As Expected, Ectopically Expressed Wt-VASP Was Phosphorylated at Serine157

3

Chen, VASP in SMC growth and inhibition by NO. ATVB/2004/013995:R1

Online Supplement

Methods

DNA synthesis. DNA synthesis was measured as described 1 by 3H-thymidine incorporation in response to stimulation by FBS. Briefly, cells (3X104/well) were seeded in 12-well plates overnight, and starved in serum-free DMEM. After 48-72 hours, cells were stimulated with either 10% FBS (for rat SMCs) or 15% FBS (for mouse SMCs) in the presence or absence of appropriate reagents [the NO donor, S-nitroso-n-acetylpenicillamine (SNAP) or cGMP analogues], and then 3H-thymidine (1mci per well) was added after 18 hours. 10 hours later, cells were rinsed with PBS, pH 7.4, cold 10% trichloroacetic acid (TCA) was added and the DNA was extracted with 0.1M NaOH. The radioactivity of the extract was measured by scintillation counting. There were at least three independent experiments for each condition, and each sample had at least three replicates in each experiment.

Protein extraction, immunoprecipitation and Western blotting. SMCs were lysed in HEB buffer [25 mM Hepes, pH 7.5; 5 mM EDTA; 5 mM EGTA; 150 mM NaCl; 100 mM Na4P2O7; 50 mM NaF; 1 mM benzamidine; 1% (v/v) Triton X-100; 10% (v/v) glycerol; 0.1% (v/v) b-mercaptoethanol; 1 mg/ml pepstain-A; 5 mg/ml leupeptin and 5 mg/ml aprotinin] 2. The cell lysates were boiled for 5 minutes in SDS-sample buffer and subjected to 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes (Bio-Rad, Richmond, CA). Blots were probed with primary antibodies (at the concentrations recommended by manufacturers or references) at 4oC overnight. The proteins were detected by horseradish peroxidase-conjugated secondary antibodies with the use of a standard enhanced-chemiluminescence (ECL) according to the manufacturer’s protocol (Amersham BioSciences, Piscataway, NJ).

Results

Characterization of expression of wt-VASP and its non-phosphorylatable mutants (S157A-VASP and S239A-VASP) in SMCs from VASP-/- mice. To investigate the potential roles of serine157 and serine239 phosphorylation in serum-induced proliferation and NO-induced growth inhibition, we expressed wt-VASP and non-phosphorylatable VASP mutants (S157A-VASP and S239A-VASP) using LXSN in VASP-/- mouse SMCs. We selected clones with VASP expression levels similar to those of primary SMCs.

As expected, ectopically expressed wt-VASP was phosphorylated at serine157 in response to FBS, phorbol-12-myristate-13-acetate (PMA), and the PKA-activating reagent, forskolin, and at serine239 in response to the NO donor, SNAP (Figure I). As reported before 3 and shown in figure I, SNAP induced phosphorylation at serine157 and forskolin induced phosphorylation at serine239 in wt-VASP expressing SMCs. Only serine157, but not serine239, was detected in response to FBS and PMA in wt-VASP expressing SMCs.

Consistent with the lack of serine157 phosphorylation, none of the stimuli caused a VASP gel shift in SMCs expressing S157A-VASP (Figure I). However, S157A-VASP could still be phosphorylated at serine239 in response to SNAP, but not to forskolin (Figure I). Serine157 in S239A-VASP expressing SMCs was still phosphorylated in response to FBS, PMA and forskolin, bur not to SNAP (Figure I). These observations suggest that in murine VSMCs serine239 phosphorylation in response to forskolin requires phosphorylation of serine157, and that serine157 phosphorylation in response to SNAP requires phosphorylation of serine239.

References

1. Chen, L., Daum, G., Forough, R., Clowes, M. M., Walter, U. and Clowes, A. W. Overexpression of human endothelial nitric oxide synthase in rat vascular smooth muscle cells and in balloon-injured carotid artery. Circ. Res. 1998; 82: 862-870.

2. Seger, R., N. G. Ahn, J. Posada, E. S. Munar, A. M. Jensen, J. A. Cooper, M. H. Cobb and E. G. Krebs. Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells. J. Biol. Chem. 1992; 267:14373-14381.

3. Butt, E., Abl, K., Krieger, M., Palm, D., Hoppe, V. and Walter, U. cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimilate phosphoprotein (VASP) in Vitro and in intact human platelets. J. Biol. Chem. 1994; 269: 14509-14517.