Identification of novel in vitro protein kinase A phosphorylation sites on recombinant non-muscle myosin light chain kinase: Nano-liquid chromatography tandem mass spectrometry methodology

Protein phosphorylation is a key event in endothelial cell signaling and barrier regulation. We previously demonstrated that the non-muscle myosin light chain kinase (nmMLCK) isoform present in endothelium (1914aa) is a multi-functional enzyme which drives the participation of the actin cytoskeleton in vascular barrier regulation, trafficking of inflammatory cells into the lung, and susceptibility to sepsis-induced acute lung injury. The activity of the nmMLCK isoform is differentially regulated by both Ser/Thr and Tyr phosphorylation; however, cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA)-mediated nmMLCK phosphorylation exerts paradoxical effects on kinase activity depending on the exact Ser/Thr sites. To address this conundrum, we mapped in vitro phosphorylation sites of nmMLCK catalyzed by the catalytic unit of PKA using data-dependent nano-liquid chromatography tandem mass spectrometry. High mass-accuracy MS and optimized biochemical protocols identified 26 novel nmMLCK1 PKA phosphorylation sites, including 11 located within the unique nmMLCK N-terminus and four (T335, S365, S947, and T1230) located within putative SH3-binding domains, as well as the known PKA-mediated nmMLCK phosphorylation site (S1208). Further structure/function analysis of the functional effects of these novel phosphorylation events may provide important insights into the regulation of nmMLCK activity and barrier regulation by cAMP-dependent processes.