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Localized measurement of kinase activation in oocytes of Xenopus laevis

Nature Biotechnology volume 17, pages 759–762 (1999)Cite this article

Abstract

We have combined a rapid cytoplasmic sampling technique with capillary electrophoresis to measure the activation of protein kinase C (PKC) in a small region (approximately 60 μm) of a Xenopus oocyte. The phosphorylation of a fluorescent PKC substrate was measured following addition of a pharmacological or physiological stimulus to an oocyte. When substrates for cdc2 kinase (cdc2K), PKC, and protein kinase A (PKA) were comicroinjected into an oocyte, all three substrates could be identified on the electropherogram after cytoplasmic sampling. With this new method, it should be possible to measure simultaneously the activation of multiple different kinases in a single cell, enabling the quantitative dissection of signal transduction pathways.

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Figure 1: Electrophoresis of F-PKC and PF-PKC dissolved in a buffer solution or microinjected into an oocyte.
Figure 2: Yolk platelet proteins interact with and alter the electrophoretic mobility of F-PKC.
Figure 3: Components for sampling and CE.
Figure 4: Time course of F-PKC phosphorylation in oocytes exposed to PMA or LPA.
Figure 5: Detection limits for F-PKC in oocytes.
Figure 6: Separation of F-PKC, F-PKA, and F-cdc2K from an oocyte.

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Acknowledgements

The authors are grateful to Jay Boniface and Augie Sanchez for advice on purification and fluorescent labeling of peptide, and to Hong Ngoc Dao, Cuong Vu, and Veronica Luzzi for technical assistance. The authors also thank Lee Moritz, Richard Busby, and Ron Hulme for fabrication of devices and for helpful discussions on equipment design. This work was supported by the Arnold and Mabel Beckman Foundation, the Searle Scholars Program, and the National Institutes of Health (GM57015 and RR13314 to N.L.A., K08AI01513 to C.E.S.). C.-L.L. was supported by a fellowship from the National Defense Medical Center, Taiwan R.O.C.

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Authors and Affiliations

  1. Institute of Aerospace Medicine, National Defense Medical Center, Taipei, R.O.C., Taiwan

    Chao-Lin Lee

  2. Department of Physiology and Biophysics, University of California-Irvine, Irvine, 92697-4560 , CA

    James Linton, Joseph S. Soughayer, Christopher E. Sims & Nancy L. Allbritton

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  1. Chao-Lin Lee
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Correspondence to Nancy L. Allbritton.

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Lee, CL., Linton, J., Soughayer, J. et al. Localized measurement of kinase activation in oocytes of Xenopus laevis . Nat Biotechnol 17, 759–762 (1999). https://doi.org/10.1038/11691

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