Modulation of Fatty Acid Synthase Degradation by Concerted Action of p38 MAP Kinase, E3 Ligase COP1, and SH2-Tyrosine Phosphatase Shp2^*

Plasmids, Antibodies, and Reagents

The different Shp2 plasmids including the wild-type, catalytically inactive C459S, constitutively active D61A and Shp2-ΔSH2 (deletion of amino acids 1–209 containing N-SH2 and C-SH2 domain regions) were subcloned in the vector pEF-HA (17). Similarly, N-SH2 (1–106 aa) or C-SH2 (107–220 aa) region was also cloned into the pEF-HA vector. pCDNA3-HA-COP1 was described previously (18). Antibodies used were Shp2 (B-1 and C-18), FASN (H-300), COP1 (K-16), p38 (C-20), Ub (A-5), and HA (rabbit IgG) from Santa Cruz Biotechnology, p-p38 (28B10, Cell Signaling), β-actin (AC-15, Sigma), anti-HA (mouse IgG, Roche). Anti-phosphothreonine-proline/phosphoserine-proline antibody (anti-pTP/pSP, ab9344) was from Abcam (Hong Kong) Ltd. Alex Flour 488, 568 (goat anti-rabbit or mouse), and Vectashield DAPI were from Invitrogen. The proteasome inhibitors MG132 and LAC, PI3K inhibitor LY294002, ERK inhibitor PD98059, p38 kinase inhibitor SB203580, JNK kinase inhibitor SP600125, and MEK inhibitor U0126 were from Calbiochem. Okadaic acid (an inhibitor of the serine/threonine phosphatase, #5934) was from Cell Signaling. Human Shp2 siRNA (sc-36488), COP1 siRNA (sc-45541), and Scrambled/Control siRNA-A (sc-37007) were from Santa Cruz Biotechnology. We also designed siRNA to target the Shp2 cDNA sequence: Shp2 siRNA, 5′-gcaguuaaauugugcgcugua-3′ and Mismatch siRNA, 5′-gcaguuaaacggugcgcugua-3′, were custom-synthesized commercially. The siRNA transfections were performed according to the siRNA Transfection Protocol from the instructions for Lipofectamine 2000.

Immunoprecipitation and Western Blot

For Western blotting analysis, anti-β-actin was used at a 1:5000 dilution, other primary antibodies were used at a 1:1000 dilution. For determination of protein ubiquitination, cells were lysed in the cell lysis buffer A (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, and protease inhibitors) on ice. Lysates from cell cultures or tissues (500–1000 μg) were pre-cleared by incubation with protein A/G plus-agarose (Santa Cruz Biotechnology) and then incubated with 1–2 μg of anti-Shp2, or anti-FASN or anti-HA(m) and normal rabbit IgG (from Santa Cruz Biotechnology) for 2 h at 4 °C. Protein A/G plus-agarose (30–40 μl) were added into the mixture and incubated with agitation for an additional 4 h at 4 °C. The immunoprecipitated samples were extensively washed with the cell lysis buffer and subjected to the Western blotting analysis.

Immunofluorescence Staining

p38^+/+ and p38^−/− MEFs were directly seeded into the uncoated 35-mm dishes at a density of 1.5 × 10⁵ cells. After 24 h of transfection, cells were washed in PBS once, fixed with 4% freshly prepared formaldehyde in PBS for 8–10 min, and then washed three times with PBS. Cells were permeabilized with 0.1% saponin/PBS for 15 min, blocked in 2.5% NGS (normal goat serum) in 0.1% saponin/PBS for 30 min-24 h, incubated in the primary antibody (FASN, dilution 1:200; Shp2, dilution 1:200; COP1, dilution 1:100) diluted in blocking solution for 1 h, washed three times with blocking solution, and then incubated in the second antibody (Alexa 568 anti-rabbit, or Alexa 488 anti-mouse, dilution 1:500) in blocking solution for 30 min. Then these were washed twice with blocking solution and once with PBS. DAPI was added for DNA staining. Pictures were taken with a Fluoview 1000 Olympus Laser Point Scanning Confocal Microscope.

