C, D), or SGI-1776 (E, F) for 120 min. Chemotaxis assays with odorants were performed for another 120 min. Chemical structures of the PIM inhibitors are shown in between the dose-dependent chemotactic indices of animals in response to AWC odorants (A, C, E) butanone (1:1000), benzaldehyde (1:200), IAA (1:100), and 2,3-pentanedione (1:10000), or AWA odorants (B, D, F) diacetyl (1:1000), pyrazine (10 mg/ml), and TMT (1:1000). Each data point represents the mean of at least six independent experiments with 150–200 animals per assay. Error bars indicate SEM and asterisks statistically significant differences (p < 0.01) between control and drug-exposed animals.

To examine whether DHPCC-9 affects the olfactory functions of also AWA neurons, we performed chemotaxis assays with AWA-sensed attractive odorants pyrazine, diacetyl and 2,4,5-trimethylthiazole (TMT). No significant suppression was observed for pyrazine or diacetyl by DHPCC-9, but the response to TMT, which can be sensed also by AWC neurons (Bargmann et al., 1993), was slightly reduced (∼15%) compared with controls (Fig. 3B).

To confirm that the observed effects of DHPCC-9 were dependent on its ability to inhibit endogenous PRK activity and were not just because of some unspecific off-target effects, we repeated the chemotaxis analyses by using the two other tested PIM inhibitors, SGI-1776 and AZD-1208. AZD-1208 efficiently targets all three PIM family members (Keeton et al., 2014) and is more selective than SGI-1776, which in addition to PIM-1 and PIM-3 also inhibits FLT-3 and haspin (Mumenthaler et al., 2009). These two inhibitors appeared to be more toxic than DHPCC-9, so animals were exposed to maximally 100 µm concentrations of them. When animals pre-exposed to either AZD-1208 or SGI-1776 were assayed for their chemotactic responses to attractive odorants sensed by either AWA or AWC neurons, very similar dose-dependent data were obtained as with DHPCC-9 (Fig. 3C–F). Again, chemotaxis to the AWC^ON-sensed butanone was significantly suppressed by both AZD-1208 and SGI-1776. Chemotaxis to benzaldehyde and TMT was also reduced (∼20%) compared with controls, whereas no significant effects were observed in animals treated with AWC^OFF-sensed 2,3-pentanedione or AWA-sensed pyrazine and diacetyl. Overall, our data suggest that the observed suppressive effects of all the drugs on olfactory sensations via AWC^ON neurons are because of their shared ability to inhibit activities of the PRKs.

Inhibition of butanone sensing is reversible and dependent on odorant dosage

To determine whether PRK inhibition resulted in permanent or only temporary changes in neuronal activity, animals were exposed for 120 min to 200 µm DHPCC-9 or 100 µm SGI-1776, and then allowed to recover for up to 180 min before assaying their chemotaxis to butanone. As shown in Figure 4, A and B, drug-exposed animals quickly regained their ability to sense butanone, with complete recovery within 120 or 180 min after exposure to DHPCC-9 or SGI-1776, respectively. These results indicate that the suppressive effects of the PIM inhibitors on olfactory sensations are reversible. Furthermore, the rapid inhibitory responses as well as recovery times suggest that the PIM inhibitors have direct, although as yet uncharacterized effects on olfactory signaling in the AWC^ON neurons.

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Figure 4.

Inhibition of butanone sensing is reversible and dependent on odorant dosage. Animals exposed for 120 min to 200 μm DHPCC-9 (A, C) or 100 μm SGI-1776 (B, D) were assayed for chemotaxis to butanone (1:1000) after indicated intervals of recovery times (A, B), or to indicated dilutions of butanone (C, D). Each data point represents the means and SEM of four independent experiments with 150–200 animals per assay.

To investigate the effects of the PIM inhibitors on butanone sensing in more detail, drug-exposed animals and their controls were assayed for their chemotaxis to a wide range of butanone dilutions. As shown in Figure 4, C and D, high concentrations of butanone were able to overcome the inhibitory effects of both DHPCC-9 and SGI-1776, whereas at lower concentrations, the chemotactic behavior of the animals was lost even without inhibition. Thus, these results confirmed that the 1:1000 dilution used in our other assays had been optimal to reveal the suppressive effects of the PIM inhibitors on butanone sensing.

