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Physiological regulation of molting in decapods is predominantly coordinated by two hormones, the peptide molt-inhibiting hormone (MIH), and steroid molting hormones termed ecdysteroids. MIH is produced and secreted by the X-organ/sinus gland complex located in the eyestalk ganglia, and negatively regulates the production of ecdysteroids in the molting gland (Y-organ, YO). MIH signaling begins with a cAMP-dependent triggering phase followed by a cGMP-dependent summation phase which ultimately leads to inhibition of mTORC1. Previous work revealed that two cGMP-dependent protein kinases (PKG1 and PKG2) have opposing roles in modulating ecdysteroidogenesis via MIH signaling in YOs. Specifically, PKG1 plays a dominant role in MIH signaling by inhibiting ecdysteroid synthesis, while PKG2 counters that inhibition and maintains basal ecdysteroidogenesis in the intermolt YO. This study aims to use sample multiplexing alongside phosphopeptide enrichment in LC-MS/MS to identify potential substrates of PKG in the YO. Transcript expression of PKG1 is two orders of magnitude greater than that of PKG2 in the intermolt YO, and preliminary proteomic data identified peptides from PKG1 but not PKG2. This data indicates that protein expression may roughly follow transcript expression for the two PKG isozymes, providing a basis for the differential effects of their opposing roles. Supported by NSF grants to DM (IOS-1922701) and LT (IOS-1922718 & IOS-23221487). 

ABSTRACT 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 with 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 with 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.