Shp2 Negatively Regulates FASN Protein Levels

To explore the biological significance for Shp2 interaction with FASN, we transfected 293T cells with a Shp2 expression construct and examined its effect on FASN protein content. As shown in Fig. 2A, with progressively increasing amounts of Shp2, FASN protein levels decreased correspondingly, indicating a negative correlation between Shp2 and FASN expression. To assess whether Shp2 affects FASN stability, we measured the protein amounts at different time points following cycloheximide treatment. The half-life of FASN shown was shortened from 15.4 to 9.7 h in cells transfected with HA-Shp2 (Fig. 2B). In contrast, Shp2 knockdown mediated by Shp2-specific siRNA resulted in increased amounts of FASN protein (Fig. 2C), indicating a role of Shp2 in facilitating FASN degradation in cells.

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FIGURE 2.

Shp2 facilitates FASN degradation. A, overexpression of Shp2 decreases FASN protein levels. 293T cells were transfected with 1, 2, or 3 μg of HA-Shp2 plasmids, and 48 h later, transfected cells were lysed for immunoblot with antibodies to FASN, HA, or β-actin. B, half-life of FASN in 293T cells transfected with or without HA-Shp2. 24 h after transfection, the cells was treated with cycloheximide (100 μg/ml) and harvested in SDS lysis buffer at the indicated time points. Relative amounts of FASN were quantified and normalized to β-actin, and plotted (the experiment repeated three times). C, Shp2 knockdown by siRNA leads to increased level of FASN protein. 293T cells were transfected with Shp2-specific siRNA, and transfected cells were lysed 48 h later for immunoblot with antibodies to FASN, Shp2, or β-actin. Left panel, SC-siRNA (from Santa Cruz Biotechnology); Right panel, Syn-siRNA (designed sequences, custom-synthesized commercially). D and E, Western blotting analysis showing high levels of FASN protein in Shp2 conditional knock-out mouse tissues, liver (D) and pancreas (E). F, H&E staining of pancreas sections from control or pancreas-specific Shp2 knock-out mouse (Pdx-Shp2KO) (19).

In previous experiments, we generated liver-specific and pancreas-specific Shp2/Ptpn11 knock-out mice, using the cre-loxP system (19, 20). We measured FASN protein levels in these Shp2-deficient mouse organs. Results shown here demonstrate that Shp2 ablation in the liver led to elevated FASN protein levels, as compared with wild-type mouse liver (Fig. 2D). Similar results were obtained with conditional knock-out of Shp2 in the pancreas (Fig. 2E). H&E staining of pancreatic sections revealed increased fat tissue when Shp2 was deleted (Fig. 2F).

Shp2-mediated Degradation of FASN Is Independent of Its Phosphatase Activity

We asked if degradation of FASN was indeed controlled by the ubiquitination pathway. As shown in Fig. 3A, the ubiquitination of FASN in coimmunoprecipitates was dramatically increased following treatment with proteasome inhibitor MG132 or lactacystin, compared with the controls. This result is consistent with a previous report on regulation of FASN by ubiquitin-mediated degradation in prostate cancer (13). To determine the molecular mechanism for Shp2 regulation of FASN protein levels, we transiently expressed wild-type (WT), catalytically inactive mutant (C459S), constitutively active mutant (D61A), and a N+C SH2 domain deletion mutant (Shp2-ΔSH2) in 293T cells. As shown in Fig. 3B, expression of the WT and two mutants (C459S or D61A) of Shp2 significantly decreased the protein level of FASN, compared with the vector control. However, the deletion mutant Shp2-ΔSH2 did not have any effect on FASN expression. These results suggest a mechanism for Shp2 control of FASN protein level through its SH2 domain in a phosphatase-independent fashion.

