Fluorescent tetradecanoylphorbol acetate: A novel probe of phorbol ester binding domains

Margit Balázs, Jánòs Szöllösi, William C. Lee, Richard P. Haugland, Anthony P. Guzikowski, Mack J. Fulwyler, Sandor Damjanovich, Burt G. Feuerstein, Harrihar A. Pershadsingh

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

Protein kinase C (PKC) has a prominent role in signal transduction of many bioactive substances. We synthesized the fluorescent derivative, phorbol‐13‐acetate‐12‐N‐methyl‐N‐4‐(N,N′‐di(2‐hydroxyethyl)amino)‐7‐nitrobenz‐2‐oxa‐1,3‐diazole‐aminododecanoate (N‐C12‐Ac(13)) of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) to monitor the location of phorbol ester binding sites and evaluate its potential use as a probe of PKC in viable cells. The excitation maximum wavelength of N‐C12‐Ac(13) is close to 488 nm, facilitating its use in argon‐ion laser flow and imaging cytometry. When incubated with 100 nM N‐C12‐Ac(13) at 25°C, P3HR‐1 Burkitt lymphoma cells accumulated the dye rapidly, reaching maximum fluorescence within 25 min, 20‐fold above autofluorescence. Addition of unlabeled TPA significantly decreased the fluorescence of N‐C12‐Ac(13) stained cells in a dose‐dependent manner indicating specific displacement of the bound fluoroprobe. Competitive displacement of [3H]‐phorbol‐12,13‐dibutyrate ([3H]‐PBu2) from rat brain cytosol with N‐C12‐Ac(13) gave an apparent dissociation constant (Kd) of 11 nM. N‐C12‐Ac(13) possessed biological activity similar to TPA. Like TPA (final concentration 65 nM) N‐C12‐Ac(13), at a lower concentration (51 nM), induced expression of Epstein‐Barr viral glycoprotein in P3HR‐1 cells, differentiation of promyelocytic HL60 cells, and caused predicted changes in the mitotic cycle of histiocytic DD cells. Microspectrofluorometric images of single cells labeled with N‐C12‐Ac(13) showed bright fluorescence localized intracellularly and dim fluorescence in the nuclear region, consistent with dye binding mainly to cytoplasmic structures and/or organelles and being mostly excluded from the nucleus. Because of the high level of non‐specific binding of N‐C12‐Ac(13), this probe is not ideal for visualizing PKC in intact cells, but would be a valuable fluoroprobe to investigate the kinetic properties of purified PKC. Also, knowledge gained from these studies allows us to predict structures of fluorescent phorbols likely to have less non‐specific binding and, consequently, be potentially useful for monitoring PKC in viable cells.

Original languageEnglish (US)
Pages (from-to)266-276
Number of pages11
JournalJournal of Cellular Biochemistry
Volume46
Issue number3
DOIs
StatePublished - Jul 1991
Externally publishedYes

Keywords

  • flow cytometry
  • fluorescence anisotropy
  • fluorescence recovery after photobleaching
  • image cytometry
  • protein kinase C

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Balázs, M., Szöllösi, J., Lee, W. C., Haugland, R. P., Guzikowski, A. P., Fulwyler, M. J., Damjanovich, S., Feuerstein, B. G., & Pershadsingh, H. A. (1991). Fluorescent tetradecanoylphorbol acetate: A novel probe of phorbol ester binding domains. Journal of Cellular Biochemistry, 46(3), 266-276. https://doi.org/10.1002/jcb.240460311