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Meeting Abstract

P1-30   -   Characterization of cGMP-dependent protein kinase in the crustacean molting gland Head, TB*; Mykles, DL; Colorado State University; Colorado State University

Growth in crustaceans requires the periodic shedding of the exoskeleton, a complex physiological process that is regulated by several molecular pathways. Molt-inhibiting hormone (MIH) synthesized and secreted by the X-organ/sinus gland complex (XO) in the eyestalk inhibits the synthesis of the pro-molting hormones, ecdysteroids, by the molting gland (Y-organ, YO). Such inhibition is believed to occur through a cAMP/Ca2+-dependent triggering phase, followed by a nitric oxide/cGMP-dependent summation phase in which cGMP-dependent protein kinase (PKG) indirectly inhibits the mTOR-dependent protein synthesis required for ecdysteroid synthesis. To date, downstream targets of PKG in the YO remain unknown, although we hypothesize that the tuberous sclerosis complex may be a target. Transcriptomic analysis of the Gecarcinus lateralis YO revealed two distinct isoforms of PKG. Two highly conserved cyclic nucleotide-binding domains and one serine/threonine kinase domain were identified in both isoforms, with a dimerization domain unique to each isoform. Both isoforms show unique expression across the molt stages, with one isoform peaking in expression during intermolt and dropping to low levels of expression during all premolt and postmolt stages, while the other remained at relatively low expression across all molt stages. Comparative analysis of PKG sequences revealed conservation of the two PKG isoforms in several decapod crustaceans. Further, in vitro inhibition of PKG in the Carcinus maenas YO using Rp-8-Br-PET-cGMPs at 100 µM or 10 µM in combination with 50 nM MIH showed stimulation of ecdysteroid secretion from the YO relative to a control with 50 nM MIH alone. This data confirms the role of PKG as a downstream messenger of MIH to inhibit ecdysteroidogenesis by the YO. Proteomic analysis of phosphoproteins in PKG-inhibited and control YOs using LC-MS will be used to identify possible targets of PKG. Supported by NSF (IOS-1922701 and IOS-1456942).