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. 2017 Aug 24;7(1):9302.
doi: 10.1038/s41598-017-09434-4.

A Novel Mechanism of ERK1/2 Regulation in Smooth Muscle Involving Acetylation of the ERK1/2 Scaffold IQGAP1

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Free PMC article

A Novel Mechanism of ERK1/2 Regulation in Smooth Muscle Involving Acetylation of the ERK1/2 Scaffold IQGAP1

Susanne Vetterkind et al. Sci Rep. .
Free PMC article

Abstract

Ceramide, a bioactive lipid and signaling molecule associated with cardiovascular disease, is known to activate extracellular signal regulated kinases 1 and 2 (ERK1/2). Here, we determined that the effect of ceramide on ERK1/2 is mediated by ceramide signaling on an ERK scaffold protein, IQ motif containing GTPase activating protein 1 (IQGAP1). Experiments were performed with aortic smooth muscle cells using inhibitor screening, small interfering RNA (siRNA), immunoprecipitation (IP), immunoblots and bioinformatics. We report here that C6 ceramide increases serum-stimulated ERK1/2 activation in a manner dependent on the ERK1/2 scaffold IQGAP1. C6 ceramide increases IQGAP1 protein levels by preventing its cleavage. Bioinformatic analysis of the IQGAP1 amino acid sequence revealed potential cleavage sites for proteases of the proprotein convertase family that match the cleavage products. These potential cleavage sites overlap with known motifs for lysine acetylation. Deacetylase inhibitor treatment increased IQGAP1 acetylation and reduced IQGAP1 cleavage. These data are consistent with a model in which IQGAP1 cleavage is regulated by acetylation of the cleavage sites. Activation of ERK1/2 by ceramide, known to increase lysine acetylation, appears to be mediated by acetylation-dependent stabilization of IQGAP1. This novel mechanism could open new possibilities for therapeutic intervention in cardiovascular diseases.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Ceramide increases serum-induced ERK1/2 phosphorylation. Aortic smooth muscle cells were treated with ceramide C6 for 6 hours; control cells were treated with diluent alone. Cells were stimulated with either 12-deoxyphorbol 13-isobutylate 20-acetate (DPBA) or serum (FBS) for 5 minutes, or left unstimulated. Cell lysates were analyzed by western blotting and densitometry. (A) Typical immunoblots (cropped) show ceramide enhanced the increases in ERK1/2 phosphorylation after FBS, but not after DPBA stimulation. (B) Statistical analysis of 7 independent experiments. *control + FBS versus C6 + FBS: p = 0.024, # C6 + DPBA vs. C6 + FBS: p = 0.025.
Figure 2
Figure 2
SiRNA knockdown demonstrates a role for IQGAP1 in ceramide-induced ERK1/2 activation. (A) Aortic smooth muscle cells were treated with control, IQGAP1, or KSR1 siRNA. 5 days after transfection, cells were treated with ceramide (6 hours, 50 µg/ml). Additionally, cells were stimulated with FBS (10%) for 5 minutes before preparation of cell extracts. Cell lysates were analyzed for expression of IQGAP1, KSR1, phospho-ERK1/2 and total ERK1/2 using specific antibodies. GAPDH staining is shown as reference. Cropped gels are shown. (B) The statistical analysis of 5 independent experiments shows a significant decrease of relative ERK1/2 phosphorylation after knock down of IQGAP, but not KSR1. Statistical analysis of the degree of siRNA-induced knock down are shown in (C) for IQGAP1 and in (D) for KSR1.
Figure 3
Figure 3
Ceramide stabilizes IQGAP1 by inhibiting its cleavage. (A) IQGAP1 antibodies raised against the N-terminal part of the protein (C-9 and H-109) show the same pattern of additional bands, whereas an antibody raised against the C-terminal region of IQGAP1 does not detect these bands. (B,C) VSM cells were treated with either C6 ceramide (50 µmol/L, 6 hours) or the ceramide synthase inhibitor, fumonisin B1 (15 µM, 24 hours) or control treated. Cells were stimulated with serum (10% FBS, 5 minutes) before preparation of whole cell lysates and western blot analysis. H109 was used to detect IQGAP1. (B) Expression levels of full length IQGAP1 were analyzed by normalization of the full length IQGAP1 band to GAPDH. Normalized full length IQGAP1 was then normalized to the untreated control. (C) Cleaved fragments of IQGAP1 are shown as a percentage of total IQGAP1 (=sum of full length IQGAP1 plus cleaved fragments) normalized to the untreated control.
