Recombinant human hyaluronan synthase 3 is phosphorylated in mammalian cells

Hyaluronan is a ubiquitous component of vertebrate extracellular and cell-associated matrices that serves as a key structural component of skin, cartilage, eyes and joints, and plays important roles in dynamic cellular processes, including embryogenesis, inflammation, wound healing and metastasis. Hyaluronan is synthesized by three homologous hyaluronan synthases designated HAS1, HAS2 and HAS3 that differ in their tissue distribution, regulation and enzymatic characteristics. Some progress has been made in characterizing regulation of HAS transcripts and in distinguishing the enzymatic properties of the various HAS isoforms, but essentially nothing is known about their possible regulation by posttranslational modification. Using [32P]P(i) radiolabelling of a recombinant FLAG (DYKDDDDK) epitope-tagged version of human HAS3 expressed in COS-7 cells, we show that HAS3 is serine-phosphorylated and that this phosphorylation can be enhanced by a number of effectors--most significantly by a membrane-permeable analogue of cAMP. By employing a novel FLAG-tagged phosphorylated reference protein derived from EGFP (enhanced green fluorescent protein), we were able to estimate the stoichiometry of FLAG-HAS3 phosphorylation. It was approx. 0.11 in unstimulated cells and increased to as much as 0.32 in cells stimulated with 8-(4-chlorophenylthio)-cAMP.