PRK-1 is more important than PRK-2 for olfactory responses via AWC neurons

To evaluate the contribution of individual PRK family members to the olfactory responses, we acquired mutant strains for both of them. The prk-1(pk86) strain contains a Tc1 transposon inserted in the coding region of the prk-1 gene. Based on our sequencing data, this insertion had resulted in a complex rearrangement of nucleotides from exon 2 to exon 3, replacing Lys123 with a stop codon. Thus, the mutated gene is expected to produce a truncated, catalytically inactive protein. In the prk-2(ok3069) strain in turn, there is a 617 bp deletion from the first intron to the third exon of the prk-2 gene (Zheng et al., 2011), which is expected to destroy the catalytic activity of the protein. The prk-1(pk86) and prk-2(ok3069) mutant animals are viable, but we noticed that their populations propagate abnormally slow. To analyze the effects of the mutations on the reproductive capacity in more detail, brood size assays were performed. When we measured the mean amounts of eggs laid by synchronized gravid adult individuals, the average brood size was reduced in both mutant strains, but more remarkably in the prk-1(pk86) strain (Fig. 5A).

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Figure 5.

PRK-1 expression and activity is required for butanone sensing. A, Reproductive capacities of wild-type (WT; n = 13), prk-1(pk86) (n = 5), and prk-2(ok3069) (n = 9) animals, as determined by the amounts of eggs laid by each parent. Shown in the box plots are mean (cross), median, quartiles (boxes), and range (whiskers). The asterisks indicate statistically significant differences (p < 0.01) between wild-type and mutant animals. B, Wild-type and mutant animals were tested for chemotaxis toward butanone (1:1000) or IAA (1:100). tax-2 was used as a chemotaxis-deficient control strain. For butanone and IAA, the box plot data are derived from five and three independent experiments, respectively, with 100–150 animals per assay. The asterisks indicate statistically significant differences (p < 0.01) between wild-type and mutant animals. C, D, The same strains were also exposed for 120 min to 200 μm DHPCC-9 and then tested as above toward butanone (C) or IAA (D). The asterisks indicate statistically significant differences (p < 0.01) between wild-type and mutant animals.

We then analyzed the chemotaxis of the prk mutants toward AWC-sensed attractive odorants, butanone and IAA. There both prk-1(pk86) and prk-2(ok3069) mutant animals responded normally to IAA, but only the prk-1(pk86) animals were significantly defective in their responses to butanone (Fig. 5B). The tax-2(p691) strain was used there as a chemotaxis-defective negative control, because it lacks expression for the TAX-2 cyclic nucleotide-gated channel ß subunit required to detect AWC-sensed odorants (Bargmann, 2006).

In another independent set of experiments, we pretreated wild-type and prk mutant strains with the PIM inhibitor DHPCC-9, after which we conducted chemotaxis assays to butanone and IAA. As expected based on previous results, the prk-1 mutation and/or DHPCC-9 blocked responses to butanone, but not to IAA (Fig. 5C,D). These data suggest that most of the effects of the PIM inhibitor on butanone sensing were mediated via inhibition of PRK-1 activity.

PRK-1 regulates also repulsive olfactory responses via AWB neurons

To determine whether PRKs are involved in the sensation of volatile repellents by AWB neurons, wild-type animals were exposed for 120 min to increasing concentrations of DHPCC-9 or SGI-1776, and then tested for their ability to sense 1-octanol and move away from it. After 120 min incubation, the avoidance indices were calculated. As shown in Figure 6A, more than half of the control-treated animals tried to avoid 1-octanol. By contrast, the avoidance responses were significantly and dose-dependently reduced in the drug-exposed populations, suggesting that the PIM inhibitors are able to diminish olfactory sensing also toward repellents via AWB neurons.

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Figure 6.

PRK-1 is essential also for sensing of olfactory repellents via AWB neurons. A, Wild-type animals were treated for 120 min with indicated concentrations of DHPCC-9 or SGI-1776. Aversion assays with 1-octanol (1:100) were performed for another 120 min. Shown are dose-dependent avoidance indices. Each data point represents the mean and SEM of at least four independent experiments with 100–150 animals per assay. The asterisks indicate statistically significant differences (p < 0.01) between control- and drug-exposed animals. B, WT, prk-1(pk86), and prk-2(ok3069) animals were similarly tested for aversion to 1-octanol. Shown are box plot data collected from three independent experiments with 100–150 animals per assay. The asterisks indicate statistically significant differences (p < 0.01) between wild-type and mutant animals.

When the olfactory aversion assays were repeated with mutant strains, prk-1(pk86) mutants showed a random chemotaxis toward either 1-octanol or the ethanol control, whereas the prk-2(ok3069) mutants avoided 1-octanol similarly to the wild-type animals (Fig. 6B). These data suggest that PRK-1 is more essential than PRK-2 for the observed effects of pan-PIM inhibitors on both attractive and repulsive olfactory responses.

