Prostaglandin E2 is a pleiotropic bioactive lipid that controls cytoskeletal alterations, although the precise G-protein coupled EP receptor signalling mechanisms remain ill defined. We adopted a phosphoproteomic approach to characterize post-receptor downstream signalling substrates using antibodies that selectively recognize and immunoprecipitate phosphorylated substrates of a number of kinases. Using human synovial fibroblasts in monolayer cell culture, PGE2 induced rapid and sustained changes in cellular morphology and reduction in cytoplasmic volume that were associated with disassembly of the phalloidin-stained stress fibres as judged by light and confocal microscopy. Furthermore, PGE2 induced a rapid dephosphorylation of myosin light chain II (MLC) at S19 under basal or cytokine-induced conditions that was linked to an activation of myosin light chain phosphatase. The use of specific synthetic EP agonists suggested that the response was mediated by EP2 receptors, as other EP agonists did not manifest the same effect on MLC phosphorylation. In addition, PGE2 induced sustained Y118 dephosphorylation of phospho-paxillin and loss of focal adhesions as observed by confocal microscopy and Western analysis. Phosphoproteomic analysis of PGE2/GPCR/PKA phosphosubstrates identified a unique, non-redundant, phosphorylated (>30-fold) site on rho-associated coiled coil-containing kinase 2 (ROCK2) at S1379. Analysis of ROCK2 mutant behaviour (e.g. S1379A) in overexpression studies revealed that PGE2-dependent phosphorylation of ROCK2 resulted in the inhibition of the kinase, since induced MLC phosphorylation was no longer blocked by PGE2 nor could PGE2 induce disassembly of stress fibres. Thus, PGE2-dependent blockade of actomyosin fibre formation, characteristic of myofibroblasts, may be mediated through specific ROCK2 S1379 phosphorylation. J. Cell. Biochem. 115: 1516–1527, 2014. © 2014 Wiley Periodicals, Inc.