Analysis of protein kinase A activity in insulin-secreting cells using a cell-penetrating protein substrate and capillary electrophoresis.
Analysis of protein kinase A activity in insulin-secreting cells using a cell-penetrating protein substrate and capillary electrophoresis.
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使用细胞穿透蛋白底物和毛细管电泳分析胰岛素分泌细胞中的蛋白激酶 A 活性。
DOI：
10.1007/s00216-010-3776-7
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发表时间：
2010
影响因子：
4.3
通讯作者：
Aspinwall,CraigA
中科院分区：
文献类型：
--
作者：
Rauf,Femina;Huang,Yiding;Muhandiramlage,ThusithaP;Aspinwall,CraigA
A cell-penetrating, fluorescent protein substrate was developed to monitor intracellular protein kinase A (PKA) activity in cells without the need for cellular transfection. The PKA substrate (PKAS) was prepared with a 6×histidine purification tag, an enhanced green fluorescent protein (EGFP) reporter, an HIV-TAT protein transduction domain for cellular translocation and a pentaphosphorylation motif specific for PKA. PKAS was expressed inEscherichia coliand purified by metal affinity chromatography. Incubation of PKAS in the extracellular media facilitated translocation into the intracellular milieu in HeLa cells, βTC-3 cells and pancreatic islets with minimal toxicity in a time and concentration dependent manner. Upon cellular loading, glucose-dependent phosphorylation of PKAS was observed in both βTC-3 cells and pancreatic islets via capillary zone electrophoresis. In pancreatic islets, maximal PKAS phosphorylation (83 ± 6%) was observed at 12 mM glucose, whereas maximal PKAS phosphorylation (86 ± 4%) in βTC-3 cells was observed at 3 mM glucose indicating a left-shifted glucose sensitivity. Increased PKAS phosphorylation was observed in the presence of PKA stimulators forskolin and 8-Br-cAMP (33% and 16%, respectively), with corresponding decreases in PKAS phosphorylation observed in the presence of PKA inhibitors staurosporine and H-89 (40% and 54%, respectively).FigureAnalysis of PKA activity via chimeric fluorescent kinase substrate proteins
