• Prostate-apoptosis response-4 phosphorylation in vascular smooth muscle.

Prostate-apoptosis response-4 phosphorylation in vascular smooth muscle.

Archives of biochemistry and biophysics (2012-12-12)
Justin A MacDonald, Lori D Moffat, Abdulhameed Al-Ghabkari, Cindy Sutherland, Michael P Walsh

The protein prostate-apoptosis response (Par)-4 has been implicated in the regulation of smooth muscle contraction, based largely on studies with the A7r5 cell line. A mechanism has been proposed whereby Par-4 binding to MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase, MLCP) blocks access of zipper-interacting protein kinase (ZIPK) to Thr697 and Thr855 of MYPT1, whose phosphorylation is associated with MLCP inhibition. Phosphorylation of Par-4 at Thr155 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition and contraction. We tested this "padlock" hypothesis in a well-characterized vascular smooth muscle system, the rat caudal artery. Par-4 was retained in Triton-skinned tissue, suggesting a tight association with the contractile machinery, and indeed Par-4 co-immunoprecipitated with MYPT1. Treatment of Triton-skinned tissue with the phosphatase inhibitor microcystin (MC) evoked phosphorylation of Par-4 at Thr155, but did not induce its dissociation from the contractile machinery. Furthermore, analysis of the time courses of MC-induced phosphorylation of MYPT1 and Par-4 revealed that MYPT1 phosphorylation at Thr697 or Thr855 preceded Par-4 phosphorylation. Par-4 phosphorylation was inhibited by the non-selective kinase inhibitor staurosporine, but not by inhibitors of ZIPK, Rho-associated kinase or protein kinase C. In addition, Par-4 phosphorylation did not occur upon addition of constitutively-active ZIPK to skinned tissue. We conclude that phosphorylation of Par-4 does not regulate contraction of this vascular smooth muscle tissue by inducing dissociation of Par-4 from MYPT1 to allow phosphorylation of MYPT1 and inhibition of MLCP.

Product Number
Product Description

Octoxinol 10, European Pharmacopoeia (EP) Reference Standard
Triton X-305 solution, 70% in H2O
Triton X-102
GF 109203X, synthetic, ≥90% (HPLC)
Triton X-100 solution, BioUltra, for molecular biology, ~10% in H2O
Triton X-100, laboratory grade
Triton X-100, for molecular biology
Triton X-100, BioXtra
Triton X-100, peroxide- and carbonyl-free
Triton X-45