The involvement of CaMKKI in activating AMPKα in yesso scallop Patinopecten yessoensis under high temperature stress

Abstract

Calcium/calmodulin dependent protein kinase kinase (CaMKK), a highly conserved protein kinase, is involved in the downstream processes of various biological activities by phosphorylating and activating 5′-AMP-activated protein kinase (AMPK) in response to the increase of cytosolic-free calcium (Ca²⁺). In the present study, a CaMKKI was identified from Yesso scallop Patinopecten yessoensis. Its mRNA was ubiquitously expressed in haemocytes and all tested tissues with the highest expression level in mantle. The expression level of PyCaMKKI mRNA in adductor muscle was significantly upregulated at 1, 3 and 6 h after high temperature treatment (25 °C), which was 3.43-fold (p < 0.05), 5.25-fold (p < 0.05), and 5.70-fold (p < 0.05) of that in blank group, respectively. At 3 h after high temperature treatment (25 °C), the protein level of PyAMPKα, as well as the phosphorylation level of PyAMPKα at Thr170 in adductor muscle, and the positive co-localized fluorescence signals of PyCaMKKI and PyAMPKα in haemocyte all increased significantly (p < 0.05) compared to blank group (18 °C). The pull-down assay showed that rPyCaMKKI and rPyAMPKα could bind each other in vitro. After PyCaMKKI was silenced by siRNA, the mRNA and protein levels of PyCaMKKI and PyAMPKα, and the phosphorylation level of PyAMPKα at Thr170 in adductor muscle were significantly down-regulated (p < 0.05) compared with the negative control group receiving an injection of siRNA-NC. These results collectively suggested that PyCaMKKI was involved in the activation of PyAMPKα in response to high temperature stress and would be helpful for understanding the function of PyCaMKKI-PyAMPKα pathway in maintaining energy homeostasis under high temperature stress in scallops.