Elsevier

Fish & Shellfish Immunology

Volume 73, February 2018, Pages 279-287
Fish & Shellfish Immunology

Full length article
Identification and analysis of an MKK4 homologue in response to the nucleus grafting operation and antigens in the pearl oyster, Pinctada fucata

https://doi.org/10.1016/j.fsi.2017.12.023Get rights and content

Highlights

  • PfMKK4 exhibits all the typical features of MKK4 gene.

  • PfMKK4 wide distributes in tissues and developmental stages.

  • PfMKK4 gene was expressed throughout the whole developmental stages, while the highest level was detected in the polar body stage.

  • PfMKK4 may be involved in post-graft processes.

  • PfMKK4 is involved in immune response in Pinctada fucata.

Abstract

The mitogen-activated protein kinase kinase 4 (MKK4) is a key component of the c-Jun N-terminal kinase (JNK) signaling pathway and regulates multiple cellular activities. However, little is known about the roles of this kinase in pearl oyster. In this study, we identified an MKK4 homologue in Pinctada fucata by using a transcriptome database. Sequence analysis and protein structure prediction showed that PfMKK4 is highly conserved to MKK4 from other vertebrate and invertebrate species. Phylogenetic analysis revealed that PfMKK4 has the closest relationship with that from Crassostrea gigas. QPCR was used to investigate expression profiles in different healthy adult tissues and developmental stages of P. fucata. We found that PfMKK4 was ubiquitously expressed in all tissues and developmental stages examined except for in D-shaped larvae. Gene expression analysis suggested that PfMKK4 is involved in the response to the nucleus insertion operation. Lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid [poly(I:C)] stimulation in vivo reduced PfMKK4 mRNA expression at 6 h, 48 h and 48 h, 72 h, respectively. LPS and poly(I:C) induced PfMKK4 phosphorylation in a primary mantle cell culture. These results contribute to better understanding of the potential role played by PfMKK4 in protecting the pearl oyster from injury caused by grafting or disease.

Introduction

The mitogen-activated protein kinase (MAPK) signaling pathway is a highly conserved and universal signaling modules in eukaryotes [[1], [2], [3]]. It includes three major classes: the p38 MAP kinase pathway, the extracellular signal-regulated kinase (ERK) pathway and the c-Jun N-terminal kinase (JNK) pathway [4]. MAPK is made up of three functionally associated protein kinases, namely MPK kinase kinases (MKKKs), MPK kinases (MKKs) and MPKs. MAPKKKs are serine/threonine kinases phosphorylating two amino acids in the S/T-X3–5-S/T motif of the MKK activation loop. The transmission of the MAPK signal is catalyzed by a family of dual-specificity kinases. In vertebrate, the MKK family consists of seven members MKK1–7 [5], six MKKs are found in Danio rerio [6] and five MKKs in Xenopus tropicalis [7]. In invertebrate, Four MKKs were found in Drosophila melanogaster [8], and five MKKs genes were identified recently in Patinopecten yessoensis [3]. MKKs, as the hub of the cascade, act as dual-specificity kinases to activate MPKs through double phosphorylation of the T-X-Y motif in the activation loop and, in turn, are activated by Ser/Thr phosphorylation by MAPKKKs, eventually, to accomplish the transmission of signals [9,10].

The JNK-MAPK signaling pathway plays critical roles in many physiological and biochemical processes, including cell growth, cell survival, differentiation, neuronal function and the immune response [7,11]. The activation of JNK is achieved by the MKK4 or MKK7 in upstream of JNK pathway [12,13]. The activated JNK induce the expression of JNK pathway-target genes through phosphorylate the transcription factors such as c-Jun, ATF2, p53, Elk1 [[12], [13], [14]]. In Drosophila, the JNK signal cascade plays critical roles in innate immunity [15]. The Drosophila immune deficiency(Imd) pathway controls primarily Gram-bacteria‐specific responses through JNK, caspase and nuclear factor-k B [[16], [17], [18]]. The Drosophila immune deficiency (Imd) pathway bifurcates into the JNK pathway at the level of Tak1/Tab2 [[19], [20], [21]]. On the one hand, Tak1/Tab2 cause the phosphorylatio of MKK4 and MKK7 that lead to the activation of JNK, which in turn activate downstream transcription factors (such as activating protein-1) by phosphorylation and induce the transcription of a subset of immune response genes [18]. On the other hand, Tak1/Tab2 can also activate Drosophila IKKβ or IKKγ that results in the rapid expression of antimicrobial peptides [14,22,23].

The pearl oyster Pinctada fucata is one of the most important bivalve mollusks for seawater pearl production. However, the outbreaks of diseases caused by bacteria and viruses are becoming worse. Moreover, the nucleus grafting operation often leads to injury and causes mass mortality of pearl oysters. In invertebrates, the Toll and IMD signal cascades are two evolutionarily conservative pathways and play major roles in innate immunity against invading pathogens [24,25]. Previously, our work relates to immunization and the characterization of immune-related genes which participate in the innate immune response to pathogenic infection in P. fucata [[26], [27], [28]]. The functions of JNK pathway from arthropods to mammals has been reported. However, little is known about its role in oysters. The functions of MKK4 and MKK7 are typically associated with the JNK signaling pathway and their roles in oyster immunity still remain unidentified. In this paper, we describe a P. fucata MKK4 gene, namely PfMKK4, which shares significant structural and functional homology with other MKK4 family members. We examine the expression pattern of MKK4 in tissues and development stages. Furthermore, we analyze the expression of PfMKK4 in a nucleus grafting operation and immune response. The results provide us with a way of understanding the potential role of PfMKK4 in the nucleus grafting operation and immunity in P. fucata.

Section snippets

Experimental animals

Pearl oysters were collected from Daya Bay in Shenzhen, Guang-dong Province, China. The bacterial challenge experiments were performed in Daya Bay and pearl oysters were acclimated in indoor cement ponds at ambient seawater temperature before experimentation. Nucleus grafting operation experiments were performed in Xuwen, Guang-dong Province, and pearl oysters were collected directly from the sea.

Gene discovery and cloning of PfMKK4

BLAST analysis of available transcriptome data for the pearl oyster revealed that a 2964 bp

Sequence analysis of PfMKK4 cDNA

The complete cDNA sequence of PfMKK4 has been obtained by assembling EST from a transcriptome database and verified by PCR (GenBank accession number MF197560). The PfMKK4 cDNA consisted of 2964 bp, with an ORF of 1194 bp, encoding a putative protein of 397 amino acids with a calculated molecular mass of ∼45.25 kDa and a theoretical isoelectric point (pI) of 7.55, a 5′-untranslated region of 235 bp, and a 3′-untranslated region of 1535 bp. A serine/threonine protein kinases catalytic (STKc)

Discussion

Although the JNK pathway has been reported to conserve in regulating innate immunity ranging from arthropods to mammals, our understanding of JNK pathway function in invertebrates, especially in pearl oyster, remains limited [[33], [34], [35]]. MKK4 is one of key members of the JNK signal pathway that can activate JNK or p38 MAPKs in response to diverse extracellular stimuli. MKK4 is involved in a variety of physiological and pathophysiological processes [4]. In this study, for the first time

Acknowledgments

This work was supported by the National Natural Science Foundation of China (41376159, 41406178), the Earmarked Fund for Modern Agro-industry Technology Research System (CARS-47), Science and Technology Planning Project of Guangdong Province, China (2014B030301064), the PhD Start-up Fund of Natural Science Foundation of Guangdong Province, China (2015A030310362), and the Project of Guangzhou Science and Technology (201707010179).

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