mTOR complex protein expressions and rictor phosphorylation at Ser1235.

Cardiomyocytes were pretreated for 30 min with 20 nM rapamycin and then stimulated with 10 nM IGF-1 in presence or absence of 10 nM E2 for 24 h. A, shown are representative western blots for mTOR, rictor and raptor and B,C,D, quantitative analysis with mean ± SEM of fold stimulation by IGF-1 of at least 3 independently performed experiments (B,C,D). * p < 0.05, **p < 0.0095. Exposure to rapamycin lead to downregulation of mTOR and rictor, which were more pronounced under culture conditions without E2. E, Western blots for GSK3β-pS9, GSK3β, rictor pS1235 and rictor and F,G,H,I quantitative analysis of at least 3 independently performed experiments indicate that rictor phosphorylation at S1235 by GSK-3β which has been reported to interfere with Akt-substrate binding to mTORC2, thereby downregulating mTORC2 activity. IGF-1 induced strong phosphorylation of GSK-3β at Ser9 in the absence and presence of E2, however, rapamycin pretreatment only reduced this increased phosphorylation in E2 co-treated cardiomyocytes, indicating increased activity of GSK-3β. This higher activity was not associated with increased phosphorylation of rictor at S1235.