CD44 is the principle transmembrane receptor for the extracellular matrix glycosaminoglycan hyaluronan. This receptor:ligand interaction plays an essential role in a number of physiological events including tumour progression, lymphocyte homing into inflammatory sites and tissue morphogenesis during development. In previous studies we have shown that serine phosphorylation is a critical control mechanism for CD44-dependent cell migration. Here we have investigated the target phosphorylation residues by mutating them individually or in combination. These studies demonstrate that Ser325 is the principle CD44 phosphorylation site and that mutation of this residue blocks CD44-mediated cell migration but not hyaluronan binding. In addition, we show that an upstream Ser323 residue is required as part of the kinase consensus site. To further characterize the role of CD44 phosphorylation, phosphorylated and non-phosphorylated peptides spanning the Ser325 region were synthesised and linked to a 16 amino acid Penetratin sequence to mediate efficient plasma membrane translocation. Peptides containing a phosphoserine at residue 325 are efficient blockers of CD44-mediated cell migration but do not reduce CD44 expression or its ability to bind hyaluronan. These data strongly argue that CD44 adhesion and migration are regulated by distinct mechanisms and that migration requires the specific interaction of intracellular component(s) with phosphorylated CD44 receptors.

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