Hyaluronan-CD44 Interaction with Rac1-dependent Protein Kinase N-γ Promotes Phospholipase Cγ1 Activation, Ca2+ Signaling, and Cortactin-Cytoskeleton Function Leading to Keratinocyte Adhesion and Differentiation*

In this study we have investigated hyaluronan (HA)-CD44 interaction with protein kinase N-γ (PKNγ), a small GTPase (Rac1)-activated serine/threonine kinase in human keratinocytes. By using a variety of biochemical and molecular biological techniques, we have determined that CD44 and PKNγ kinase (molecular mass ∼120 kDa) are physically linked in vivo. The binding of HA to keratinocytes promotes PKNγ kinase recruitment into a complex with CD44 and subsequently stimulates Rac1-mediated PKNγ kinase activity. The Rac1-activated PKNγ in turn increases threonine (but not serine) phosphorylation of phospholipase C (PLC) γ1 and up-regulates PLCγ1 activity leading to the onset of intracellular Ca2+ mobilization. HA/CD44-activated Rac1-PKNγ also phosphorylates the cytoskeletal protein, cortactin, at serine/threonine residues. The phosphorylation of cortactin by Rac1-PKNγ attenuates its ability to cross-link filamentous actin in vitro. Further analyses indicate that the N-terminal antiparallel coiled-coil (ACC) domains of PKNγ interact directly with Rac1 in a GTP-dependent manner. The binding of HA to CD44 induces PKNγ association with endogenous Rac1 and its activity in keratinocytes. Transfection of keratinocytes with PKNγ-ACCcDNA reduces HA-mediated recruitment of endogenous Rac1 to PKNγ and blocks PKNγ activity. These findings suggest that the PKNγ-ACC fragment acts as a potent competitive inhibitor of endogenous Rac1 binding to PKNγ in vivo. Most important, the PKNγ-ACC fragment functions as a strong dominant-negative mutant that effectively inhibits HA/CD44-mediated PKNγ phosphorylation of PLCγ1 and cortactin as well as keratinocyte signaling (e.g. Ca2+ mobilization and cortactin-actin binding) and cellular functioning (e.g. cell-cell adhesion and differentiation). Taken together, these findings strongly suggest that hyaluronan-CD44 interaction with Rac1-PKNγ plays a pivotal role in PLCγ1-regulated Ca2+ signaling and cortactin-cytoskeleton function required for keratinocyte cell-cell adhesion and differentiation.