Quantitative phosphoproteomic analysis reveals unique cAMP signaling pools emanating from AC2 and AC6 in human airway smooth muscle cells.
Quantitative phosphoproteomic analysis reveals unique cAMP signaling pools emanating from AC2 and AC6 in human airway smooth muscle cells.
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DOI：
10.3389/fphys.2023.1149063
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发表时间：
2023
影响因子：
4
通讯作者：
中科院分区：
文献类型：
--
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人类气道平滑肌（HASM）是β AR激动剂的主要靶点，用于控制哮喘和慢性阻塞性肺病（COPD）的气道过度收缩。β AR激动剂通过腺苷酸环化酶（AC）诱导cAMP的产生，激活PKA并引起支气管扩张。在人气道平滑肌细胞中表达的其他几种G蛋白偶联受体（GPCR）通过cAMP抑制细胞外信号，但这些受体引起不同的细胞反应。一些G-蛋白偶联受体偶联到不同的腺苷酸环化酶异构体与不同的定位，部分解释了这种区室化，但鲜为人知的是下游网络的结果。我们使用定量磷酸化蛋白质组学来确定由两种腺苷酸环化酶异构体产生的cAMP的下游信号网络，其在人气道平滑肌中的定位存在差异。在使用毛喉素短暂刺激腺苷酸环化酶活性后，通过LC-MS/MS分析磷酸肽，并将过表达AC 2（位于非筏膜中）或AC 6（位于脂筏膜中）的细胞与对照人气道平滑肌之间的差异进行比较。滴定AC 2和AC 6过表达的程度以产生大致相等的毛喉素刺激的cAMP产生。14个差异磷酸化蛋白（DPP）由AC 2活性产生，34个差异磷酸化蛋白由AC 6活性产生。使用STRING蛋白相互作用工具对这些命中的分析表明，AC 2信号传导与RNA/DNA结合蛋白和微管/纺锤体蛋白的修饰更相关，而AC 6信号传导与调节自噬、钙-钙调蛋白（Ca 2 +/CaM）信号传导、Rho GTP酶和细胞骨架调节的蛋白质相关。一种蛋白质，OFD 1，在相反的方向调节，与丝氨酸899磷酸化增加AC 6条件下的1.5倍，但降低到0.46倍，由AC 2。总之，定量磷酸化蛋白质组学是一个强大的工具，破译复杂的信号网络产生的谨慎的信号事件发生在cAMP车厢。我们的数据显示了AC 2和AC 6活性产生的cAMP池的关键差异，并暗示了这两个隔室调节不同的细胞反应。
Human airway smooth muscle (HASM) is the primary target of ßAR agonists used to control airway hypercontractility in asthma and chronic obstructive pulmonary disease (COPD). ßAR agonists induce the production of cAMP by adenylyl cyclases (ACs), activate PKA and cause bronchodilation. Several other G-protein coupled receptors (GPCR) expressed in human airway smooth muscle cells transduce extracellular signals through cAMP but these receptors elicit different cellular responses. Some G-protein coupled receptors couple to distinct adenylyl cyclases isoforms with different localization, partly explaining this compartmentation, but little is known about the downstream networks that result. We used quantitative phosphoproteomics to define the downstream signaling networks emanating from cAMP produced by two adenylyl cyclases isoforms with contrasting localization in uman airway smooth muscle. After a short stimulus of adenylyl cyclases activity using forskolin, phosphopeptides were analyzed by LC-MS/MS and differences between cells overexpressing AC2 (localized in non-raft membranes) or AC6 (localized in lipid raft membranes) were compared to control human airway smooth muscle. The degree of AC2 and AC6 overexpression was titrated to generate roughly equal forskolin-stimulated cAMP production. 14 Differentially phosphorylated proteins (DPPs) resulted from AC2 activity and 34 differentially phosphorylated proteins resulted from AC6 activity. Analysis of these hits with the STRING protein interaction tool showed that AC2 signaling is more associated with modifications in RNA/DNA binding proteins and microtubule/spindle body proteins while AC6 signaling is associated with proteins regulating autophagy, calcium-calmodulin (Ca2+/CaM) signaling, Rho GTPases and cytoskeletal regulation. One protein, OFD1, was regulated in opposite directions, with serine 899 phosphorylation increased in the AC6 condition 1.5-fold but decreased to 0.46-fold by AC2. In conclusion, quantitative phosphoproteomics is a powerful tool for deciphering the complex signaling networks resulting from discreet signaling events that occur in cAMP compartments. Our data show key differences in the cAMP pools generated from AC2 and AC6 activity and imply that distinct cellular responses are regulated by these two compartments.
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影响因子：
5.9
作者：
Horvath M;Petrvalska O;Herman P;Obsilova V;Obsil T
通讯作者：
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