Expression of tyrosine phosphatases in relation to PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori

Abstract

Protein tyrosine phosphorylation is a reversible, dynamic process regulated by the activities of tyrosine kinases and tyrosine phosphatases. Although the involvement of tyrosine kinases in the prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs) has been documented, few studies have been conducted on the involvement of protein tyrosine phosphatases (PTPs) in PTTH-stimulated ecdysteroidogenesis. In the present study, we investigated the correlation between PTPs and PTTH-stimulated ecdysteroidogenesis in Bombyx mori PGs. Our results showed that the basal PTP enzymatic activities exhibited development-specific changes during the last larval instar and pupation stage, with high activities being detected during the later stages of the last larval instar. PTP enzymatic activity was stimulated by PTTH treatment both in vitro and in vivo. Pretreatment with phenylarsine oxide (PAO) and benzylphosphonic acid (BPA), two chemical inhibitors of tyrosine phosphatase, reduced PTTH-stimulated enzymatic activity. Determination of ecdysteroid secretion showed that treatment with PAO and BPA did not affect basal ecdysteroid secretion, but greatly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated PTP activity is indeed involved in ecdysteroid secretion. PTTH-stimulated phosphorylation of the extracellular signal-regulated kinase (ERK) and 4E-binding protein (4E-BP) was partially inhibited by pretreatment with either PAO or BPA, indicating the potential link between PTPs and phosphorylation of ERK and 4E-BP. In addition, we also found that in vitro treatment with 20-hydroxyecdysone did not affect PTP enzymatic activity. We further investigated the expressions of two important PTPs (PTP 1B (PTP1B) and the phosphatase and tension homologue (PTEN)) in Bombyx PGs. Our immunoblotting analysis showed that B. mori PGs contained the proteins of PTP1B and PTEN, with PTP1B protein undergoing development-specific changes. Protein levels of PTP1B and PTEN were not affected by PTTH treatment. The gene expression levels of PTP1B and PTEN showed development-specific changes. From these results, we suggest that PTTH-regulated PTP signaling may crosstalk with ERK and target of rapamycin (TOR) signaling pathways and is a necessary component for stimulation of ecdysteroid secretion.