USP17 regulates Ras activation and cell proliferation by blocking RCE1 activity

Abstract

The proto-oncogene Ras undergoes a series of post-translational modifications at its carboxyl-terminal CAAX motif that are essential for its proper membrane localization and function. One step in this process is the cleavage of the CAAX motif by the enzyme Ras-converting enzyme 1 (RCE1). Here we show that the deubiquitinating enzyme USP17 negatively regulates the activity of RCE1. We demonstrate that USP17 expression blocks Ras membrane localization and activation, thereby inhibiting phosphorylation of the downstream kinases MEK and ERK. Furthermore, we show that this effect is caused by the loss of RCE1 catalytic activity as a result of its deubiquitination by USP17. We also show that USP17 and RCE1 co-localize at the endoplasmic reticulum and that USP17 cannot block proliferation or Ras membrane localization in RCE1 null cells. These studies demonstrate that USP17 modulates Ras processing and activation, at least in part, by regulating RCE1 activity.

FIGURE 1.

USP17 blocks the Ras/MEK/ERK pathway. A, Ba/F3 cells expressing tetracycline-regulated USP17 were cultured for 48 h with and without tetracycline (Tet), cultured in the absence of fetal calf serum (FCS) for 4 h, and treated with IL-3 (100 units/ml) for the time periods specified and lysates immunoblotted with phospho-specific antibodies for ERK and MEK. In addition, to show equal loading, the same immunoblots were subsequently immunoblotted with antibodies specific for ERK2 and MEK1/2. The bottom panel shows USP17 expression. B, Ba/F3 cells expressing tetracycline regulated USP17 were cultured as above and treated with IL-3 for the time periods specified. Pulldowns were performed using GST-tagged Raf-RBD fusion protein. The resultant pulldowns and protein lysates were immunoblotted with pan-Ras antibody. C, MDA-MB-231 cells transfected as indicated were cultured without serum for 4 h and stimulated with serum for the periods specified. The resultant Raf-RBD pulldowns (upper panel), and protein lysates (lower panel) were immunoblotted with a pan-Ras antibody. In addition RNA was extracted from parallel transfections, and RT-PCR was performed as indicated.

FIGURE 2.

USP17 blocks Ras membrane localization. WT MEF cells transiently transfected with vectors expressing GFP-H-Ras, USP17, or USP17CS were fixed. The localization of GFP-H-Ras (top row) and USP17 or USP17CS (middle row), as well as both together (bottom row) was then assessed by confocal microscopy. In panels 1 and 3, the yellow arrows indicate membrane localization of GFP-H-Ras.

FIGURE 3.

USP17 deubiquitinates RCE1. 293T cells were transiently transfected with vectors expressing either RCE1-FLAG, HA-ubiquitin, USP17, or USP17CS. The lysates were extracted and immunoprecipitated where indicated with the FLAG epitope antibody and immunoblotted for: USP17, HA epitope-tagged ubiquitin, and FLAG epitope-tagged RCE1 (A); HA epitope-tagged CYLD, FLAG epitope-tagged RCE1, and HA epitope-tagged ubiquitin (B); and HA epitope-tagged K63 ubiquitin, FLAG epitope-tagged RCE1, and USP17 (C). D, RCE1^-/- (panels 1-3) and WT (panels 4-6) MEF cells transiently transfected with the indicated expression vectors were fixed. The localization of GFP-H-Ras (top row) and USP17 or USP17CS (middle row), as well as both together (bottom row) was then assessed by confocal microscopy. The yellow arrows indicate membrane localization of GFP-H-Ras.

FIGURE 4.

USP17 blocks RCE1 activity. 293T cells were transiently transfected with either empty vector (EV) or vectors expressing RCE1-FLAG, USP17, USP17CS, shRNA1, or scrambled shRNA. Membrane fractions were extracted and incubated with a RCE1-specific quenched fluorescent peptidyl substrate, and activity was determined by monitoring hydrolysis of this substrate in the presence of each membrane fraction in a time course assay (A and B, top panels). Concurrently, protein lysates were extracted from a sample of the transfection, and equal amounts of protein were immunoprecipitated and analyzed as in Fig. 2 to determine that equal amounts of RCE1 were present in each sample assessed for RCE1 activity (A, lower panel, and B, lower left panel). RNA was extracted and RT-PCR was performed as indicated to check shRNA knockdown (B, lower right panel). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 5.

USP17 and RCE1 co-localize. A, Cos7 cells were transiently transfected with HA-RCE1. RCE1 was visualized by confocal using an anti-HA antibody (first panel), and an anti-calnexin antibody (second panel) was used to visualize the ER. An overlay is also provided (third panel). B, the endogenous distribution of USP17 was examined in HeLa cells. USP17 was visualized using a USP17 specific monoclonal antibody (first panel), and an anti-calnexin antibody (second panel) was used to visualize the ER. An overlay is also provided (third panel). C, Cos7 cells were transiently transfected with USP17CS-FLAG and HA-RCE1. USP17CS was visualized using an anti-FLAG antibody (second panel) and HA-RCE1 (third panel) and the ER (first panel) were visualized as above. An overlay is also provided (fourth panel).

FIGURE 6.

USP17 regulation of growth is RCE1-dependent. WT and RCE1^-/- MEF cells were infected with either bicistronic retroviral vectors expressing EGFP and either USP17-FLAG or USP17CS-FLAG or with empty vector (EV). The cells were sorted upon EGFP expression to produce populations of >99% positive cells, and then the proliferation of each population was monitored by trypan blue exclusion assay. The results are illustrated as fold increase of cell numbers over time.