ABSTRACT
The cellular senescence-inhibited gene (CSIG) is implicated in important biological processes, including cellular senescence and apoptosis. Our work showed that CSIG is involved in the myristoylation of the serine/threonine protein phosphatase PPM1A. Previous research has shown that myristoylation is necessary for PPM1A to dephosphorylate Smad2 and Smad3. However, the control and the biological significance of the myristoylation remain poorly understood. In this study, we found that CSIG knockdown disturbs PPM1A myristoylation and reduces the dephosphorylation by PPM1A of its substrate Smad2. By regulating PPM1A myristoylation, CSIG is involved in modulating the signaling of transforming growth factor β (TGF-β). Further study of the mechanism indicated that CSIG facilitates the interaction between N-myristoyltransferase 1 (NMT1) and PPM1A. Taking the data together, we found that CSIG is a regulator of PPM1A myristoylation and TGF-β signaling. By promoting the myristoylation of PPM1A, CSIG enhanced the phosphatase activity of PPM1A and further inhibited TGF-β signaling. This work not only extends the biological significance of CSIG but also provides new ideas and a reference for the study of the regulatory mechanism of myristoylation.
INTRODUCTION
The cellular senescence-inhibited gene (CSIG) protein, also known as ribosomal L1 domain containing 1 (RSL1D1), is a nucleolar protein with a ribosomal L1 domain at its N terminus and a lysine-rich domain at its C terminus (1). Previous studies have indicated that CSIG is involved in various biological processes, including cellular senescence and apoptosis (2–5). CSIG also regulates the nucleolar localization of p33ING1 (inhibitor of growth family member 1) and nucleostemin (NS) via physical interactions (4, 6). Recent research has shown that CSIG regulates the MDM-p53 pathway under nucleolar stress. To date, whether CSIG regulates protein myristoylation remains unknown (7).
PPM1A is a phosphatase belonging to the serine/threonine PPM family of protein phosphatases and has magnesium/manganese ion dependency. PPM1A has a catalytic subunit that can be located in the nucleus or the cytoplasm (8, 9). PPM1A can specifically bind to its substrates and catalyze the dephosphorylation of these substrates. The main substrates of PPM1A are p38 mitogen-activated protein kinase (MAPK), Smad2 and Smad3, MAPK kinase 4 (MKK4), and MKK6 (10–12). Studies indicate that PPM1A can be modified by myristoylation. Moreover, the dephosphorylation of the substrate AMPKα (5′ AMP-activated protein kinase, subunit alpha) by PPM1A indicates a clear relationship with myristoylation (13).
Protein myristoylation is a modification that occurs at the N termini of proteins. Myristoylation involves irreversible covalent bonding. The main reaction that occurs during myristoylation is the combination of myristic acid, a C14 saturated fatty acid, with glycine residues in the N-terminal region of the protein, a reaction catalyzed by N-myristoyltransferase (NMT) (14). Some studies have indicated that myristoylation occurs in a number of eukaryotic cells and viruses. There has not been much functional research on myristoylation, and studies have focused mainly on the mechanisms by which myristoylation affects protein-protein interactions, the membrane-binding ability of proteins, and protein localization in cells. Myristoylation also influences the assembly and replication processes of viruses (15–17).
The transforming growth factor β (TGF-β) signaling pathway is involved in pathogenesis in a variety of diseases, such as cancer and some autoimmune diseases (18). TGF-β signaling is also involved in the normal developmental processes of many organs, playing an important role in individual development and disease-related processes (19, 20). The PPM1A protein participates in signal transduction in the TGF-β signaling pathway by dephosphorylating the phosphorylated Smad2 (p-Smad2) or p-Smad3 protein; therefore, the enzymatic activity of PPM1A can influence the regulatory effect of the TGF-β signaling pathway (12, 21).
In this study, we found that CSIG, cloned by our laboratory, is associated with PPM1A. When CSIG is expressed at normal levels, the dephosphorylation activity of PPM1A is maintained, and PPM1A is able to dephosphorylate p-Smad2 and terminate TGF-β signaling. However, when small interfering RNA (siRNA) is used to knock down CSIG expression, the N-myristoylation of PPM1A is affected. The phosphatase activity of PPM1A decreases significantly, resulting in increased p-Smad2 levels and enhancement of TGF-β signal transduction.
Our research in this study explores, for the first time, the effect of decreasing CSIG expression levels on N-myristoylation and the phosphatase activity of PPM1A. In addition, this process may affect the TGF-β signaling pathway. Thus, we further explore the biological function of CSIG and provide important clues and evidence for the role of N-myristoylation.
