Protein kinase-like ER kinase (PERK) regulates autophagy of hemocytes in antiviral immunity of Pacific oyster Crassostrea gigas

The maintenance of cellular homeostasis is an important process for successful immune defense against pathogenic invading, in which unfolded protein response (UPR) pathway regulates endoplasmic reticulum (ER) homeostasis upon exposure to environmental changes. Protein kinase-like ER kinase (PERK) is an important ER stress sensor to be activated during the UPR to regulate cells homeostasis. In the present study, one PERK homologue was identified from Pacific oyster Crassostrea gigas (designated as CgPERK). The cDNA of CgPERK was of 4307 bp with a 3174 bp open reading frame (ORF) encoding a polypeptide of 1058 amino acids. There were two conserved protein kinases domains and two conserved autophosphorylation sites at Lys618 and Thr980 in CgPERK. The mRNA transcript of CgPERK was constitutively expressed in all the tested tissues including mantle, adductor muscle, hepatopancreas, gill, gonad and labial palp with the highest expression level in hemocytes (31.15-fold compared to mantle). The CgPERK protein was found to be located mainly in the cytoplasm of hemocytes. The mRNA expression level of CgPERK in hemocytes was significantly up-regulated and reached the highest level (5.25-fold compared to seawater group, p < 0.01) at 48 h after the oysters were stimulated with poly(I: C). Meanwhile, a significant increase of fluorescence autophagosome spots in hemocytes was also observed at 36 h post stimulation. After the mRNA expression of CgPERK was knocked down (0.49-fold compared to dsGFP group, p < 0.01) by injection of CgPERK dsRNA, the mRNA expression of autophagy related 12 (ATG12) in hemocytes was significantly decreased at 12 h post poly(I: C) stimulation, which was 0.53-fold (p < 0.01) compared to dsGFP-injected oysters. When the CgPERK was inhibited by its inhibitor GSK2656157 stimulation, the autophagosomes rate of hemocytes decreased significantly at 12 h post poly(I: C) stimulation, which was 0.34-fold (p < 0.01) of that of DMSO group. Collectively, these results suggested that CgPERK, as an UPR initiator, was involved in autophagosomes formation upon poly(I: C) stimulation by regulating the expression of ATG12, and ER stress stimulated the autophagosome formation on an ATG protein-dependent manner in oysters.