Differential Effects of Ceramide and Sphingosine 1-Phosphate on ERM Phosphorylation PROBING SPHINGOLIPID SIGNALING AT THE OUTER PLASMA MEMBRANE
Differential Effects of Ceramide and Sphingosine 1-Phosphate on ERM Phosphorylation PROBING SPHINGOLIPID SIGNALING AT THE OUTER PLASMA MEMBRANE
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DOI：
10.1074/jbc.m110.141028
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发表时间：
2010-10-15
影响因子：
4.8
通讯作者：
Hannun, Yusuf A.
中科院分区：
文献类型：
--
作者：
Canals, Daniel;Jenkins, Russell W.;Hannun, Yusuf A.
ERM蛋白通过最C-末端苏氨酸残基的磷酸化来调节，将它们从活化形式转换为失活形式。然而，人们对这一规定的控制知之甚少。我们小组先前的工作表明，分泌的酸性鞘磷脂酶作用于ERM去磷酸化的上游，这表明鞘磷脂（SM）水解参与了ERM的调节。为了确定特定脂质的作用，我们采用重组细菌鞘磷脂酶（bSMase）作为SM在质膜代谢的直接探针。bSMase诱导ERM去磷酸化的快速剂量和时间依赖性降低。如使用重组鞘磷脂酶D所示，ERM去磷酸化是由神经酰胺产生驱动的，而不是由鞘磷脂消耗驱动的。在质膜上产生的神经酰胺足以进行ERM调节，并且不需要细胞内SM水解，如使用特异性结合SM的Venus标记的胞溶素探针所观察到的。有趣的是，使用细菌神经酰胺酶水解质膜bSM酶诱导的神经酰胺导致ERM过度磷酸化和细胞表面突起的形成。质膜神经酰胺水解的影响是由于鞘氨醇1-磷酸的形成，因为ERM磷酸化被鞘氨醇激酶抑制剂阻断并由鞘氨醇1-磷酸诱导。两者合计，这些结果表明了一种新的调节机制的ERM磷酸化的鞘脂与神经酰胺和鞘氨醇1-磷酸的相反的行动。该方法还定义了一个工具包，以探测鞘脂信号在质膜。
ERM proteins are regulated by phosphorylation of the most C-terminal threonine residue, switching them from an activated to an inactivated form. However, little is known about the control of this regulation. Previous work in our group demonstrated that secretion of acid sphingomyelinase acts upstream of ERM dephosphorylation, suggesting the involvement of sphingomyelin (SM) hydrolysis in ERM regulation. To define the role of specific lipids, we employed recombinant bacterial sphingomyelinase (bSMase) as a direct probe of SM metabolism at the plasma membrane. bSMase induced a rapid dose-and time-dependent decrease in ERM dephosphorylation. ERM dephosphorylation was driven by ceramide generation and not by sphingomyelin depletion, as shown using recombinant sphingomyelinase D. The generation of ceramide at the plasma membrane was sufficient for ERM regulation, and no intracellular SM hydrolysis was required, as was visualized using Venus-tagged lysenin probe, which specifically binds SM. Interestingly, hydrolysis of plasma membrane bSMase-induced ceramide using bacterial ceramidase caused ERM hyperphosphorylation and formation of cell surface protrusions. The effects of plasma membrane ceramide hydrolysis were due to sphingosine 1-phosphate formation, as ERM phosphorylation was blocked by an inhibitor of sphingosine kinase and induced by sphingosine 1-phosphate. Taken together, these results demonstrate a new regulatory mechanism of ERM phosphorylation by sphingolipids with opposing actions of ceramide and sphingosine 1-phosphate. The approach also defines a tool kit to probe sphingolipid signaling at the plasma membrane.