Thrombin activates MAPKAP2 kinase in vascular smooth muscle

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Thrombin activates MAPKAP2 kinase in vascular smooth muscle Colleen M. Brophy, MD, David Woodrum, BS, Mary Dickinson, BS, Arthur Beall, PhD Journal of Vascular Surgery Volume 27, Issue 5, Pages 963-969 (May 1998) DOI: 10.1016/S0741-5214(98)70279-4 Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Thrombin-induced contraction of bovine carotid artery smooth muscle. Strips of bovine carotid artery smooth muscle were equilibrated in a muscle bath, and the maximal contractile response to high extracellular KCl (110 mM, K) was determined. The strips were washed (W) and re-equilibrated in normal bicarbonate buffer. The strips were then exposed to bovine thrombin (300 U/ml, T). Contractions induced by thrombin were 107 ± 5.4% (n = 8) of the magnitude of the response to KCl. The contractions were sustained for at least 30 minutes (data not shown). The magnitude of the contractile response (grams) is depicted on the y axis and time (minutes) on the x axis. Journal of Vascular Surgery 1998 27, 963-969DOI: (10.1016/S0741-5214(98)70279-4) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Thrombin-induced activation of MAPKAP2 kinase in carotid artery. Strips of bovine carotid artery smooth muscle were stimulated with thrombin (THR; 300 U/ml for 10 minutes) or remained unstimulated (CONT). The strips were homogenized, and MAPKAP2 kinase was immunoprecipitated from 400 μg of protein. The activity of MAPKAP2 kinase was determined with a specific substrate peptide in a phosphotransferase assay. The results are reported as the amount of 32P transferred to the peptide. Thrombin stimulation led to a significant increase in the activity of MAPKAP2 kinase (*p < 0.05, paired t test; n = 6). Journal of Vascular Surgery 1998 27, 963-969DOI: (10.1016/S0741-5214(98)70279-4) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 Thrombin stimulation leads to an increase in the phosphorylation of multiple isoforms of HSP27. Strips of bovine carotid artery smooth muscle were phosphorylated with [32P]-orthophosphate and remained in buffer (A and C, control [CONT]) or were stimulated with thrombin (300 U/ml) for 10 minutes (B and D, THR). The strips were homogenized and the proteins separated on two-dimensional gels and transferred to Immobilon-P. The blots were exposed on a PhosphorImager (A and B), and the extent of phosphorylation was determined with ImageQuant software. The blots were then probed with anti-HSP27 antibodies (C and D). Thrombin stimulation led to significant (p < 0.05, ANOVA on ranks; n = 4) increases in the phosphorylation of the myosin light chains (m; 2.8 ± 0.8-fold increase over control) and four different 27 kd proteins (A, 3.0 ± 0.8-fold; B, 1.6 ± 0.6-fold; C, 1.7 ± 0.5-fold; and D, 2.3 ± 0.9-fold increase over control). The four 27 kd phosphoproteins were immunoreactive with anti-HSP27 antibodies (C and D). An immunoreactive isoform of HSP27 was not phosphorylated (NP). The relative mobility of the proteins is indicated by the molecular weight markers on the left, and the direction of the isoelectric gradient is indicated by the + and − at the top of the gels. Journal of Vascular Surgery 1998 27, 963-969DOI: (10.1016/S0741-5214(98)70279-4) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 4 Recombinant HSP27 (0.5 μg) was phosphorylated in vitro by MAPKAP2 kinase (0.1 U). The proteins were separated on two-dimension gels, and autoradiographs were obtained. The relative mobility and isoelectric focusing points of recombinant HSP27 phosphorylated in vitro by MAPKAP2 kinase were similar to the patterns that occurred in carotid artery smooth muscle stimulated by thrombin in situ. The relative mobility of the proteins is indicated by the molecular weight markers on the left, and the direction of the isoelectric gradient is indicated by the + and − at the top. Journal of Vascular Surgery 1998 27, 963-969DOI: (10.1016/S0741-5214(98)70279-4) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 5 Phosphopeptide maps of the HSP27 isoforms. The spots (A, B, C, and D) corresponding to the various isoforms of HSP27 (HSP27A, HSP27B, HSP27C, and HSP27D) were excised from two-dimension gels. Proteolytic digests of the proteins were performed according to the method of Cleveland et al.,21 the peptides were separated on a 15% SDS-PAGE gel, and autoradiographs were obtained. The patterns of phosphorylated peptides from each of the isoforms were similar. This autoradiograph is representative of three separate experiments. Journal of Vascular Surgery 1998 27, 963-969DOI: (10.1016/S0741-5214(98)70279-4) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Quantitation of immunoreactive HSP27. Two-dimensional immunoblots of bovine carotid artery smooth muscles stimulated with thrombin (THR, 300 U/ml for 10 minutes) compared with unstimulated (CONT) were quantitated with a PhosphorImager. There was no change in the amount of total immunoreactive HSP27 in the stimulated arteries compared with the unstimulated arteries (data not shown). Stimulation with thrombin led to a decrease in the amount of the nonphosphorylated isoform of HSP27 when expressed as a percentage of the total amount of HSP27 (*p < 0.05, paired t test; n = 6). Journal of Vascular Surgery 1998 27, 963-969DOI: (10.1016/S0741-5214(98)70279-4) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions