Hormone Signaling Linked to Silkmoth Sex Pheromone Biosynthesis Involves Ca2+/Calmodulin-dependent Protein Kinase II-mediated Phosphorylation of the Insect PAT Family Protein Bombyx mori Lipid Storage Droplet Protein-1 (BmLsd1)

Atsushi Ohnishi,J Joe Hull,Misato Kaji,Kana Hashimoto,Jae Min Lee,Kazuhide Tsuneizumi,Takehiro Suzuki,Naoshi Dohmae,Shogo Matsumoto

doi:10.1074/jbc.m111.250555

Abstract

Species-specific sex pheromones released by female moths to attract conspecific male moths are synthesized de novo in the pheromone gland (PG) via the fatty acid biosynthetic pathway. This pathway is regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN), a 33-amino acid peptide that originates in the subesophageal ganglion. In the silkmoth, Bombyx mori, cytoplasmic lipid droplets, which store the sex pheromone (bombykol) precursor fatty acid, accumulate in PG cells. PBAN stimulates lipolysis of the stored lipid droplet triacylglycerols (TAGs) and releases the precursor for final modification. PBAN exerts its physiological function via the PG cell-surface PBAN receptor, a G protein-coupled receptor that belongs to the neuromedin U receptor family. The PBAN receptor-mediated signal is transmitted via a canonical store-operated channel activation pathway utilizing Gq-mediated phospholipase C activation (Hull, J. J., Kajigaya, R., Imai, K., and Matsumoto, S. (2007) Biosci. Biotechnol. Biochem. 71, 1993-2001; Hull, J. J., Lee, J. M., Kajigaya, R., and Matsumoto, S. (2009) J. Biol. Chem. 284, 31200-31213; Hull, J. J., Lee, J. M., and Matsumoto, S. (2010) Insect Mol. Biol. 19, 553-566). Little, however, is known about the molecular components regulating TAG lipolysis in PG cells. In the current study we found that PBAN signaling involves phosphorylation of an insect PAT family protein named B. mori lipid storage droplet protein-1 (BmLsd1) and that BmLsd1 plays an essential role in the TAG lipolysis associated with bombykol production. Unlike mammalian PAT family perilipins, however, BmLsd1 activation is dependent on phosphorylation by B. mori Ca(2+)/calmodulin-dependent protein kinase II rather than protein kinase A.

Highlights

JULY 8, 2011 VOLUME 286 NUMBER 27 processes that comprise sex pheromone biosynthesis in female moths must be precisely regulated
In most moth species these processes are regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN),4 a 33-amino acid peptide that originates in the subesophageal ganglion and that is characterized by a core C-terminal FSPRLamide sequence [1, 2]
We further report that BmLsd1 is a lipid droplets (LDs)-associated insect PAT family protein that plays an essential role in bombykol biosynthetic TAG lipolysis after phosphorylation by B. mori Ca2ϩ/calmodulin-dependent protein kinase II (BmCaMKII)

Summary

EXPERIMENTAL PROCEDURES

Insects—Larvae of the inbred p50 strain of B. mori, kindly provided by T. Trimmed PGs were homogenized in a cytoplasmic fraction buffer (25 mM Hepes (pH 7.5), 50 mM NaCl, 50 mM NaF, 5 mM EDTA, 10 nM okadaic acid, 0.1 mM Na3VO4, 1 mM p-amidinophenylmethanesulfonyl fluoride hydrochloride (p-APMSF, Wako Chemicals, Osaka, Japan), and 10 ␮g/ml each of aprotinin and leupeptin) for 1 min on ice. PG cell homogenates were centrifuged at 15,000 ϫ g for 10 min to separate soluble and insoluble fractions. Analysis of PG Protein Phosphorylations—For SDS-PAGE, cytoplasmic, membrane, and fat-cake fractions were incubated with 80 mM Tris buffer (pH 8.8) containing 1% SDS and 2.5% 2-mercaptoethanol in boiling water for 5 min and developed by the method of Laemmli [25].

Primer sequence

Primer sequenceb

RESULTS

DISCUSSION