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

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 Py CaMKKI mRNA in adductor muscle was significantly upregulated at 1, 3 and 6 h after high temperature treatment (25 degrees 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 degrees C), the protein level of Py AMPK alpha, as well as the phosphorylation level of Py AMPK alpha at Thr170 in adductor muscle, and the positive co-localized fluorescence signals of Py CaMKKI and Py AMPK alpha in haemocyte all increased significantly (p < 0.05) compared to blank group (18 degrees C). The pull-down assay showed that rPyCaMKKI and rPyAMPK alpha could bind each other in vitro. After Py CaMKKI was silenced by siRNA, the mRNA and protein levels of Py CaMKKI and Py AMPK alpha, and the phosphorylation level of Py AMPK alpha 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 Py CaMKKI was involved in the activation of Py AMPK alpha in response to high temperature stress and would be helpful for understanding the function of Py CaMKKI- Py AMPK alpha pathway in maintaining energy homeostasis under high temperature stress in scallops.