RESULTS
PPM1A interacts with CSIG in cells.CSIG is a senescence-associated gene cloned by our lab. As shown in Fig. 1A, when we proceeded to identify the CSIG complex with H1299 cells stably transfected with pIRES-FLAG-HA-CSIG, we determined, via mass spectrometric detection, that PPM1A interacts with CSIG. Since PPM1A is a phosphatase that possesses many substrates and is involved in some important signaling pathways in cells, we then verified the interaction between CSIG and PPM1A by coimmunoprecipitation (co-IP). Both stably transfected and transiently transfected H1299 cells were examined (Fig. 1B through E). This interaction was also detected in normal HaCaT cells (Fig. 1F and G). All the results show that CSIG interacts with PPM1A. Once the interaction was confirmed, we needed to study which regions of CSIG bind to PPM1A. For this purpose, we transfected cells with truncated CSIG mutants. The co-IP result demonstrated that the N terminus of CSIG (CSIG-NT, containing the ribosomal L1 domain) interacts with PPM1A (Fig. 1H).
Association of CSIG and PPM1A. (A) Coomassie blue-stained gel of successive IPs using FLAG and HA beads. Proteins were extracted from H1299 cells stably transfected with FLAG-HA-CSIG. All the bands in lane FLAG-HA-CSIG were cut for sequencing via mass spectrometric analysis, and PPM1A was identified from the marked band. (B and C) Co-IP of CSIG and PPM1A in stably transfected H1299 cells, as described above. (D and E) Co-IP of CSIG and PPM1A in H1299 cells transiently transfected with PPM1A-FLAG. (F and G) The interaction between PPM1A and CSIG was confirmed by co-IP in normal HaCaT cells. (H) (Top) Diagrams of CSIG and its truncated mutants. (Bottom) Co-IP of CSIG and PPM1A in HaCaT cells. NT, N-terminal region; ΔN, N-terminally deleted.
CSIG is necessary for the dephosphorylation of p-Smad2 by PPM1A.After confirming the interaction, we explored whether CSIG influences the activity of PPM1A by interacting with PPM1A. Since PPM1A is a protein phosphatase, we decided to explore the effect of CSIG on PPM1A by studying the dephosphorylation activity of PPM1A. We designed an experiment to determine the dephosphorylation activity of PPM1A by examining the phosphorylation level of the downstream Smad2 protein. First, we determined that in U2OS cells, TGF-β treatment led to increases in p-Smad levels. However, overexpression of PPM1A led to the inhibition of these increases (Fig. 2A). In addition, we found that overexpression of PPM1A in U2OS cells can significantly reduce the level of Smad2 phosphorylation (Fig. 2B, lanes 5 and 6). However, following transfection with siRNA against CSIG (siCSIG), PPM1A overexpression cannot reduce the level of Smad2 phosphorylation detectably (Fig. 2B, lanes 7 and 8). This result is consistent with that in HaCaT cells. (Fig. 2C), suggesting that CSIG is necessary for the dephosphorylation of p-Smad2 by PPM1A.
CSIG is required for PPM1A to dephosphorylate p-Smad2. (A) Western blot of p-Smad2 with or without PPM1A-FLAG overexpression in U2OS cells. Both cells treated with TGF-β1 (2 ng/ml, 1 h) and untreated cells were examined. (B) Western blot of p-Smad2 with or without PPM1A-FLAG overexpression in the presence of siCSIG (lanes 7 and 8) or siNC (lanes 5 and 6) in U2OS cells. Both cells treated with 2 ng/ml TGF-β1 for 1 h (lanes 5 to 8) and untreated cells (lanes 1 to 4) were examined. (C) Western blot of p-Smad2 with or without PPM1A-FLAG overexpression in the presence or absence of siCSIG in HaCaT cells. Cells were treated with TGF-β1 (2 ng/ml, 1 h).
CSIG knockdown perturbs PPM1A myristoylation.Because myristoylation is necessary for the dephosphorylation activity of PPM1A, we wondered whether CSIG affects the myristoylation of PPM1A. To investigate this question, a G2A mutant of PPM1A was constructed. The G2A mutant cannot be modified by myristoylation (13, 22). The level of Smad2 phosphorylation was significantly reduced upon overexpression of PPM1A only; however, upon treatment with siCSIG and overexpression of PPM1A or its G2A mutant, the phosphorylation of Smad2 was seen not to be inhibited (Fig. 3A). We thus verified that PPM1A myristoylation is necessary for the dephosphorylation of Smad2 in our system.
