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J. Biol. Chem., Vol. 279, Issue 28, 29654-29669, July 9, 2004

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Hyaluronan-CD44 Interaction with Rac1-dependent Protein Kinase N- Promotes Phospholipase C1 Activation, Ca²⁺ Signaling, and Cortactin-Cytoskeleton Function Leading to Keratinocyte Adhesion and Differentiation^*

Lilly Y. W. Bourguignon, Patrick A. Singleton, and Falko Diedrich

From the Department of Medicine, University of California, San Francisco, Endocrine Unit (111N), San Francisco Veterans Affairs Medical Center, San Francisco, California 94121

In this study we have investigated hyaluronan (HA)-CD44 interaction with protein kinase N- (PKN), 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 PKN kinase (molecular mass 120 kDa) are physically linked in vivo. The binding of HA to keratinocytes promotes PKN kinase recruitment into a complex with CD44 and subsequently stimulates Rac1-mediated PKN kinase activity. The Rac1-activated PKN in turn increases threonine (but not serine) phosphorylation of phospholipase C (PLC) 1 and up-regulates PLC1 activity leading to the onset of intracellular Ca²⁺ mobilization. HA/CD44-activated Rac1-PKN also phosphorylates the cytoskeletal protein, cortactin, at serine/threonine residues. The phosphorylation of cortactin by Rac1-PKN attenuates its ability to cross-link filamentous actin in vitro. Further analyses indicate that the N-terminal antiparallel coiled-coil (ACC) domains of PKN interact directly with Rac1 in a GTP-dependent manner. The binding of HA to CD44 induces PKN association with endogenous Rac1 and its activity in keratinocytes. Transfection of keratinocytes with PKN-ACCcDNA reduces HA-mediated recruitment of endogenous Rac1 to PKN and blocks PKN activity. These findings suggest that the PKN-ACC fragment acts as a potent competitive inhibitor of endogenous Rac1 binding to PKN in vivo. Most important, the PKN-ACC fragment functions as a strong dominant-negative mutant that effectively inhibits HA/CD44-mediated PKN phosphorylation of PLC1 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-PKN plays a pivotal role in PLC1-regulated Ca²⁺ signaling and cortactin-cytoskeleton function required for keratinocyte cell-cell adhesion and differentiation.

Received for publication, April 1, 2004

^* This work was supported by United States Public Health Service Grants P01 AR39448, R01 CA66163, and R01 CA 78633 from the National Institutes of Health and a Veterans Affairs Merit Review grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence and reprint requests should be addressed: Endocrine Unit (111N), Dept. of Medicine, University of California, San Francisco, and Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-221-4810 (ext. 3321); Fax: 415-383-1638; E-mail: lillyb{at}itsa.ucsf.edu.

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