PIM inhibitors do not affect gustatory sensations

We next examined whether PRKs are essential also for the attractive and aversive gustatory responses by the ASE and ASH neurons, respectively. Similarly to AWC neurons, also the ASE neurons are functionally asymmetric: the ASER (right) neuron preferentially detects potassium ions, whereas the ASEL (left) neuron detects sodium ions (Pierce-Shimomura et al., 2001). When we placed animals exposed to 200 µm DHPCC-9 or 100 µm SGI-1776 on plates containing drops of either 2.5 m NaCl or KCl, and calculated the chemotactic indices after 120 min incubation, more than half of the animals were attracted to either salt, but the PIM inhibitors did not significantly interfere with these responses compared with controls (Fig. 7A).

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Figure 7.

PIM inhibitors do not affect responses to gustatory attractants or repellents. Wild-type animals were treated for 120 min with 200 μm DHPCC-9 or 100 μm SGI-1776 and assayed for responses to gustatory attractants (A) or repellents (B–D). A, Shown are box plots of chemotactic indices of control- or drug-exposed animals to 2.5 m NaCl or 2.5 m KCl. Data were collected from three independent experiments with 150–200 animals per assay. B–D, Indicated concentrations of CuSO₄ were pipetted across the midlines of the assay plates. Control- or drug-exposed animals were placed on one side and drops of ethanol (B), butanone (C), or IAA (D) to the other side. After 120 min of incubation, chemotactic indices to the odorants were calculated. Shown are box plot data collected from four independent experiments with 150–200 animals per assay. The asterisks indicate statistically significant differences (p < 0.01) between control- and drug-exposed animals.

To investigate the repulsive responses by ASH neurons, midlines of distilled water or aqueous CuSO₄ solution (50 or 100 mM) were drawn onto assay plates. Control- or drug-exposed animals were placed on one side of the midline, whereas aliquots of ethanol, butanone or IAA were dropped on the other side. After 120 min incubation, chemotactic indices were calculated. As shown in Figure 7B, the initially mild chemotaxis toward the neutral odorant ethanol was further reduced by increasing concentrations of CuSO₄ in the midline, whereas the PIM inhibitors did not have significant effects. Both butanone (Fig. 7C) and IAA (Fig. 7D) efficiently attracted the animals to cross the midline of water, but the chemotactic responses were strongly diminished by the presence of 50 mM CuSO₄ and completely blocked by 100 mM CuSO₄. As observed also in our olfactory assays (Fig. 3A,C,E), exposures to either 200 µm DHPCC-9 or 100 µm SGI-1776 efficiently inhibited the positive chemotaxis toward butanone, but had less effects on IAA sensing (Fig. 7C,D).

PRK-1 is expressed in amphid and phasmid neurons

To confirm that PRK kinases are present in those amphid neurons, whose activities are suppressed by the PIM inhibitors, we wanted to analyze the PRK expression patterns in more detail. Unfortunately there are no antibodies available against PRKs to allow direct detection of these proteins. However, using the prk-1::GFP reporter strain, we were able to confirm previous data on prk-1 promoter-driven GFP expression in the intestine as well as in the head and tail neurons (Fig. 8A,B). A higher magnification revealed that the reporter expression is detected in a few amphid neurons and in one phasmid neuron. To obtain further evidence for the presence of PRK-1 expression in amphid neurons, we crossed the prk-1::GFP strain with the odr-1::RFP marker strain, where fluorescence is specifically detected in AWB and AWC neurons. Imaging of animals coexpressing these reporters revealed colocalized expression patterns for prk-1::GFP and odr-1::RFP in these two types of neurons on both sides of the heads (Fig. 8C–K). These results support our observations from the olfactory assays, suggesting that PRK-1 regulates olfactory responses to volatile attractants and repellents via AWB and AWC neurons.

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Figure 8.

Expression pattern of prk-1. A, B, Lateral bright-field (BF) and fluorescent views of a representative adult animal from the transgenic prk-1::GFP strain expressing GFP in amphid and phasmid neurons and in the intestine. Areas indicated by rectangles have been magnified to show GFP expression in the amphid and phasmid neurons (white arrowhead). C–H, Lateral views from different focal planes of a double transgenic animal expressing the prk-1 promoter-driven GFP (C, F) and the odr-1 promoter-driven RFP marker (D, G) in AWC (*) and AWB (Δ) neurons, as well as their colocalization in the merged picture (E, H). I–K, Dorsal view from another double transgenic animal to demonstrate that the prk-1 and odr-1 promoter-driven markers are expressed on both sides of the head.