CSIG is essential for PPM1A myristoylation. (A) Shown are Western blots of p-Smad2 with overexpression of PPM1A-FLAG (lanes 3 and 4) or PPM1A-G2A-FLAG (lanes 5 and 6) in the presence or absence of siCSIG in HaCaT cells treated with TGF-β1 (2 ng/ml, 1 h) and Western blots of p-Smad2 with vector and siNC in HaCaT cells that were either left untreated or treated with 2 ng/ml TGF-β1 for 1 h (lanes 1 and 2). (B to D) Gels with Cy3 fluorescence. (B) Cells were tagged with YnMyr (50 μM, 12 h). YnMyr (50 μM, 6 h) was used as a negative control for the tagging process, and proteins were labeled by click chemistry. Cells transfected with or without siCSIG were examined. A Coomassie blue-stained gel was used as a control for protein volume. (C) Cells were tagged with YnMyr (50 μM, 12 h), and proteins were labeled by the click method before PPM1A immunoprecipitation. Cells transfected with or without siCSIG were examined (left two lanes). IgG was used as a negative control of PPM1A immunoprecipitation (right two lanes). A Western blot of PPM1A was used as a control for protein volume. (D) Cells were first transfected with PPM1A-G2A-FLAG and then tagged with YnMyr (50 μM, 12 h). Proteins were labeled by the click method before FLAG immunoprecipitation. Cells transfected with or without siCSIG were examined (anti-FLAG lanes). IgG was used as a negative control for FLAG immunoprecipitation (four right lanes). Cells transfected with PPM1A-FLAG were used as positive controls (anti-PPM1A lanes).
To verify that CSIG can directly affect the myristoylation of PPM1A, we adopted click chemistry to directly detect PPM1A myristoylation. HaCaT cells were tagged with YnMyr (an analogue of myristic acid), and the protein was then subjected to copper-catalyzed azide-alkyne cycloaddition (CuAAC) labeling (14, 22–24) (Fig. 3B). When cells were transfected with siCSIG, the level of PPM1A myristoylation was significantly reduced (Fig. 3C); however, CSIG knockdown had little effect on the myristoylation level of total protein in cells (Fig. 3B). The results indicated that CSIG enhanced or maintained PPM1A myristoylation. In addition, we also verified, by use of a click chemistry assay, that the G2A mutant cannot be modified by myristoylation (Fig. 3D).
CSIG knockdown can alter the localization of PPM1A in cells.According to the results presented in Fig. 3, CSIG promoted the myristoylation of PPM1A. Myristoylation can affect the hydrophilicity or hydrophobicity of the protein, indicating that protein myristoylation can respond to the conformation and localization of proteins. Therefore, we wondered whether CSIG alters the localization of PPM1A in cells by inhibiting PPM1A myristoylation. We separated the nucleoproteins and cytoplasmic proteins and detected PPM1A by Western blotting. As shown in Fig. 4A, CSIG does affect the location of PPM1A; PPM1A, which was originally located in the nucleus (Fig. 4A, lanes 1 and 2), was detected in the cytoplasm in this experiment (Fig. 4A, lanes 5 and 6). Moreover, the G2A mutant of PPM1A was also detected in the cytoplasm (Fig. 4A, lanes 3, 4, 7, and 8). To gain more insight, immunofluorescence was performed with enhanced green fluorescent protein (EGFP)-labeled PPM1A (PPM1A-EGFP) and PPM1A-G2A-EGFP in U2OS cells and HaCaT cells. The same results were observed by confocal microscopy. Additionally, following CSIG knockdown, most PPM1A proteins were seen to aggregate around the nucleus (Fig. 4B).
CSIG is essential for PPM1A to be distributed predominantly in the nucleus. (A) Western blot analysis of PPM1A and PPM1A-G2A with nuclear (lanes 1 to 4) and cytoplasmic (lanes 5 to 8) proteins of HaCaT cells. Cellular overexpression of PPM1A-FLAG in the presence of siCSIG (lanes 2 and 6) and cellular overexpression of PPM1A-G2A-Myc in the presence of siNC (lanes 3 and 7) were detected. Cellular overexpression of PPM1A-FLAG in the presence of siNC and cellular overexpression of PPM1A-G2A-Myc in the presence of siCSIG were detected as controls (lanes 1, 4, 5, and 8). (B) Immunofluorescence of PPM1A or PPM1A-G2A (green) and CSIG (red) in HaCaT cells and U2OS cells. U2OS cells overexpressing PPM1A-EGFP or PPM1A-G2A-EGFP (top two rows) or overexpressing PPM1A-EGFP in the presence of siNC or siCSIG (two center rows) were detected. U2OS cells overexpressing PPM1A-G2A-EGFP in the presence of siNC or siCSIG (bottom two rows) were detected as negative controls.
