Protein kinase A activity is regulated by actomyosin contractility during cell migration and is required for durotaxis.

Dynamic subcellular regulation of protein kinase A (PKA) activity is important for the motile behavior of many cell types, yet the mechanisms governing PKA activity during cell migration remain largely unknown. The motility of SKOV-3 epithelial ovarian cancer (EOC) cells has been shown to be dependent both on localized PKA activity and, more recently, on mechanical reciprocity between cellular tension and extracellular matrix rigidity. Here, we investigated the possibility that PKA is regulated by mechanical signaling during migration. We find that localized PKA activity in migrating cells rapidly decreases upon inhibition of actomyosin contractility (specifically, of myosin ATPase, Rho kinase, or myosin light-chain kinase activity). Moreover, PKA activity is spatially and temporally correlated with cellular traction forces in migrating cells. Additionally, PKA is rapidly and locally activated by mechanical stretch in an actomyosin contractility-dependent manner. Finally, inhibition of PKA activity inhibits mechanically guided migration, also known as durotaxis. These observations establish PKA as a locally regulated effector of cellular mechanotransduction and as a regulator of mechanically guided cell migration.

Aminoacyl-tRNA synthetase dependent angiogenesis revealed by a bioengineered macrolide inhibitor.

Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans.

Secreted Threonyl-tRNA synthetase stimulates endothelial cell migration and angiogenesis.

Aminoacyl-tRNA synthetases classically regulate protein synthesis but some also engage in alternative signaling functions related to immune responses and angiogenesis. Threonyl-tRNA synthetase (TARS) is an autoantigen in the autoimmune disorder myositis, and borrelidin, a potent inhibitor of TARS, inhibits angiogenesis. We explored a mechanistic link between these findings by testing whether TARS directly affects angiogenesis through inflammatory mediators. When human vascular endothelial cells were exposed to tumor necrosis factor-α (TNF-α) or vascular endothelial growth factor (VEGF), TARS was secreted into the cell media. Furthermore, exogenous TARS stimulated endothelial cell migration and angiogenesis in both in vitro and in vivo assays. The borrelidin derivative BC194 reduced the angiogenic effect of both VEGF and TARS, but not a borrelidin-resistant TARS mutant. Our findings reveal a previously undiscovered function for TARS as an angiogenic, pro-migratory extracellular signaling molecule. TARS thus provides a potential target for detecting or interdicting disease-related inflammatory or angiogenic responses.