DOI: 10.5061/dryad.1g1jwsv7b
title: Data from: Two cGMP-dependent protein kinases have opposing effects on molt-inhibiting hormone regulation of Y-organ ecdysteroidogenesis

description: Decapod crustaceans regulate molting through steroid molting hormones,
 ecdysteroids, synthesized by the molting gland (Y-organ, YO).
 Molt-inhibiting hormone (MIH), a neuropeptide synthesized and secreted by
 the eyestalk ganglia, negatively regulates YO ecdysteroidogenesis. MIH
 signaling is mediated by cyclic nucleotide second messengers.
 cGMP-dependent protein kinase (PKG) is the presumed effector of MIH
 signaling by inhibiting mechanistic Target of Rapamycin Complex 1
 (mTORC1)-dependent ecdysteroidogenesis. Phylogenetic analysis of PKG
 contiguous sequences in CrusTome as well as 35 additional species in NCBI
 RefSeq, identified 206 PKG1 sequences in 108 species and 59 PKG2 sequences
 in 53 species. These included four PKG1α splice variants in the N-terminal
 region that were unique to decapods, as well as PKG1β and PKG2 homologs.
 In vitro assays using YOs from the blackback land crab (Gecarcinus
 lateralis) and green shore crab (Carcinus maenas) determined the effects
 of MIH ± PKG inhibitors on ecdysteroid secretion. A general PKG inhibitor,
 Rp-8-Br-PET-cGMPS, countered the effects of MIH, as ecdysteroid secretion
 increased in PKG-inhibited YOs compared to C. maenas YOs incubated with
 MIH alone. By contrast, a PKG2-specific inhibitor, AP-C5
 (4-[4-(1H-Imidazol-1-yl)phenyl]-N-2-propyn-1-yl-2-pyrimidinamine),
 enhanced the effects of MIH, as ecdysteroid secretion decreased in G.
 lateralis and C. maenas YOs incubated with AP-C5 and MIH compared to YOs
 incubated with MIH alone. These data suggest that both PKG1 and PKG2 are
 activated by MIH, but have opposing effects on mTORC1-dependent
 ecdysteroidogenesis. A model is proposed in which the dominant role of
 PKG1 is countered by PKG2, resulting in low ecdysteroid production by the
 basal YO during intermolt.
description: ## Two cGMP-dependent protein kinases have opposing effects on
 molt-inhibiting hormone regulation of Y-organ ecdysteroidogenesis
 [https://doi.org/10.5061/dryad.1g1jwsv7b](https://doi.org/10.5061/dryad.1g1jwsv7b) ## Description of the data and file structure This dataset consists of Fasta files of all sequences used for phylogenetic characterization of PKG in crustaceans, and various alignments of isoforms are included; an Excel file with metadata for all sequences used is included and Tree files used to build the PKG phylogenetic tree is included. Sequencing data for PKG1 isoform validation and PKG2 in G. lateralis and C. maenasAssay data comes from *in vitro* assays of paired Y-organs from *G. lateralis* and *C. maenas* incubated with MIH or MIH ± PKG inhibitors. Hemolymph ecdysteroid (20E) was used to assess the molt stage of the animals, media 20E was measured from the incubation of each YO with the described treatments. Fasta files include peptide and nucleotide sequences used in this manuscript. Unaligned and aligned files are provided. All alignments were performed using MAFFT-dash. Sample metadata including the source of each sequence is provided in "PKGSequencesAccessionInfo.xlsx" ### Files and variables #### File: PKGSequencesAccessionInfo.xlsx **Description:** Metadata for all sequences used throughout the manuscript ##### Variables * Isoform (Type) * Contig ID * Taxon * Species * Amino Acid sequence * Source * Accession * Reference #### File: GlCm\_PKG2\_Peptide\_Aligned.fa **Description:** Alignment of *G. lateralis* and *C. maenas* PKG2 protein sequences #### File: Glateralis\_PKG\_Nucleotide\_Aligned.fasta **Description:** Alignment of *G. lateralis* PKG1 and PKG2 nucleotide sequences #### File: 240426\_TreeCode.txt **Description:** Code