CSIG regulates TGF-β signaling by influencing PPM1A myristoylation.Smad2 is a member of the R-Smad proteins, which are involved in signal transduction in the TGF-β pathway. In addition, the dephosphorylation of Smad2 by PPM1A has important effects on the TGF-β pathway (25). Therefore, we studied this process by detecting changes in the expression of the downstream target gene p15INK4b and the feedback-regulated Smad7 gene of the TGF-β pathway (26, 27). The levels of p15INK4b and Smad7 mRNAs were reduced when PPM1A was overexpressed (Fig. 5A and B, group 3). However, when the G2A mutant was used, the levels of p15INK4b and Smad7 mRNAs were significantly higher than the level with PPM1A overexpression (Fig. 5A and B, group 4). Following CSIG knockdown, the levels of p15INK4b and Smad7 mRNAs increased further (Fig. 5A and B, group 6), and overexpression of PPM1A or the G2A mutant following CSIG knockdown could not decrease p15INK4b and Smad7 mRNA levels significantly (Fig. 5A and B, groups 7 and 8). However, after the cells were treated with SB431542 (an inhibitor of the TGF-β receptor that blocks TGF-β signaling), the effects of PPM1A overexpression and CSIG knockdown on p15INK4b and Smad7 mRNA levels were weakened significantly (Fig. 5A and B, +SB431542). The addition of SB431542 blocked all the effects, indicating that CSIG regulates p15INK4b and Smad7 just through TGF-β signaling.
CSIG is essential for PPM1A to regulate p15INK4b and Smad7 through the TGF-β pathway. (A and B) Relative mRNA levels of the cyclin-dependent kinase inhibitor p15INK4b (A) and Smad7 (B) detected by RT-PCR in HaCaT cells. HaCaT cells were transfected with siNC alone (group 2), siNC and PPM1A-FLAG (group 3), or siNC and PPM1A-G2A-FLAG (group 4) separately, and TGF-β1 (2 ng/ml, 1 h) was added before the cells were harvested. HaCaT cells were transfected with siCSIG alone (group 6), siCSIG and PPM1A-FLAG (group 7), or siCSIG and PPM1A-G2A-FLAG (group 8) separately, and TGF-β1 (2 ng/ml, 1 h) was added before the cells were harvested. siNC and a FLAG-tagged vector were used as a negative control (group 1). siCSIG and a FLAG-tagged vector were used as a control for the siCSIG treatments (group 5). Additionally, cells subjected to the same treatments but also treated for 4 h with 5 μM SB431542 (an inhibitor of the TGF-β receptor that blocks TGF-β signaling) before being harvested were examined. Data are presented as means ± SD. **, P < 0.01. (C) Percentages of cells detected by FCM. HaCat cells with overexpression of PPM1A-FLAG (second and fourth lanes) or PPM1A-G2A-Myc (third and fifth lanes) in the presence or absence of siCSIG were detected. TGF-β1 (2 ng/ml, 1 h) was added before the cells were harvested. Cells without PPM1A overexpression or TGF-β1 stimulation were detected as negative controls (first lane). Data are presented as means ± SD. Asterisks indicate significant differences from the data in the second lane. (D) IP of Smad2 and PPM1A in HaCaT cells. Cells were cultured in the presence (right three lanes) or absence (left three lanes) of siCSIG (top) or in the presence (left three lanes) or absence (right three lanes) of stimulation with 2 ng/ml TGF-β1 for 1 h (bottom). Cell lysates were detected by Western blotting before IP.
Because p15INK4b has important effects on the cell cycle, we hypothesized that the decrease in PPM1A myristoylation following CSIG knockdown could affect the normal cell cycle process via the TGF-β pathway. To verify this hypothesis, fluorescence confocal microscopy (FCM) was performed (Fig. 5C). Both siCSIG transfection and the G2A mutation can increase the ratio of cells in the G0/G1 phase over those for the control and the PPM1A overexpression group. Taken together with previous experimental results, these results led us to conclude that CSIG knockdown regulates the TGF-β pathway and affects the cell cycle by influencing myristoylation.