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FIGURE 3.

FASN degradation is controlled by ubiquitin E3 ligase COP1. A, FASN protein was degraded by ubiquitinylation. 293T cells treated with proteasome inhibitors Lactacystin (20 μm) or MG132 (20 μm) for 6 h were lysed for coimmunoprecipitation with FASN antibody, and followed by immunoblot with ubiquitin antibody. B, lysates from 293T cells transfected with empty vector, three mutants CS, DA, and ΔSH2, or wild-type Shp2, were analyzed by immunoblotting. C, the same Shp2 constructs were co-transfected with His-ubiquitin into 293T cells. The assay was performed for precipitation of the tagged ubiquitin with Ni²⁺-NTA-agarose beads, as previously described (31). D, overexpression of COP1 decreased protein level of FASN. 293T cells transfected with COP1 expression vector were lysed for immunoblot with antibodies to FASN, COP1, or β-actin. E, COP1 knockdown by siRNA leads to increase in FASN protein amounts. 293T cells were transfected with siRNA-COP1, and at 48 h after transfection, cells were lysed for immunoblot with antibodies to FASN, COP1, or β-actin. F, 293T cells were transfected with pEF-HA, or pEF-HA-N-SH2, or pEF-HA-C-SH2 or pEF-HA-Shp2-ΔSH2 plasmid. Lysates from the cells at 48 h after transfection were used for immunoprecipitation with HA(m) antibody, then detected by immunoblotting with antibodies to HA(r), FASN, and COP1.

We next determined whether Shp2 expression affected the FASN level through protein degradation via the ubiquitin-proteasome system. Wild-type and mutant Shp2 expression constructs were co-transfected with His₆-tagged ubiquitin into 293T cells. Cell lysates were prepared in a denaturing 6 m guanidinium-HCl buffer, and His₆-tagged ubiquitin was precipitated with Ni²⁺-NTA-agarose beads. Immunoblotting of precipitates showed markedly increased levels of FASN protein ubiquitination in 293T cells transfected with WT, CS, or DA mutants of Shp2, compared with the empty vector or the mutant Shp2-ΔSH2 (Fig. 3C). Taken together, these results suggest a role of Shp2 in control of FASN degradation by ubiquitination, which is independent of the catalytic activity but requires its SH2 domains.

FASN Degradation Is Mediated by an E3 Ligase COP1

COP1 is an E3 ubiquitin ligase that is involved in the ubiquitination of various protein substrates to trigger their proteasomal degradation (21). The first identified direct mammalian substrate of COP1 is p53 tumor suppressor (22). COP1 also targets JUN and shows that COP1 acts as a bridge between JUN, DET1, and the large E3 ligase complex that contains CUL4A and DDB1 (23). It has also been reported putative roles for COP1 in cell metabolism including glucose (24) and lipid metabolism (18). Importantly, COP1 can promote the degradation of acetyl-CoA carboxylase (ACC), a rate-limiting enzyme in fatty acid synthesis (18, 21). To test if COP1 as an ubiquitin E3 ligase also mediates FASN degradation, we transiently transfected the COP1 expression plasmid into 293T cells and showed that COP1 was able to significantly decrease the protein level of FASN (Fig. 3D). In contrast, down-regulation of endogenous COP1 by siRNA caused accumulation of FASN protein (Fig. 3E). Thus, overexpression or knockdown of COP1 had reciprocal effects on the protein level of FASN (Fig. 3, D and E), indicating that COP1 directly affects FASN degradation.