used to align, trim, and build the phylogenetic tree #### File: Cmaenas\_PKG\_Nucleotide\_Aligned.fasta **Description:** Alignment of *C. maenas* PKG1 and PKG2 nucleotide sequences #### File: GlCm\_PKG1\_Peptide\_Aligned.fasta **Description:** Alignment of *G. lateralis* and *C. maenas* PKG1 (all isoforms) protein sequences #### File: 240426\_V11KpicSmartGapRenamed.treefile **Description:** Tree file created from "240426_TreeCode.txt" #### File: AlignmentScript.txt **Description:** Alignment code used to align decapod, human, and mouse sequences #### File: GlCm\_Human\_Mouse\_Pep\_Aligned.fasta **Description:** Alignment of decapod, human, and mouse PKG proteins #### File: 240627\_PKG1b\_Aligned.fasta **Description:** Alignment of decapod PKG1β isoforms #### File: 240626\_PKG1a3groupAligned.fasta **Description:** Alignment of decapod PKG1α3 isoforms #### File: 240626\_PKG1a1groupAligned.fasta **Description:** Alignment of decapod PKG1α1 isoforms #### File: PKG1insertionContigs\_Aligned.fasta **Description:** Alignment of PKG1ins isoforms, and their matching sequences that lack the insertion #### File: 240626\_PKG1a2groupAligned.fasta **Description:** Alignment of decapod PKG1α2 isoforms #### File: 240628\_PKG1a4\_Aligned.fasta **Description:** Alignment of decapod PKG1α4 isoforms #### File: PKG1insertionContigs.fasta **Description:** Raw protein sequences used to make the alignment PKG1insertionContigs_Aligned.fasta #### File: GlCm\_Human\_Mouse\_Nucl\_Aligned.fasta **Description:** Alignment of decapod, human, and mouse PKG nucleotide sequences #### File: 240426\_V11KpicSmartGap.fasta **Description:** Trimmed alignment generated by ClipKit using the KpicSmartGap parameter. This file was used in IQ tree to generate the treefile #### File: PKGpeptide.fasta **Description:** All PKG protein sequences used #### File: 240426\_V11input.fasta **Description:** Input of all PKG protein sequences used to generate the phylogenetic tree #### File: PKGnucleotide.fasta **Description:** All PKG sequences used, in nucleotide #### File: 240426\_V11aligned.fasta **Description:** Alignment of all PKG protein sequences used before trimming with ClipKit #### File: SupplementaryMaterial1\_AssayData.xlsx **Description:** ELISA data from *in vitro* Y-organ assays, 20E quantified ##### Variables * Exp. * Experiment * Hemo 20E (pg/µl) * Quantity (picograms per microliter) of 20 hydroxyecdysone (20E) in circulating hemolymph for each animal before *in vitro *assays, used to determine molt stage * Control 20E (pg/µl) * Quantity (picograms per microliter) of 20E secreted by the control YO into the media over the course of 4.5 hours * Experimental 20E (pg/µl) * Quantity (picograms per microliter) of 20E secreted by the experimental YO into the media over the course of 4.5 hours * %Control_Control * Percent of the control, for the control samples (Control 20E / Control 20E *100) -&gt; represents the dark gray control bars in Figure 1 in the associated publication * %Control_Experimental * Percent of 20E secreted by the experimental YOs relative to the paired control (Experimental 20E / Control 20E *100) * Mean * Mean value for each variable for each experiment * SEM * Standard error of the mean for each variable for each experiment #### Experiments * Gl_MIH (*G. lateralis* MIH Assay) * Control: No treatment * Experimental: 50 nM MIH * Gl_Rp8 (*G. lateralis* MIH ± Rp-8-Br-PET-cGMPs Assay) * Control: 50 nM MIH * Experimental: 50 nM MIH + 100 µM Rp-8-Br-PET-cGMP * Gl_AP-C5 (*G. lateralis* MIH ± AP-C5 Assay) * Control: 50 nM MIH * Experimental: 50 nM MIH + 100 µM AP-C5 * Cm_MIH (*C. maenas* MIH Assay) * Control: No treatment * Experimental: 50 nM MIH * Cm_Rp8 (*C. maenas* MIH ± Rp-8-Br-PET-cGMPs Assay) * Control: 50 nM MIH * Experimental: * 50 