| Comprising a catalytic a subunit and two regulatoryβand y subunits, AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis as well as cell proliferation and polarity. To date, the mechanism underlying the autoregulation of AMPK kinase activity remains elusive. Using tissues collected from mice ablated with AMPK a2, we showed that the stability ofβsubunit depended on the presence of a. In the absence ofα1 or a2 subunit, the decrease inβwas due to an accelerated protein degradation, which could be rescued by the a complementation. Similarly, siRNA knocking down ofβdecreased a expression in cultured cells. Experiments with a2 truncations revealed that residues 412-426 of murine a2 were critical for the binding ofβsubunit. Exhibited little effect onαβinteraction, a 12-a.a truncation at the C-terminal (aa.541-552α2△12) of a2 resulted in decreased kinase activity. This truncation could act as a domain negative mutant to abate endogenous AMPK activity in phosphorylating the downstream targets such as acetyl-CoA carboxylase. The C-terminus of AMPKα2 subunit forms a specialαβdomain with a hydrophobic center similar to that of MARK3 C-terminus. The deletion of the a helix possibly disrupts the conformation of this domain therefore affects the kinase activity. The mutations of highly conserved Leu550 and Leu546 that involved in the formation of hydrophobic center to glutamate similarly decreased kinase activity. Taken together, the interaction ofαβis important in maintaining the AMPK stability, whereas the conformation integrity of C-terminal tail of the a is necessary for the kinase activity of AMPK. Endothelial dysfunction is closely associated with vascular diseases such as atherosclerosis and hypertension. Physiological range of shear stress is critical in maintaining endothelial functions such as anti-inflammation, anti-proliferation and regulating vessel tones. Multiple mechanisms have been elucidated to be involved in the vascular protective effects mediated by shear stress. However, whether shear stress can regulate the redox state of endothelial cells and the underlying mechanism is still unknown. In the present study, we proved that laminar shear can induce the endothelial cell (ECs) mitochondrial biogenesis and antioxidant availability which is mediated by SIRT1, a well known anti-aging molecule. Another pivotal molecule involved in the shear stress benefits is CaMKKβ, an AMP-activated protein kinase (AMPK) kinase ablation of which resulted in impaired anti-oxidant activity in ECs as genomically knockdown of CaMKKP led to decreased SOD2 and Nrf2 expression, two major genes in the antioxidant system. Loss of CaMKKP also attenuated flow-induced activation of AMPK, therefore blocked eNOS activation, which is critical in endothelial-dependent vessel dilation. This is proved in the ex vivo experiment that arteries dissected from CaMKKP and AMPK knockout mice responsed poorly to flow compared to there wild type littermates. In conclusion, shear stress can induce SIRT1 mediated ECs mitochondrial biogenesis; the vascular protective effect of shear stress is also mediated through CaMKKβ-AMPK pathway, blocking of which leads to decreased antioxidant activity and impared response to flow induced vessel dilation.
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