Shp2 protein contains one PTPase domain and two SH2 domains (N-SH2 and C-SH2). Since the SH2 domains are required for promoting degradation of FASN (Fig. 3, B and C), next we want to investigate whether one or both of SH2 domain(s) are able to directly interact with FASN and/or COP1. We generated and transfected three constructs HA-N-SH2, HA-C-SH2, and HA-Shp2-ΔSH2 into 293T cells. Coimmunoprecipitation/Western blot assays showed that N-SH2 domain of Shp2 complexed with both FASN and COP1, whereas either C-SH2 domain or Shp2-ΔSH2 (mainly PTPase domain) could not bind to FASN and COP1 (Fig. 3F, IP), suggesting that FASN degradation mediated by COP1 is involved in the direct interaction of Shp2 N-SH2 domain with FASN and COP1. However, three deletion mutants including N-SH2 domain did not lead to FASN protein degradation (Fig. 3F, Input), indicating that full-length Shp2 is required for this pathway.

p38 MAP Kinase Is Involved in FASN Degradation

To probe the molecular mechanism underlying FASN degradation, we treated cells with small molecule inhibitors LY294002 for PI3K, PD98059 for ERK, SB203580 for p38 MAP kinase, SP600125 for JNK, and U0126 for MEK, respectively. p38 MAP kinase inhibitor SB203580 significantly increased FASN protein level, SP600125 and U0126 caused a decrease, and LY294002 and PD98058 did not have a significant effect on FASN protein levels (Fig. 4A). To establish a role of p38, we examined FASN expression in p38^+/+ and p38^−/− MEF cells. FASN protein level was much higher in p38^−/− MEF than in p38^+/+ MEF cells (Fig. 4B), consistent with the experimental results using the chemical inhibitors shown in Fig. 4A. Notably, the FASN protein levels were lower in JNK1^−/− MEFs and JNK1/2 double knock-out (JNK1/2^−/−) cells than in wild-type cells (Fig. 4B).

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FIGURE 4.

p38 MAP kinase is involved in FASN degradation. A, FASN protein was affected by kinase inhibitors. Hela cells were treated with 10 μm of different inhibitors LY294002, PD98059, SB203580, SP600125, or U0126 for 24 h, and then lysed for immunoblotting. B, comparison of FASN protein levels in p38^−/− and p38^+/+ MEFs, JNK1^−/− and JNK1^+/+ MEFs, JNK1/2^−/− and JNK1/2^+/+ MEF cells. C, FASN mRNA levels in p38^+/+ and p38^−/− MEFs were determined by qRT-PCR. Each value represents the mean ± S.E. of three independent experiments with triplicates each. Differences between two groups were analyzed using the t test (two-tailed and unpaired), p value = 0.4317. D, activated/phosphorylated p38 increased FASN degradation mediated by Shp2. 293T cells were transfected with Shp2 with or without p38α+MKK6Be. After 48 h, transfected cells were lysed for immunoblotting with antibodies to FASN, Shp2, β-actin, or p-P38.

We tested whether p38-mediated regulation of FASN occurs at the transcription and/or mRNA stability level. qRT-PCR analysis detected similar levels of FASN mRNA levels in p38^+/+ and p38^−/− MEF cells (Fig. 4C), thus excluding the possibility that p38 regulates FASN gene transcription or mRNA stability. We also investigated possible concerted functions of p38 and Shp2 in controlling degradation of FASN protein. As shown in Fig. 4D, the decrease in FASN protein levels was more severe in 293T cells transfected with p38/MKK6Be and Shp2 (lane 4), than in cells transfected with either p38/MKK6Be or Shp2 alone.

