Directional sensing of a phorbol ester gradient requires CD44 and is regulated by CD44 phosphorylation

Abstract

Cancer progression is associated with enhanced directional cell migration, both of the tumour cells invading into the stroma and stromal cells infiltrating the tumour site. In cell-based assays to study directional cell migration, phorbol esters are frequently used as a chemotactic agent. However, the molecular mechanism by which these activators of protein kinase C (PKC) result in the establishment of a polarized migratory phenotype is not known. Here we show that CD44 expression is essential for chemotaxis towards a phorbol ester gradient. In an investigation of CD44 phosphorylation kinetics in resting and stimulated cells, Ser316 was identified as a novel site of phosphorylation following activation of PKC. PKC does not phosphorylate Ser316 directly, but rather mediates the activation of downstream Ser316 kinase(s). In transfection studies, a phosphorylation-deficient Ser316 mutant was shown to act in a dominant-negative fashion to impair chemotaxis mediated by endogenous CD44 in response to a phorbol ester gradient. Importantly, this mutation had no effect on random cell motility or the ability of cells to migrate directionally towards a cocktail of chemoattractants. These studies demonstrate that CD44 functions to provide directional cues to migrating cells without affecting the motility apparatus.

Hyaluronan: A constitutive regulator of chemoresistance and malignancy in cancer cells

“…P-glycoprotein-targeted siRNA also decreased the rate of cell migration (7), which agrees with the observation that both P-glycoprotein and CD44 need to be active in T lymphocytes for their proper migration to lymph nodes (75). Most likely these effects are due to alterations in CD44 expression, and therefore its migration-inducing activity (76,77), by inhibition of P-glycoprotein expression.…”

The Wnt5A/Protein Kinase C Pathway Mediates Motility in Melanoma Cells via the Inhibition of Metastasis Suppressors and Initiation of an Epithelial to Mesenchymal Transition

“…This was not a simple phosphorylation, but actually involved a phosphorylation switch from a serine 325, to a serine 291, and the serine 291 phosphorylation was critical for directional cell motility. Recently another serine residue on the CD44 molecule, serine 316 has also been shown to require PKC activity for phosphorylation (indirectly) and subsequent directional cell migration along a phorbol ester gradient (45). We show here that, in addition to these observations, phorbol ester treatment of melanoma cells with low levels of CD44 dramatically increased CD44 expression, as did treatment with recombinant Wnt5A.…”

An Integrated Phosphoproteomics Work Flow Reveals Extensive Network Regulation in Early Lysophosphatidic Acid Signaling

“…In addition, LPA, but not HB-EGF, induced an about 3-fold up-regulation of Ser(P) 697 and Ser(P) 704 on the type I transmembrane glycoprotein CD44. Phosphorylation of Ser 697 has been functionally linked to cell motility upon phorbol ester treatment (58). Furthermore, CD44 co-localizes and co-immunoprecipitates with the EGFR (59), which has also been implicated in LPA-triggered A498 cell migration and invasion (13).…”