An Investigation of Kinases Responsible for Site-specific Phosphorylation of p70s6k1 in Skeletal Muscle Following Chronic Functional Overloading
Open Access
- Author:
- Martin, Tony Dewayne
- Graduate Program:
- Physiology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- January 24, 2014
- Committee Members:
- Leonard Shelton Jefferson Jr., Thesis Advisor/Co-Advisor
Scot R Kimball, Thesis Advisor/Co-Advisor
Ralph Lauren Keil, Thesis Advisor/Co-Advisor - Keywords:
- MAPK signaling
muscle hypertrophy
tenotomy - Abstract:
- ABSTRACT The protein kinase p70S6K1 is a known regulator of cell growth as deletion of its gene results in smaller cell size and consequently reduced organ and body sizes. Its activity is modulated through multisite phosphorylation, some of which is mediated by the mechanistic target of rapamycin complex 1 (mTORC1) pathway. The present project was designed to investigate the roles of mTORC1 and that of other signaling pathways in the phosphorylation of p70S6K1 in an animal model of induced hypertrophic growth of skeletal muscle. Within 24 h of male Sprague Dawley rats undergoing unilateral tenotomy to induce chronic functional overloading of the plantaris muscle, phosphorylation of the Thr389 and the Thr421/Ser424 sites on p70S6K1 was significantly elevated compared to a control plantaris from the contralateral leg. To further investigate regulation of phosphorylation of these sites on p70S6K1, a cell culture model system was employed for easier manipulation of signaling pathways. In HEK293E cells deprived of serum for 2.5 h to impair cell growth, treatment with insulin-like-growth factor 1 (IGF-1) for 30 min resulted in a robust stimulation of the phosphorylation of the Thr389 and the Thr421/Ser424 sites on p70S6K1. In this model system, rapamycin, an inhibitor of mTORC1, and TORIN2, an inhibitor of both mTORC1 and mTORC2, each prevented IGF-1-induced stimulated phosphorylation of the Thr389 site on p70S6K1 but not that of the Thr421/Ser424 sites. The JNK inhibitor SP600125 attenuated the IGF-1-induced stimulated phosphorylation of the Thr421/Ser424 sites on p70S6K1 but not that of the Thr389 site, and in combination with TORIN2 it blocked the effect of IGF1 on both sites. In contrast, inhibitors of the MAP kinase signaling pathway including UO126 and SB203580 had no effect on the IGF-1-induced stimulated phosphorylation of the Thr 389 and the Thr421/Ser424 sites whereas anisomycin, an activator of the JNK pathway specifically stimulated phosphorylation of the Thr421/Ser424 sites but not the Thr389 site. To assess a possible role for JNK in mediating phosphorylation of p70S6K1 on the Thr421/Ser424 sites in vivo, rats were treated with SP600125 6 h prior to tissue harvest following tenotomy. SP600125 attenuated the tenotomy-induced elevated phosphorylation of the Thr421/Ser424 sites on p70S6K1 but had no effect on that of Thr389. Phosphorylation of JNK was elevated following tenotomy and this was prevented by treatment with SP600125, providing additional evidence of a role for the JNK signaling pathway in mediating regulation of p70S6K1 in this experimental model of hypertrophic growth. Taken together, the results from these two experimental model systems demonstrate that both the mTORC1 and JNK signaling pathways contribute to the stimulation of cell growth by mediating site-specific phosphorylation of p70S6K1.