Prostaglandins influence protein phosphorylation in established insect cell line

Abstract

Prostaglandins (PGs) are oxygenated metabolites of arachidonic acid and two other C20 polyunsaturated fatty acids. Among other actions in invertebrates, PGs act in ovarian development, renal functions, immunity, hemocyte migration, and gene/protein expression. Reversible phosphorylation is a major mechanism of regulating protein functions in eukaryotic cells and for some mammalian proteins it is influenced by PGs. We posed the hypothesis that PGs influence protein phosphorylation within insect cells, which we tested with the established insect cell line, BCIRL‐HzAM1. After 20, 30, or 40 min incubations in the presence of one of three PGs (at 15 μM), PGA2, PGE1, or PGF2α, separate sets of cells were processed for analysis by two‐dimensional electrophoresis followed by tandem mass spectrometry. We recorded significant phosphorylation changes in 31 proteins, decreases in 15, and increases in 15, and one protein with increased or decreased phosphorylation, depending on PG treatment. Increasing PG exposure times led to changes in fewer proteins, 20 min incubations led to changes in 16 proteins, 30 min to changes in 13, and 40 min to changes in 2 proteins. The proteins were identified by bioinformatic analyses, including transcript description, calculated molecular weights and isoelectric points, MOlecular Weight SEarch score, total ion score, numbers of peptides, percent protein coverage, E‐value, and highest peptide score. The data presented in this paper firmly support our hypothesis that PGs influence protein phosphorylation within insect cells and adds a novel PG‐signaled function to insect biology.

Prostaglandin A2 induces apoptosis in three cell lines derived from the fall armyworm, Spodoptera frugiperda

“…We exposed SfNS cells to selected concentrations of PGA 2 (10 μM or 30 μM) for 24 h and processed the cells according to the manufacturer's procedures. These PG concentrations were selected based on our work with other experimental protocols involving related insect cell lines (Stanley et al, 2020; Stanley et al, 2012). Cells were fixed using 1% paraformaldehyde in phosphate‐buffered saline (PBS) on ice for 30 min, then washed with PBS, centrifuged (300 g , 5 min, 4°C), resuspended in 70% (v/v) ice‐cold ethanol for 30 min.…”

“…PGs mediate a wide range of physiological processes in insects (Stanley & Kim, 2019), such as the release of egg‐laying behavior in some, but not all, cricket species, aspects of development, mediating several aspects of humoral and hemocytic immunity, and Malpighian tubule physiology. PGs also influence gene expression and protein phosphorylation in an established insect cell line (Stanley et al, 2020; Stanley et al, 2012). As PGs act in apoptosis in mammals, we posed the hypothesis that they also influence apoptosis in insect cells.…”

Eicosanoid Signaling in Insect Immunology: New Genes and Unresolved Issues

“…After genome databases became available, considerable efforts to identify one or more genes encoding COXs in insect genomes led to a conundrum. While PG actions including releasing cricket egg-laying behavior, modulating primary urine formation in isolated Malpighian tubules, signaling several aspects of insect immunity and post-translational protein phosphorylation [6,7,18], genes encoding insect COXs proved very elusive. For example, Varvas et al [19] characterized two genes encoding COXs in two crustacean species, but not from insects.…”

An ovary‐specific mucin is associated with choriogenesis mediated by prostaglandin signaling in Spodoptera exigua

“…In addition, PGE 2 receptor identified in S. exigua use cAMP as a secondary messenger to mediate physiological processes and its downstream signal components including protein kinases (Kim et al, 2020). Indeed, PGs can phosphorylate various proteins in insect cell lines (Stanley et al, 2020). These findings suggest that PGs might activate trans elements for the expression of chorion genes.…”