May 2007
Volume 48, Issue 13
ARVO Annual Meeting Abstract  |   May 2007
RSK-Independent Synthesis of p112BAD: Implications to Mitochondrial Protection in Human Lens Epithelial Cells
Author Affiliations & Notes
  • P. R. Cammarata
    Cell Biology & Genetics, UNT Hlth Sci Cntr, Fort Worth, Texas
  • J. M. Flynn
    Cell Biology & Genetics, UNT Hlth Sci Cntr, Fort Worth, Texas
  • J. A. Smith
    U of Virginia, Charlottesville, Virginia
  • D. A. Lannigan
    U of Virginia, Charlottesville, Virginia
  • Footnotes
    Commercial Relationships P.R. Cammarata, None; J.M. Flynn, None; J.A. Smith, None; D.A. Lannigan, None.
  • Footnotes
    Support None.
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 5647. doi:
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      P. R. Cammarata, J. M. Flynn, J. A. Smith, D. A. Lannigan; RSK-Independent Synthesis of p112BAD: Implications to Mitochondrial Protection in Human Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5647.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose:: 17-beta estradiol (E2) modulates the degree of oxidative stress-induced depolarization of mitochondrial membrane potential (MMP) in cultured human lens epithelial cells (HLE-B3), following H2O2 insult, by activation of mitogen-activated protein kinase (MAPK) (Mitochondrion 5:235-247; 2005). The p90 ribosomal S6 kinase (RSK) family of serine/threonine protein kinases are downstream effectors of MAPK, which upon activation lead to the phosphorylation of BAD at S112. Herein we determined whether E2 also activates BAD to yield p112BAD in B3 cells.

Methods:: Specific antisera and Western blot analysis was used to detect pERK and pBAD.

Results:: E2 (1 µM) addition to serum deprived B3 cells prompted the rapid phosphorylation of ERK 1/2 within 5-15 min and BAD phosphorylation within 15-30 min at S112. RSK was phosphorylated at multiple serine and threonine residues within a similar timeframe as p112BAD. The Thr359/Ser363 sites of RSK appeared E2-responsive, suggesting that E2 might play a role in modulating RSK activity. The 3-phosphoinositide-dependent protein kinase-1 (Ser221) and C-terminal kinase (Thr573) sites of RSK were highly phosphorylated in the absence of serum and both sites appeared to be E2-insensitive. B3 cells exposed to the RSK specific inhibitor, SL0101 (100 µM) prior to E2 addition did not affect upstream E2-stimulated pERK 1/2 but failed to block BAD phosphorylation at S112. Complete inhibition of RSK activity was confirmed by demonstrating that the RSK substrate, S6, was not phosphorylated in the presence of SL0101, under conditions where BAD remained phosphorylated at the S112 site. Treatment with UO126 (10 µM) blocked activation of ERK but not the E2-induced increase in p112BAD.

Conclusions:: SL0101’s and UO126’s inability to block BAD phosphorylation at S112 in parallel suggest a RSK-independent pathway contributes to p112BAD phosphorylation in HLE-B3 cells. SL0101 is currently being used in combination with several specific signal transduction inhibitors; KT5720 (PKA) and SP600125 (JNK), as well as RNA interference of ERK 1 and ERK 2 in order to identify potential RSK-independent pathways of p112BAD synthesis. Our goal is to distinguish the function of RSK from that of MAPK insofar as whether E2-mediated protection of MMP involves ERK 1/2 activation, p112BAD accumulation, or synergistic interplay resulting from ERK and BAD phosphorylation. This is a logical starting point for analysis of these complex signaling pathways, without which, interaction and cross-talk between pathways may otherwise prove difficult to dissociate.

Keywords: signal transduction • mitochondria • cell survival 

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