nM MIH + 1 µM Rp-8-Br-PET-cGMP * 50 nM MIH + 10 µM Rp-8-Br-PET-cGMP * 50 nM MIH + 100 µM Rp-8-Br-PET-cGMP * Cm_AP-C5 (*C. maenas* MIH ± AP-C5 Assay) * Control: 50 nM MIH * Experimental: * 50 nM MIH + 1 µM AP-C5 * 50 nM MIH + 10 µM AP-C5 * 50 nM MIH + 100 µM AP-C5 #### File: Glat\_PKG\_AzentaPrimerSeqs\_Aligned.fa     **Description:** Alignment of G. lateralis PKG sequences identified in transcriptomic data with that sequenced from PCR #### File: Cm\_PKG\_AzentaPrimerSeqs\_Aligned.fa    **Description:** Alignment of C. maenas PKG sequences identified in transcriptomic data with that sequenced from PCR #### File: 30-850534263.fasta    **Description:** Raw sequencing reads #### File: 30-850534263\_QSCRL.xls   **Description:** Qscores of raw sequencing reads ##### Variables * TrackingNumber * Tracking number for shipment of PCR products to be sequenced * DNAType * Type of DNA sequenced was a purified PCR product * TubeLabel * Label of the tube for each sample created by the authors * DNAName * Label of the sample, corresponding to sample names in 30-850534263.fasta * PrimerName * All primers used were premixed by the authors. Primer sequences are available in the associated publication. * TemplateName * Name of the PCR template used for sequencing * QualitySCore * "The quality score (QS) is the average QV for all peaks in the trace with an assigned base. It serves as a metric for overall sequence quality. Generally speaking, traces with QS ≥ 40 have good quality; however, it’s advisable to scan the chromatogram to confirm this. Use caution for traces with QS values around 30. Those with QS &lt; 20 are likely to have a weak signal and/or high noise." * CRL * Continuous Read Length - "The longest uninterrupted stretch of bases with a running QV average of 20 or higher is known as the continuous read length (CRL)." * QV20Plus * "Each assigned base is accompanied by a quality value (QV), which is logarithmically related to the base-calling error: QV = -10 × log (error probability) For example, an assigned base with QV = 20 has an error probability of 0.01, or a 1% chance of being incorrectly called. The QV ultimately depends on the shape and signal-to-noise ratio of the peak, and it provides the most objective metric to evaluate the confidence of the base call. Please note these values are closely related to Phred quality scores1,2. By default, Azenta Life Sciences assigns an A, T, G or C when QV ≥ 10 and an N when QV &lt; 10. QVs are embedded in the ab1 file and can be seen in chromatogram viewing software (see example below). High-quality peaks generally have a QV of 20 or higher." * SI_A * Signal Intensity Adenine * Intensity values are reported in relative fluorescence units. * SI_C * Signal Intensity Cytosine * Intensity values are reported in relative fluorescence units. * SI_G * Signal Intensity Guanine * Intensity values are reported in relative fluorescence units. * SI_T * Signal Intensity Thymine * Intensity values are reported in relative fluorescence units. * CreatedDate * Date and time sequencing files were created by Azenta Life Sciences ## Code/software For fasta files and alignments, JalView is recommended for viewing aligned sequences: [https://www.jalview.org/](https://www.jalview.org/) For viewing the tree file, FigTree is recommended [http://tree.bio.ed.ac.uk/software/figtree/](http://tree.bio.ed.ac.uk/software/figtree/) To replicate alignments, use MAFFT-dash [https://doi.org/10.1093/nar/gkz342](https://doi.org/10.1093/nar/gkz342) ## Access information Other publicly accessible locations of the data: * N/A Data was derived from the following sources: * CrusTome [https://doi.org/10.1093/g3journal/jkad098; https://github.com/invertome/crustome](https://doi.org/10.1093/g3journal/jkad098) * NCBI * UniProt