As described above, PPM1A terminates TGF-β signaling by dephosphorylating p-Smad2. CSIG interacts with PPM1A, and siCSIG can upregulate TGF-β signaling. To investigate whether the mechanism is based on the reduced PPM1A phosphatase activity or the inhibited ability of PPM1A to bind to Smad2, a co-IP experiment was performed to detect the binding of Smad2 and PPM1A. Following CSIG knockdown, the association of Smad2 with PPM1A was decreased dramatically (Fig. 5D, top). Because TGF-β induces the intracellular localization of Smad2, we examined whether TGF-β stimulation influenced the interaction of Smad2 with PPM1A. However, we did not detect significant changes (Fig. 5D, bottom). This further proves that CSIG promotes the binding of PPM1A to Smad2.
CSIG plays an important role in the interaction between PPM1A and NMT1.The myristoylation process is catalyzed by NMT (28). Whether CSIG knockdown regulates the myristoylation of PPM1A via NMT1 remains unknown. We first examined whether knocking down CSIG could alter the expression level of NMT1. However, the result indicated that CSIG knockdown does not change the expression of NMT1 in cells (Fig. 6A). Simultaneously, this experiment confirmed the earlier result that CSIG changed the total myristoylation level in cells. Therefore, our focus shifted to the combination of PPM1A with NMT1. A co-IP experiment was performed to verify that the combination of NMT1 with PPM1A is affected following CSIG knockdown. CSIG knockdown was seen to weaken the combination of PPM1A with NMT1 (Fig. 6B). From the experiments for which results are shown in Fig. 6B, we found that CSIG increases the interaction between PPM1A and NMT1. The association of CSIG with PPM1A was also confirmed. Therefore, we performed a further co-IP experiment, in which we tried to detect a CSIG-PPM1A-NMT complex. We found that wild-type PPM1A can form a CSIG-PPM1A-NMT complex, while G2A-PPM1A cannot form the complex (Fig. 6C). Since the CSIG-PPM1A-NMT complex was detected in cells, we assumed that CSIG is necessary to maintain that complex in cells. Therefore, we performed glutathione S-transferase (GST) pulldown to determine whether CSIG is required for the interaction between PPM1A and NMT1. In the absence of CSIG, the combination of PPM1A with NMT1 was difficult to detect. However, the interaction was clearly detected when CSIG was added to the reaction system (Fig. 6D). To verify that CSIG and NMT1 function in the same pathway to regulate PPM1A myristoylation, we performed rescue experiments. According to the results, under conditions of CSIG knockdown, NMT1 overexpression could not reduce the level of Smad2 phosphorylation from that with the control group (Fig. 6E, left, third and fourth lanes). In addition, CSIG overexpression could not reduce Smad2 phosphorylation in the context of NMT knockdown (Fig. 6E, right, third and fourth lanes), either. These rescue data further confirmed that CSIG is essential for the combination between PPM1A and NMT1.
CSIG is essential for the binding of PPM1A and NMT1. (A) Western blot analysis of NMT1 with overexpression of PPM1A-G2A-FLAG (lanes 5 and 6) or PPM1A-FLAG (lanes 3 and 4) and siNC or siCSIG in HaCaT cells. Overexpression of the FLAG-tagged vector in HaCaT cells (lane 2) was used as a control for transfections. Cells were either left untreated or treated with 2 ng/ml TGF-β1 for 1 h before being harvested. (B) IP of NMT1 and PPM1A in HaCaT cells. Cells were used in the presence (right three lanes) or absence (left three lanes) of siCSIG. Western blot analyses were performed with cell lysates before IP. (C) (Top) Co-IP of the CSIG-PPM1A-NMT complex in HaCaT cells. (Bottom) Co-IP of G2A-PPM1A with CSIG and NMT1 in HaCaT cells. (D) Purified GST-NMT1 proteins were incubated separately with PPM1A-FLAG with (fourth lane) or without (third lane) FLAG-HA-CSIG. The bound proteins were separated using glutathione-Sepharose and were detected by Western blotting. Purified GST proteins incubated with PPM1A-FLAG were detected as negative controls. (E) (Left) Western blot analysis of p-Smad2 with or without NMT overexpression in the presence (right two lanes) or absence (left two lanes) of siCSIG in HaCaT cells. (Right) Western blot analysis of p-Smad2 with or without CSIG overexpression in the presence (right two lanes) or absence (left two lanes) of siNMT1 in HaCaT cells.
