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Analytical and Bioanalytical Chemistry

, Volume 397, Issue 8, pp 3359–3367 | Cite as

Analysis of protein kinase A activity in insulin-secreting cells using a cell-penetrating protein substrate and capillary electrophoresis

  • Femina Rauf
  • Yiding Huang
  • Thusitha P. Muhandiramlage
  • Craig A. Aspinwall
Original Paper

Abstract

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 in Escherichia coli and 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).

Figure

Analysis of PKA activity via chimeric fluorescent kinase substrate proteins

Keywords

Capillary electrophoresis Protein kinase A Cell-penetrating peptides Fluorescent protein 

Notes

Acknowledgments

We thank Dr. Sergey N. Krylov from York University Canada for providing the plasmid for the protein kinase A substrate used for preliminary studies. This work was supported by National Institute of Health (NIH-GM074522) and National Science Foundation (NSF-0548167).

Supplementary material

216_2010_3776_MOESM1_ESM.pdf (105 kb)
ESM. 1 (PDF 104 kb)

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Femina Rauf
    • 1
  • Yiding Huang
    • 1
  • Thusitha P. Muhandiramlage
    • 1
  • Craig A. Aspinwall
    • 1
  1. 1.Department of Chemistry and BiochemistryUniversity of ArizonaTucsonUSA

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