The EGL-4 PKG acts with KIN-29 salt-inducible kinase and protein kinase A to regulate chemoreceptor gene expression and sensory behaviors in Caenorhabditis elegans

Abstract

The regulation of chemoreceptor (CR) gene expression by environmental signals and internal cues may contribute to the modulation of multiple physiological processes and behavior in Caenorhabditis elegans. We previously showed that KIN-29, a homolog of salt-inducible kinase, acts in sensory neurons to regulate the expression of a subset of CR genes, as well as sensory behaviors. Here we show that the cGMP-dependent protein kinase EGL-4 acts partly in parallel with KIN-29 to regulate CR gene expression. Sensory inputs inhibit both EGL-4 and KIN-29 functions, and KIN-29 function is inhibited in turn by cAMP-dependent protein kinase (PKA) activation. EGL-4 and KIN-29 regulate CR gene expression by antagonizing the gene repression functions of the class II HDAC HDA-4 and the MEF-2 transcription factor, and KIN-29, EGL-4, and PKA target distinct residues in HDA-4 to regulate its function and subcellular localization. While KIN-29 acts primarily via MEF-2/HDA-4 to regulate additional sensory signal-regulated physiological processes and behaviors, EGL-4 acts via both MEF-2-dependent and -independent pathways. Our results suggest that integration of complex sensory inputs via multiple signaling pathways allows animals to precisely regulate sensory gene expression, thereby appropriately modulating physiology and behavior.

Figure 1.—

cGMP signaling and EGL-4 regulate chemoreceptor gene expression. (A) The expression of str-1p∷gfp in an AWB neuron of adult animals of the indicated genotypes. Arrows indicate the AWB cell body. Images are lateral views; anterior is at left. Images were acquired using identical exposure times at ×400 magnification. Bar, 15 μm. All strains contain stably integrated copies of a str-1p∷gfp transgene. (B) Expression of str-3p∷gfp in an ASI neuron in wild-type (top) and egl-4(n479) (bottom) mutant animals. The ASI cell body is indicated by an arrowhead. Images are lateral views; anterior is at left. Images were acquired using identical exposure times at ×400 magnification. Bar, 15 μm. Strains contain stably integrated copies of a str-3p∷gfp transgene. (C) Levels of endogenous str-1 messages are downregulated in egl-4(lf) mutants and upregulated in egl-4(gf) mutants. Shown is the ratio of endogenous str-1 message to endogenous odr-10 (Sengupta et al. 1996) message as quantified by qRT–PCR in animals of the indicated genotypes. Expression of odr-10 is unaffected in egl-4 mutants (Daniels et al. 2000). The mean of the ratios from two independent experiments is shown. Error bars denote the SEM. * and ** indicate values that are different from that of wild type at P < 0.05 and P < 0.01, respectively.

Figure 2.—

Mutations in mef-2 partly suppress the egl-4(lf) defects in the regulation of body size. Shown is the mean body length of adult animals of the indicated genotypes. The body lengths of at least 20 animals were measured for each strain. * and ** indicate values that are different at P < 0.05 and P < 0.01, respectively, from that of wild type; # and ## indicate values that are different at P < 0.05 and P < 0.01, respectively, between the values compared by brackets. Statistical analyses were performed using one-way ANOVA and adjusted for multiple comparisons using Tukey's HSD post-hoc test. Error bars are the SD. All strains contain integrated copies of a str-1p∷gfp fusion gene.

Figure 3.—

kin-29 and kin-2 mutants exhibit defects in food-induced quiescence behavior. (A and B) Quiescence duration of nonfasted animals of the indicated genotypes after feeding with HB101 bacteria for 6 hr. Quiescence is defined as cessation of both locomotion and pharyngeal pumping. n = 20 each in two independent assays. Values are the mean ± SEM. * and ** indicate values that are different at P < 0.05 and P < 0.01, respectively, from that of wild type; ## indicates values that are different at P < 0.01 between the values compared by brackets in A. Statistical analyses were performed using one-way ANOVA and adjusted for multiple comparisons in A using Tukey's HSD post-hoc test. All strains contain integrated copies of a str-1p∷gfp fusion gene.

Figure 4.—

Mutations in mef-2 suppress the chemosensory behavioral and dauer formation defects of egl-4(lf) mutants. (A) Responses of adult animals of the indicated genotypes to a point source of 1 μl of a 1:1000 dilution of diacetyl or 1 μl of a 1:10,000 dilution of 2,3-pentanedione. Each data point is the average of two independent assays in duplicate with ∼100 animals in each assay. (B) Percentage of dauers formed upon exposure of animals of the indicated genotypes to 5 μl of crude pheromone extract. n = 300; two independent experiments. For both A and B, error bars equal the SEM. ** indicates values that are different at P < 0.01 from that of wild type; # and ## indicate values that are different at P < 0.05 and P < 0.01, respectively, between the values compared by brackets. Statistical analyses were performed using one-way ANOVA and Tukey's HSD post-hoc test for multiple comparisons. All strains contain integrated copies of a str-1p∷gfp fusion gene.

Figure 5.—

Localization of HDA-4 may be regulated by both PKA- and PKG-mediated phosphorylation. (A–F) The expression of stably integrated (A–D) or extrachromosomal copies (E and F) of a functional full-length gfp-tagged hda-4 transgene (van der Linden et al. 2007) in the indicated genetic backgrounds. Note cytoplasmic localization in a subset of cells in F (arrowheads). (G and H) A GFP-tagged HDA-4[S363A S462A] protein is localized to the cytoplasm (arrowheads) in the indicated genetic backgrounds. Arrows point to str-1p∷gfp expression in an AWB neuron. (A–H) Images of L1/L2 larval stage animals were acquired using identical exposure times at ×400 magnification. The area imaged in A–H is indicated by a box in the cartoon at top. Bar, 20 μm.

Figure 6.—

Model of KIN-29- and EGL-4-regulated pathways in the modulation of CR gene expression and sensory behaviors. (A) KIN-29 and EGL-4 act in partly parallel pathways to regulate str-1p∷gfp expression. Increased activity of the GSA-1 G_αs and ACY-1 adenylyl cyclase result in increased intracellular cAMP levels and activation of PKA. PKA inhibits KIN-29 activity via direct or indirect phosphorylation. EGL-4 activity is regulated by cGMP levels generated by the DAF-11 and ODR-1 receptor guanylyl cyclases. Sensory inputs downregulate DAF-11/ODR-1, but may upregulate ACY-1 activity. KIN-29 and EGL-4 may phosphorylate HDA-4 at the S198 and other residue(s) to alleviate the gene repression properties of HDA-4. Nuclear localization of HDA-4 requires phosphorylation at the S363/S462 residues by PKA and EGL-4. Proposed phosphorylation is indicated by dashed arrows. See discussion for additional details. (B) KIN-29 acts primarily via MEF-2 and HDA-4 to regulate CR gene expression, body size, dauer formation, and food-induced quiescence behaviors (van der Linden et al. 2007 and this work). EGL-4 acts via MEF-2/HDA-4 to regulate CR gene expression and via MEF-2 and the DAF-3 SMAD and DAF-5 SKI proteins to regulate additional sensory behaviors (Daniels et al. 2000).