Figure 4
Figure 4
Observed IQGAP1 cleavage products in comparison to positioning of SPC and caspase cleavage sites. (A) List of K/R-X-K/R-K/R motifs found in the IQGAP1 amino acid sequence. (B) Diagram to show full length IQGAP1 and its putative fragments. The epitopes recognized by the different antibodies used in Fig. 3 are indicated in the top row. Fragment lengths were calculated based on SDS-PAGE with a molecular weight marker (Precision Plus, BioRad) as standard. The positions of SPC cleavage motifs are indicated by black and white arrows, with the white arrows also matching furin cleavage sites. For comparison, predicted caspase cleavage sites are also shown (white arrowheads). Abbreviations: CH, calponin homology; WW, poly-proline protein-protein interaction domain; IQ, IQ motifs; GRD, GAP (GTPase activating protein) Related Domain; RGCT, RasGAP-C-terminus IQGAP1. Domain map modified after.
Figure 5
Figure 5
Acetylation and cleavage of IQGAP1. (A) Cells were treated with nicotinamide (NAM) or phenylbutyrate (PB) at 5 mmol/l for 24 hours. Cell lysates were then analyzed by western blotting. Acetylated proteins were detected with an acetylated lysine-specific antibody. Identity of the IQGAP1 band was confirmed by subsequent co-staining with an anti-IQGAP1 antibody. Cropped gels are shown. (B) Bar graph and statistical analysis of IQGAP1 acetylation in untreated, NAM treated and PB treated cells. (C) Endogenous IQGAP1 is immunoprecipitated from lysates of NAM-treated aortic smooth muscle cells with an acetylated lysine-specific antibody, but not with a GFP antibody. A cropped gel is shown. (D) Treatment with NAM or PB results in reduced cleavage of IQGAP1 relative to full length IQGAP1. Cropped gels are shown. (E) The bar graph shows full length IQGAP1 relative to the sum of full length IQGAP1 and smaller IQGAP1 fragments (normalized to control). (*p < 0.05, **p < 0.01).
Figure 6
Figure 6
Ceramide increases acetylation of IQGAP. (A) Ceramide (6 hr) increases acetylation of band co-migrating with IQGAP. Inset: Typical blot. (B) Ceramide increases acetylation by activating HATs. Treatment (1 hour) with the HAT inhibitor anacardic acid (AA) inhibits effect of ceramide to increase acetylation. NAM (1 hour) has no significant additional effect of ceramide to increase acetylation. (inset) Typical blot. Cropped gels are shown.
Figure 7
Figure 7
Effect of HDAC Inhibitor NAM on ERK1/2 activation with a proliferative versus contractile stimulus. (A) Typical blots. Cropped gels are shown. (B) Increases in ERK1/2 phosphorylation with and without NAM for DPBA versus FBS (*p < 0.05).
Figure 8
Figure 8
Model of the regulation of cleavage by acetylation in the presence of ceramide. Based on our data, we suggest a model in which IQGAP1 cleavage is regulated by acetylation at or in close proximity to cleavage sites, and in which ceramide stabilizes IQGAP1 by increasing acetylation and thus, reducing IQGAP1 cleavage. Two cleavage sites that overlap with acetylation motifs are indicated as KQKK and KMKK above the full length IQGAP1 domain map. The domain composition of the resulting IQGAP1 fragments is expected to affect their spectrum of binding partners (shown in the blue-shaded box) and thus, scaffold function. CH, calponin homology (actin binding); WW, proline-rich protein protein interaction domain (ERK1/2 binding); IQ, IQ domain (calmodulin binding); GRD, GTPase activating protein related domain (Cdc42 and Rac binding); RGCT, RasGAP-C-Terminus (beta catenin and E-cadherin binding).

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