Regulation of Acidification and Apoptosis by SHP-1 and Bcl-2

Muthusamy Thangaraju,Kamal Sharma,Brian Leber,David W Andrews,Shi-Hsiang Shen,Coimbatore B Srikant

doi:10.1074/jbc.274.41.29549

Muthusamy Thangaraju, Kamal Sharma + Show 4 more

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https://doi.org/10.1074/jbc.274.41.29549

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Abstract

Recruitment of the SH2 domain containing cytoplasmic protein-tyrosine phosphatase SHP-1 to the membrane by somatostatin (SST) is an early event in its antiproliferative signaling that induces intracellular acidification-dependent apoptosis in breast cancer cells. Fas ligation also induces acidification-dependent apoptosis in a manner requiring the presence of SHP-1 at the membrane. Moreover, we have recently reported that SHP-1 is required not only for acidification, but also for apoptotic events that follow acidification (Thangaraju, M., Sharma, K., Liu, D., Shen, S. H., and Srikant, C. B. (1999) Cancer Res. 59, 1649-1654). Here we show that ectopically expressed SHP-1 was predominantly membrane-associated and amplified the cytotoxic signaling initiated upon SST receptor activation and Fas ligation. The catalytically inactive mutant of SHP-1 (SHP-1C455S) abolished the ability of the SST agonists to signal apoptosis by preventing the recruitment of wild type SHP-1 to the membrane. Overexpression of the anti-apoptotic protein Bcl-2 in MCF-7 cells inhibited SST-induced apoptosis upstream of acidification by inhibiting p53-dependent induction of Bax as well as by raising the resting pH(i) and attenuating SST-induced decrease in pH(i). By contrast, Bcl-2 failed to prevent apoptosis triggered by direct acidification. These data demonstrate that (i) membrane-associated SHP-1 is required for receptor-mediated cytotoxic signaling that causes intracellular acidification and apoptosis, and (ii) Bcl-2 acts distal to SHP-1 and p53 to prevent SST-induced acidification but cannot inhibit the apoptotic events that ensue intracellular acidification.

Highlights

Apoptosis is a unique physiological mechanism that eliminates discrete cells in normal development, host defense, and suppression of oncogenesis
In order to obtain definitive evidence for the involvement of SHP-1 in SSTR-initiated and Fas-mediated intracellular acidification cytotoxic signaling, we evaluated the effect of the octapeptide SST analog octreotide (OCT) and Fas ligation in MCF-7 cells transfected with SHP-1 or its catalytically inactive mutant SHP-1C455S
Ectopic Expression of SHP-1, SHP-1C455S, and Bcl-2 Does Not Affect SSTR Function—We first established that overexpression of SHP-1, its inactive mutant, or Bcl-2 did not alter SSTR levels since the binding capacity determined by saturation binding analysis using [125I-Tyr3]OCT was similar in all cell lines and ranged from 356 Ϯ 34 to 389 Ϯ 65 fmol/mg protein

Summary

Introduction

Apoptosis is a unique physiological mechanism that eliminates discrete cells in normal development, host defense, and suppression of oncogenesis. The onset of nuclear catastrophe is preceded by events in non-nuclear regions of the cell including activation of proteases and ion-fluxes, and disruption of membrane potentials and cytoskeletal architecture. These are in turn regulated by other cellular components including protein kinases and phosphatases, and members of the Bcl-2 family. The non-transmembrane tyrosine phosphatases SHP-1 (SHPTP1/PTP11C/HCP/SHP/PTP-N6) and SHP-2 (PTP1D/ PTP2C/SHPTP2/Syp/corkscrew) are critical regulators of cellular function. SHP-1 is known to associate with multiple signaling molecules such as growth factor receptor tyrosine kinases, non-receptor tyrosine kinases, activated cytokine receptor, interleukin-3 receptor ␤ chain, erythropoietin receptor, interferon ␣/␤ receptor, and FC␥RIIB receptor (6 –12). SHP-1 was required for SST-induced intracellular acidification and for acidification-triggered apoptosis to occur [25]

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