DISCUSSION
Our previous work has indicated that CSIG has many important functions, including the regulation of cellular senescence and apoptosis (1). CSIG also regulates the nucleolar localization of p33ING1 and NS via physical interactions (2–5). A recent study indicated that CSIG plays an important role in regulating the MDM2-p53 pathway (7).
This study reveals that CSIG is a crucial protein that can regulate the myristoylation of PPM1A. PPM1A myristoylation is a necessary condition for the dephosphorylation of p-Smad2. Thus, when CSIG is knocked down, the myristoylation of PPM1A is perturbed, increasing the level of Smad2 phosphorylation and enhancing TGF-β signaling (Fig. 7).
Schematic diagrams demonstrating the role of CSIG in the regulation of the PPM1A myristoylation and TGF-β signaling. (Left) Under normal conditions, CSIG expression is abundant, and PPM1A, maintaining high phosphatase activity, dephosphorylates the phosphorylated Smad2 (p-Smad2), which is activated in TGF-β signaling, to terminate the TGF-β signaling. (Right) Following CSIG knockdown by siRNA transfection, PPM1A myristoylation is inhibited and the phosphatase activity of PPM1A decreases dramatically, leading to increased p-Smad2 levels and the enhancement of TGF-β signal transduction.
The TGF-β pathway participates in the regulation of numerous biological processes. A recent study added PPM1A to the TGF-β pathway as a terminator (12). In this study, CSIG was shown to participate in TGF-β signaling by regulating PPM1A myristoylation. As shown in Fig. 5, CSIG knockdown affects the cell cycle via the TGF-β pathway. This result demonstrates a novel function of CSIG in cell cycle regulation. Additionally, CSIG participates in a variety of biological processes, and some functions of CSIG are implemented via other pathways. Whether CSIG influences these process via TGF-β signaling requires further investigation.
In this study, we discovered that CSIG knockdown causes PPM1A translocation. However, the structural changes in PPM1A caused by CSIG knockdown remain unknown. Most proteins that can be myristoylated exhibit a wide range of hydrophilicities and structures (24, 29, 30). Further biophysical research is required to identify the specific changes in PPM1A conformation. Conformational change could be an important factor in the decrease in the interaction between PPM1A and Smad2. The change in hydrophilicity may reduce the affinity of PPM1A for p-Smad2 because of the conformational change in PPM1A. Additionally, CSIG knockdown can cause the translocation of PPM1A from the nucleus, while p-Smad2 is transported to the nucleus after phosphorylation. This difference in the localization of PPM1A and p-Smad2 may be another explanation for the reduction in the ability of PPM1A to bind to Smad2 upon CSIG knockdown or G2A mutation.
Although we detected a decrease in PPM1A myristoylation after CSIG knockdown, the expression of NMT1 was unchanged following CSIG knockdown. It is not clear how CSIG affects the level of PPM1A myristoylation. We determined that the combination of PPM1A with NMT1 was inhibited following CSIG knockdown. Since myristoylation occurs during the protein translation process, and previous research has shown that CSIG can regulate protein translation in cells (2), it is very likely that CSIG knockdown prevents the interaction between NMT1 and PPM1A during the translation of the PPM1A protein. It is also possible that CSIG can recruit NMT1 during PPM1A translation and facilitate the interaction between PPM1A and NMT1.
In this study, we focused on Smad2 as one of the substrates of PPM1A. To date, many other substrates of PPM1A have been reported, such as p38 MAPK, MKK4, and MKK6 in the stress-activated protein kinase (SAPK) cascade, and proteins in the bone morphogenetic protein (BMP) signaling pathway (10, 11, 31, 32). Loss of PPM1A myristoylation has been shown to perturb PPM1A-mediated dephosphorylation of AMPKα in cells (13). Whether the dephosphorylation of other PPM1A substrates is affected by PPM1A myristoylation requires further investigation. Although loss of myristoylation cannot change the enzymatic activity of PPM1A directly, we hypothesize that the translocation effect and the change in hydrophilicity may influence the interaction of PPM1A with its substrates.
TGF-β has been reported to play an important role in heart disease (33). TGF-β signaling is responsible for cardiomyocyte apoptosis and cardiac hypertrophy, and TGF-β also promotes myofibroblast formation and extracellular matrix (ECM) production, which leads to cardiac fibrosis (34–36). In addition, our lab has newly bought mice in which the CSIG gene has been conditionally knocked out from cardiac muscle. In the preliminary immunohistochemistry results with cardiac tissues obtained from the knockout mice, we observed p-Smad2 levels higher than those in the control group. However, we cannot get enough knockout mice for in vivo research at present. We expected that conditionally knocked out mice would have heart disease, and we can study the pathogenic mechanism further. Our lab will carry out further research with the knockout mouse model. Many studies have indicated that abnormal TGF-β signaling is associated with cancers and genetic diseases (18). We propose that CSIG could also be an important target of these diseases.
