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.
摘要:
钙/钙调蛋白依赖性蛋白激酶激酶(CaMKK),一种高度保守的蛋白激酶,通过磷酸化和激活5'-AMP激活的蛋白激酶(AMPK)参与各种生物活性的下游过程,以响应无胞浆钙(Ca2)的增加。在本研究中,从Yesso扇贝Patinopectenyessoensis中鉴定出一种CaMKKI。其mRNA在血细胞和所有测试组织中普遍表达,在地幔中表达水平最高。高温处理(25℃)后1、3、6h内收肌中PyCaMKKImRNA的表达水平显著上调,这是3.43倍(p<0.05),5.25倍(p<0.05),是空白组的5.70倍(p<0.05),分别。在高温处理后3小时(25°C),PyAMPKα的蛋白质水平,以及PyAMPKα在内收肌中Thr170的磷酸化水平,血细胞中PyCaMKKI和PyAMPKα的阳性共定位荧光信号均比空白组(18°C)显着增加(p<0.05)。下拉实验表明,rPyCaMKKI和rPyAMPKα在体外可以相互结合。在PyCaMKKI被siRNA沉默后,PyCaMKKI和PyAMPKα的mRNA和蛋白水平,与注射siRNA-NC的阴性对照组相比,内收肌中Thr170处PyAMPKα的磷酸化水平显着下调(p<0.05)。这些结果共同表明,PyCaMKKI参与了响应高温胁迫的PyAMPKα的激活,这将有助于了解PyCaMKKI-PyAMPKα通路在维持扇贝高温胁迫下能量稳态中的作用。