Transforming growth factor-beta inhibition of proteasomal activity: a potential mechanism of growth arrest

Although the proteasome plays a critical role in the controlled degradation of proteins involved in cell cycle control, the direct modulation of proteasomal function by growth regulatory signaling has not yet been demonstrated. We assessed the effect of transforming growth factor (TGF)-, a potent inhibitor of cell growth, on proteasomal function. TGF- selectively decreased hydrolysis of the proteasomal substrate Cbz-Leu-Leu-Leu-7-amido-4-methyl-coumarin (z-LLL-AMC) in a concentration-dependent manner but did not inhibit hydrolysis of other substrates Suc-Leu-Leu-Val-Tyr-AMC (suc-LLVY-AMC) or Cbz-Leu-Leu-Glu-AMC (z-LLE-AMC). An increase in intracellular oxidative injury occurred during incubation with TGF-. Furthermore, in vitro hydrolysis of z-LLL-AMC, but not suc-LLVY-AMC, was decreased by hydrogen peroxide. TGF- did not increase cellular expression of heat shock protein (HSP)90, a potent inhibitor of z-LLL-AMC hydrolysis in vitro. The physiological relevance of TGF- inhibition of proteasomal activity was studied by assessing the role of z-LLL-AMC hydrolysis on cyclin-dependent kinase inhibitor expression and cell growth. TGF- increased expression of p27^KIP1 but did not alter expression of p21^WAF1 or p16^INK4A. The peptide aldehyde Cbz-Leu-Leu-leucinal (LLL-CHO or MG132) potently inhibited z-LLL-AMC hydrolysis in cell extracts as well as increasing p27^KIP1 and decreasing cell proliferation. Thus growth inhibition by TGF- decreases a specific proteasomal activity via an HSP90-independent mechanism that may involve oxidative inactivation or modulation of proteasomal subunit composition and results in altered cellular expression of key cell cycle regulatory proteins such as p27^KIP1. oxidative stress; cytokine; heat shock protein; cell cycle regulation