p38 MAP Kinase Engages FASN-Shp2-COP1 Complex Formation in the Cytoplasm

It has been shown that ionizing radiation (IR) triggered an ATM-mediated COP1 serine 387 (S387) phosphorylation leading to IR-induced COP1 nuclear exclusion (25). Also, COP1 S387 phosphorylation is essential for 14-3-3σ induced COP1 nuclear export (26). Therefore, we wanted to test whether p38 MAP kinase can also phosphorylate COP1. We used a very useful tool, okadaic acid, an inhibitor of the serine/threonine protein phosphatases PP1 and PP2A, to treat p38^+/+ and p38^−/− MEFs. After treatment of okadaic acid (2 μm) for 60 min, cells were lysed for immunoprecipitation with anti-pTP/pSP antibody, and then detected by immunoblotting with antibody to COP1. The results showed that COP1 phosphorylation was easily detected in p38^+/+ MEFs, and strongly increased by okadaic acid treatment when compared with DMSO treatment. In contrast, COP1 phosphorylation was almost not detected under treatment of either okadaic acid or DMSO in p38^−/− MEFs (Fig. 5A). Moreover, we transiently transfected plasmids MKK6Be with or without p38α-WT and p38α-KR (a dominant-negative mutant) into 293T cells. Lysates were used for immunprecipitation with anti-pTP/pSP antibody and then detected with anti-COP1 antibody. Compared with control, COP1 phosphorylation was significantly increased by p38α-WT but not by p38α-KR (Fig. 5B). These suggest that p38 can phosphorylate COP1 in cells.

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FIGURE 5.

FASN regulation by the p38-COP1-Shp2 pathway. A, lysates from p38^+/+ and p38^−/− MEFs treated with okadaic acid (2 μm for 60 min) were coimmunoprecipitated with anti-pTP/pSP antibody and then detected by immunoblotting with antibody to COP1. B, 293T cells were transfected with plasmids MKK6Be with or without p38α-WT and p38α-KR (mutant). After 48 h, transfected cells were lysed for immunprecipitation with anti-pTP/pSP antibody and then detected with anti-COP1 antibody. C, endogenous COP1 binds FASN and Shp2 in p38^+/+ but not in p38^−/− MEFs. Lysates from p38^+/+ and p38^−/− MEFs were coimmunoprecipitated with antibodies to FASN, Shp2, or control IgG, and then detected by immunoblotting with antibody to COP1. D, p38^+/+ and p38^−/− MEF cells were processed for immunostaining and confocal microscopy to visualize the localization of Shp2, FASN, and COP1. E, wild-type and liver-specific Shp2 knock-out C57BL/6 mice were either fasted for 24 h, or fed regular chow food. Liver lysates from sacrificed mice were subjected to immunoblot and Co-IP experiments with indicated antibodies. F, a model for FASN regulation by the p38-COP1-Shp2 pathway.

The data shown above suggest participation of three proteins Shp2, COP1, and p38 in mediating FASN protein degradation. We then explored the biochemical mechanisms linking these proteins to examine whether endogenous COP1 binds FASN and/or Shp2 in p38^+/+ and p38^−/− MEF cells. COP1 bound both FASN and Shp2 only in p38^+/+ MEFs but not in p38^−/− MEFs, suggesting that p38 is requested for COP1 binding to its target protein FASN and in interaction with Shp2 (Fig. 5C).

We further examined localization and association of Shp2, FASN, and COP1 in p38^+/+ and p38^−/− MEF cells by immunofluorescence staining and confocal microscopy. Both Shp2 and FASN were mainly cytoplasmic, and their colocalization showed no difference between p38^+/+ and p38^−/− MEFs (Fig. 5D, upper two panels), indicating that p38 does not affect binding of FASN with Shp2. However, COP1 was largely detected in the cytoplasm in p38^+/+ MEFs, whereas increased nuclear distribution of COP1 was observed in p38^−/− MEFs, likely due to p38-dependent movement of COP1 from the nucleus to the cytoplasm, thereby increasing COP1 binding to FASN and Shp2 in the cytoplasm, as revealed by COP1 colocalization with both FASN and Shp2 (Fig. 5D, 4th and 6th panels), consistent with the coimmunoprecipitation data (Fig. 5C). These results support a model that p38-phosphorylated COP1 accumulates in the cytoplasm and subsequently binds FASN through Shp2 here as a bridge molecule, leading to FASN-Shp2-COP1 complex formation in the cytoplasm and FASN degradation mediated by ubiquitination pathway.