FKBP12, the 12-kDa FK506-binding protein, binds to the immunoppressant FK506 and rapamycin with high affinity. Although first identified in immune cells, FKBP12 is expressed in all tissues, with particularly high levels in the brain tissue. It possesses cis-trans peptidyl-prolyl isomerase or rotamase activity that is essential for efficient folding of proteins. FKBP12 is reported to modulate intracellular calcium release by affecting the activity of IP3 and ryanodine receptors. FKBP12 strongly associates with RyR1 with a stoichiometry of 4 FKBP12 molecules per tetrameric channel. FKBP12 is also required for T-cell receptor-mediated signal transduction, leading to transcription of interleukin-2. When associated with FK506, the FK506-FKBP12 complex inhibits calcineurin activity and thereby blocks the transcription of early T-cell activation genes.

The activity of mTOR was assessed without inhibitor and in the presence of rapamycin alone (Cat. No. 553210), FKBP12 alone (Cat. No. 325902), FKBP12/rapamycin complex, and Wortmannin (Cat. No. 681675), using the K-LISA™ mTOR Activity Kit (Cat. No. CBA055); For more details, see protocol. The FKBP12/rapamaycin inhibitor complex was pre-assembled by mixing 62.5 mg/ml FKBP12 and 50 mM rapamycin on ice for 10 min. The mTOR Standard (50 ml) was incubated for 15 min on ice with FKBP12 (2 ml: final concentration 2.5 mg/ml), rapamycin (2 ml; final concentration 2 mM). FKPP12/rapamycin complex (final concentration 2.5 mg/ml, 2 mM rapamycin) or wortmannin (concentration 10 mM. The kinase reaction was initiated by transferring each mixture to a corresponding well containing 2X kinase buffer and ATP. The results were assessed as outlined in the user protocol.