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"Role of calcium in nutrient stimulation of mTORC1"

Ishwarya Murali
Pharmacology and Physiology Program
B.S. 2009, SASTRA University, India

Thesis Advisor: Andrew P. Thomas, Ph.D.
Department of Pharmacology, Physiology and Neuroscience

Monday, July 31, 2017
1:30 P.M., MSB Room H609b


Diabetes and cancer are a worldwide problem today and an important predisposing factor appears to be obesity associated with nutrient overload and inactivity. In addition to dietary carbohydrate and fat, excess protein consumption resulting in increased circulating levels of amino acids (AAs) has been shown to induce insulin resistance and disturb glucose homeostasis. AA overload desensitizes insulin signaling pathways by causing persistent activation of mammalian target of rapamycin complex 1 (mTORC1) and downstream targets including S6 kinase 1 (S6K1) and ribosomal protein S6. mTORC1 is a master cell growth regulator and has a key role in cell growth and metabolism. The constitutively active mTORC1 has also been shown to play a role in several types of cancer including breast, colon, and ovarian cancer. Previous studies from our lab and other research groups have suggested the involvement of calcium [Ca2+] in AA-induced mTORC1 activation. However, the underlying molecular mechanism by which Ca2+ alters AA-induced S6K1 activation is yet to be discovered. The goal of this research project is to investigate the role of intracellular calcium [Ca2+]i in AA-induced mTORC1/S6K1 activation. This study was performed in MDA-MB-231 breast cancer cell line. Application of extracellular AAs on cells deprived of serum and AAs for 2 hours, induces an increase in [Ca2+]i (p<0.01; live cell imaging) levels, which was shown to be essential for S6K1 activation because, [Ca2+]i chelation using BAPTA-AM suppressed S6K1 phosphorylation (p<0.001; pS6/totalS6). We further investigated the source of Ca2+ and found that extracellular Ca2+ does not play a role in the AA-induced [Ca2+]i increase, because chelating extracellular Ca2+ using EGTA did not affect AA-induced S6K1/S6 phosphorylation. Ca2+ release from endoplasmic reticulum (ER) by both inositol 1,4,5-triphosphate-mediated signaling and the ER Ca2+ ATPase inhibitor, thapsigargin (Tg) did not affect AA-induced S6K1/S6 activation. It has been shown by others that mTORC1 translocate to lysosomal membrane in the presence of AAs. We examined the role of lysosomal Ca2+ in AA-induced S6K1/S6 activation using nigericin (a K+/H+ ionophore) and concanamycin A (a vacuolar ATPase inhibitor). Both nigericin and concanamycin A collapse lysosomal pH gradient, thereby indirectly releasing Ca2+ from lysosomes. Cells pretreated with nigericin and concanamycin A had decreased AA-induced S6K1/S6 phosphorylation (p<0.05 and p<0.005 respectively; pS6/totalS6) and reduced AA-induced [Ca2+]i release suggesting that Ca2+ release from lysosomes is critical for AA-induced mTORC1 activation. Taken together, our results indicate that AA-induced S6K1/S6 activation is mediated via local Ca2+ release from lysosomes.

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