MYPT1 O-GlcNAcylation controls the sensitivity of fibroblasts to sphingosine-1-phosphate mediated cellular contraction

Many intracellular proteins can be modified by N-acetylglucosamine, a posttranslational modification known as O-GlcNAc. Because this modification is found on serine and threonine side-chains, O-GlcNAc has the potential to dynamically regulate cellular signaling pathways through interplay with phosphorylation. Here, we discover and characterize one such example. First, we find that O-GlcNAc levels control the sensitivity of fibroblasts to actin contraction induced by the signaling lipid sphingosine-1-phosphate (S1P). In follow-up mechanistic investigations, we show that this O-GlcNAc dependence lies in the signaling pathway through the S1PR2 receptor and subsequent activation of the Rho and Rho kinase. This pathway typically culminates in the phosphorylation of myosin light chain (MLC), resulting in myosin activation and cellular contraction. We discovered that O-GlcNAc modification of the phosphatase subunit MYPT1 inhibits this pathway by blocking MYPT1 phosphorylation, maintaining its activity and causing the dephosphorylation of MLC. Therefore, MYTP1 O-GlcNAc levels function to regulate the sensitivity of cells to S1P-mediated cellular contraction. Finally, we demonstrate that O-GlcNAc levels alter the sensitivity of primary human dermal fibroblasts in a collagen matrix model of wound healing. Our findings have important implications for the role of O-GlcNAc in fibroblast motility and differentiation, particularly in diabetic wound healing, where increased levels of the modification may inhibit S1P-mediated healing phenotypes in fibroblasts.