In summary, we demonstrated that CSIG knockdown reduces PPM1A myristoylation and then inhibits the dephosphorylation of p-Smad2. We showed that PPM1A translocates from the nucleus to the cytoplasm and that TGF-β signaling is enhanced upon CSIG knockdown. Our study presents a new regulatory process for protein myristoylation and opens new avenues for studying the function of CSIG.
MATERIALS AND METHODS
Cell culture and transfections.U2OS cells, H1299 cells, and HaCaT cells were maintained in Dulbecco's modified Eagle medium (DMEM; Macgene) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified atmosphere under 5% CO2. Plasmid transfections were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The following plasmids were used in this study: pEGFP-N1, pPPM1A-EGFP, pPPM1A(G2A)-EGFP, pIRES-FLAG-HA-CSIG, pIRES-FLAG-HA-CSIG-NT, pIRES-FLAG-HA-CSIG-ΔN, pcDNA3-PPM1A-FLAG, pcDNA3-PPM1A(G2A)-FLAG, pCMV6-PPM1A(G2A)-Myc, and pCMV6-NMT1-Myc, which were constructed by our laboratory, and pcDNA3-PPM1A-FLAG, which was kindly provided by Takayasu Kobayashi of Tohoku University. The siRNA transfections were performed using the Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer's instructions. The sequences of the siRNAs were as follows: CSIG-1 siRNA, 5′-AGAAGGAACAGACGCCAGA-3′; CSIG-2 siRNA, 5′-UUAUCCAGCUGCUUCCGUGCUGUCG-3′; negative-control (NC) siRNA, 5′-UUCUCCGAACGUGUCACGU-3′.
Western blotting.Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Macgene) supplemented with a protease inhibitor cocktail (Amresco). Protein concentrations were evaluated with a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific). The following antibodies were used in this study: anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; bioWORLD), anti-FLAG (Sigma), anti-CSIG protein (2), anti-PPM1A (Abcam), anti-Smad2 (Cell Signaling Technology), and anti-phospho-Smad2 (Cell Signaling Technology and ABclonal Technology).
Immunoprecipitation (IP).Cells were harvested and lysed in FLAG lysis buffer (50 mM Tris-HCl [pH 7.3], 137 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM NaF, 1 mM Na3VO4, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], and a protease inhibitor cocktail [Amresco]). The supernatant was incubated with antibody overnight at 4°C; then protein G-Sepharose (GE Healthcare) was added to the samples, which were incubated for an additional 6 h at 4°C. The immunoprecipitates were washed three times with FLAG lysis buffer and were then boiled at 100°C for 10 min with protein loading buffer. The samples were resolved via SDS-PAGE and were probed using the antibodies indicated in the figures.
Immunofluorescence.Cells grown on coverslips were fixed in 4% paraformaldehyde for 10 min and were then washed with phosphate-buffered saline (PBS) and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. Samples were blocked with 10% goat serum for 1 h at room temperature and were then incubated with antibodies overnight at 4°C. The samples were washed with PBS and were incubated with an Alexa Fluor 647- or Alexa Fluor 488-conjugated secondary antibody (Abcam) for 1 h at room temperature. The samples were washed again with PBS and were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Immunofluorescence images were captured with a confocal laser scanning microscope (Olympus FV1000).
Real-time PCR.Total RNA was isolated from cells with an RNeasy minikit (Qiagen) according to the manufacturer's instructions. First-strand cDNA was synthesized using a TransScript first-strand cDNA synthesis kit (TransGen Biotech). Real-time PCR (RT-PCR) analysis was performed using SYBR Select master mix (Life Technologies) in conjunction with an ABI Prism 7500 sequence detection system. Data were analyzed using the 2−ΔΔCT method.
Cell cycle analysis.Cells were harvested with trypsin, fixed in 70% ethanol overnight at 4°C, treated with RNase A (100 μg/ml; Sigma) at 37°C for 30 min, and stained with propidium iodide (20 μg/ml; Millipore). The cells were then analyzed for DNA content using a BD FACS flow cytometer. Data were analyzed using the CellQuest and ModFit software programs.
