Dynamic regulation of the Ras pathway via proteolysis of the NF1 tumor suppressor - PubMed (original) (raw)

Dynamic regulation of the Ras pathway via proteolysis of the NF1 tumor suppressor

Karen Cichowski et al. Genes Dev. 2003.

Abstract

Mutations in the NF1 tumor suppressor underlie the familial tumor predisposition syndrome neurofibromatosis type I. Although its encoded protein, neurofibromin, functions as a Ras-GTPase activating protein (GAP), nothing is known about how it is normally regulated or its precise role in controlling Ras signaling pathways. We show here that neurofibromin is dynamically regulated by the ubiquitin-proteasome pathway. Degradation is rapidly triggered in response to a variety of growth factors and requires sequences adjacent to the catalytic GAP-related domain of neurofibromin. However, whereas degradation is rapid, neurofibromin levels are re-elevated shortly after growth factor treatment. Accordingly, Nf1-deficient mouse embryonic fibroblasts (MEFs) exhibit an enhanced activation of Ras, prolonged Ras and ERK activities, and proliferate in response to subthreshold levels of growth factors. Thus, the dynamic proteasomal regulation of neurofibromin represents an important mechanism of controlling both the amplitude and duration of Ras-mediated signaling. Furthermore, this previously unrecognized Ras regulatory mechanism may be exploited therapeutically.

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Figures

Figure 1

Neurofibromin is degraded in response to serum and growth factors. (A) Serum-starved NIH 3T3 cells were exposed to 10% serum for increasing lengths of time. Cells were lysed with 1% boiling SDS buffer and Western blots were probed with antibodies that recognize an epitope located in the C terminus of neurofibromin (top, NF1GRP-D) and an anti-p120RasGAP antibody (bottom). The lane labeled c (control) is full-length neurofibromin produced in baculovirus detected with the same neurofibromin antibody. (B) NIH 3T3 cells were unstimulated or stimulated with serum for 5 min as described above, and Western blots were probed with either an N-terminal NF1 antibody (NF1GRP-N), an antibody that recognizes an internal epitope (GAP4), or an antibody that recognizes a second, distant C-terminal epitope (NF1C). The last blot was also reprobed with the p120RasGAP antibody. (C) IMR90 cells (top) or RT4 Schwannoma cells (bottom) were stimulated as described in A, and Western blots were probed with the NF1GRP-D antibody and the p120RasGAP antibody. (D) Serum, LPA (6 μM), and PDGF (20 ng/mL) were used to stimulate cells as described in A–C.

Figure 2

Neurofibromin is degraded by the proteasome. (A) Cells were untreated or pretreated with LLnL or lactacystin prior to EGF (5 min), PDGF (5 min), or LPA treatment for the times indicated, and Western blots were probed with the NF1GRP-D and p120RasGAP antibodies. (B) NIH 3T3 cells were transfected with a control plasmid or a plasmid encoding a His-tagged ubiquitin molecule. Twenty-four hours later, cycling cells were treated with LLnL where indicated, and His-tagged proteins were purified using nickel-coupled agarose beads. Proteins were eluted, and a Western blot was probed with the C-terminal neurofbromin antibody. The NF1 control lane represents protein from a total cell lysate. (C) Baculovirus-expressed full-length tagged neurofibromin was subjected to an in vitro ubiquitination reaction or a mock reaction in the presence of LLnL. A Western blot was probed with the NF1GRP-D antibody.

Figure 3

Sequences adjacent to the GRD are required for neurofibromin degradation. (A) FLAG-tagged fusion proteins encoding the GRD of human neurofibromin or the GRD plus 80 N-terminal amino acids (GRD+80) were generated by in vitro transcription and translation reactions (IVT) in the presence of 35S-methionine. Proteins were subjected to a reticulocyte lysate (RL)-based in vitro degradation reaction in the presence of ATP or, where indicated, ATPγ, and separated by PAGE. A construct encoding the Drosophila GRD+80 protein was subjected to the same reaction (DGRD+80). (B) The GRD+80 construct was truncated to generate a GRD+60 construct that was used in the in vitro degradation assay. Alternatively, mutations resulting in triple alanine substitutions [1095–1097 (KYF to AAA) or 1098–1100 (TLF to AAA)] were introduced in the GRD+80 construct.

Figure 4

_Nf1_-deficient MEFs exhibit deregulated Ras activity. (A) Wild-type and _Nf1_-deficient MEFs were serum starved and treated with 10% or 1% serum for increasing lengths of time. Cells were lysed, and Ras–GTP was precipitated with a GST–Ras-binding domain fusion protein. Western blots were probed with a pan-isoform-specific Ras antibody. Ras–GTP levels were quantitated using a densitometer and expressed as percentage of maximal levels. Total cell lysates from these samples were probed with the same antibody to ensure equal protein loading. (B) Total cell lysates were prepared from duplicate samples as described in A and immunoblots were sequentially probed with an antineurofibromin antibody or an anti-p120RasGAP antibody.

Figure 5

_Nf1_-deficient MEFs exhibit deregulated ERK activation and possess proliferation defects. (A,B) Wild-type (solid line) and _Nf1_-deficient (broken line) MEFs were serum starved and treated with 10% (A) or 1% (B) serum for increasing lengths of time. ERK activation was measured using kinase assays performed in triplicate and quantitated using a PhosphorImager. (C) Wild-type and _Nf1_-deficient MEFS were serum starved and exposed to 10% serum. Cells from triplicate wells were counted on day 0 and at 25 and 72 h following serum treatment. (D) Serum-starved wild-type (white bars) and mutant cells (gray bars) were treated with 0.1%, 0.5%, or 1% serum. Cells in triplicate wells were counted 72 h after serum addition. Fold increase in cell number ratios were calculated as described in Materials and Methods. (E) Nf1+/+ (white bars), Nf1+/− (light-gray bars), and _Nf1_−/− (black bars) MEFs were grown in 1% serum in triplicate wells for 72 h and analyzed as described in D. Differences in B, D, and E as described in the text were found to be statistically significant at or better than α = 0.01 using a two-tailed T test.

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Dynamic regulation of the Ras pathway via proteolysis of the NF1 tumor suppressor - PubMed (original) (raw)