Immunohistochemistry.Paraffin sections were immersed in xylene twice, for 10 min each time, and were soaked in decreasing concentrations of ethanol for 7 min at each concentration. In addition, the sections were soaked twice in ultrapure water for 7 min each time. A pressure cooker was used to repair the antigen, and the sections were washed with PBS three times after cooling. Then the paraffin sections were blocked for 10 min in methanol with 3% H2O2 at room temperature. After blocking, the paraffin sections were incubated with antibodies (10 μg/ml) overnight at 4°C. Staining was performed with an immunohistochemical staining kit (GT Vision) according to the manufacturer's instructions.
Detection of myristoylation levels.Myristoylation levels were detected by a procedure described previously (22, 37). Promastigotes were cultured in RPMI medium–10% fetal calf serum (FCS) containing 50 mM YnMyr (an analogue of myristic acid) for 12 h. Cells were collected, washed twice with PBS, and then lysed by sonication in BC500 buffer (25 mM Tris-HCl [pH 7.3], 500 mM NaCl, 0.5% Triton X-100, and 20% glycerol).
Proteins were first precipitated with chloroform-methanol (MeOH/CHCl3/double-distilled water [ddH2O] ratio, 4:1:3) at −20°C for 1 h and then resuspended at 1 mg/ml in 1% SDS in PBS. Then click reagents [100 μM 6-carboxytetramethylrhodamine (TAMRA)–azide–polyethylene glycol (PEG)–biotin (AzTB), 1 mM CuSO4, 1 mM tris(2-carboxyethyl)phosphine (TCEP), 100 μM tris(benzyltriazolylmethyl)amine (TBTA) (final concentrations)] were added to the samples, and the samples were first vortexed for 1 h at room temperature and then quenched by the addition of 10 mM EDTA. Proteins were precipitated again with 10 volumes of MeOH (overnight at −80°C), washed with ice-cold MeOH, air- dried, and resuspended in 2% SDS and 10 mM EDTA in PBS. In addition, gel analysis was performed immediately after the addition of protein loading buffer. Samples were separated by SDS-PAGE and were scanned with Cy3 filters to detect the fluorophore TAMRA using an Ettan DIGE scanner and a Typhoon FLA laser scanner (GE Healthcare).
Protein purification and GST pulldown.To purify FLAG-HA-CSIG protein, the corresponding plasmid was transfected into H1299 cells, and 48 h later, the cells were lysed by sonication in BC500 buffer (25 mM Tris-HCl [pH 7.3], 500 mM NaCl, 0.5% Triton X-100, and 20% glycerol). The soluble extracts were immunoprecipitated with antihemagglutinin (anti-HA) affinity gel (Roche). The beads were washed with BC500 buffer once and were further washed with BC100 buffer (25 mM Tris-HCl [pH 7.3], 100 mM NaCl, 0.5% Triton X-100, and 20% glycerol) twice. Proteins were then eluted with a HA peptide (Roche). The soluble extracts were immunoprecipitated with Anti-FLAG M2 affinity gel (Sigma). The beads were washed with BC500 buffer once and were further washed with BC100 buffer three times. Proteins were then eluted with a FLAG peptide (Sigma). GST and GST-NMT1 were expressed in Escherichia coli Rosetta (DE3) cells induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Cell pellets were resuspended in BC500 buffer containing DTT (1 mM) and were then disrupted by sonication. Cleared cell lysates were incubated with glutathione-Sepharose (GE Healthcare). After extensive washing, the bound protein was eluted with reduced glutathione (Amresco).
To perform GST pulldown analysis, purified GST and GST-NMT1 were incubated with purified PPM1A-FLAG protein with or without FLAG-HA-CSIG protein overnight at 4°C. Glutathione-Sepharose was added, and the mixture was incubated for 5 h. The beads were washed with BC100 buffer and were boiled. The samples were resolved by SDS-PAGE and analyzed by Western blotting.
Statistical analysis.Data are presented as means ± standard deviations (SD). The Student t test was used to analyze statistical differences between groups. A two-tailed P value of <0.05 was considered significant.
ACKNOWLEDGMENTS
We are grateful to Wenhui Zhao and Jianyuan Luo for helpful discussions.
This work was supported by grants from the National Natural Science Foundation of China (81772949) and the National Basic Research Programs of China (2013CB530801).
FOOTNOTES
- Received 17 August 2018.
- Returned for modification 30 August 2018.
- Accepted 5 September 2018.
- Accepted manuscript posted online 10 September 2018.
- Copyright © 2018 American Society for Microbiology.
