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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

Received for publication, April 1, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the epidermis, extracellular matrix (ECM)¹ components form an integral part of hemidesmosomes and mediate keratinocyte attachment to the underlying basement membrane. Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix (ECM) of most mammalian tissues including epidermis and dermis (1-3) and HA has been implicated in skin epidermal function (1-3). However, the cellular and molecular mechanism by which keratinocytes respond to HA is not understood.

The predominant receptor for HA on the cell surface of keratinocytes is CD44 (1-12). CD44 is encoded by a single gene that contains 19 exons (5). The most common form, CD44s (CD44 standard form), contains exons 1-5 (N-terminal 150 amino acids), exons 15 and 16 (membrane proximal 85 amino acids), exon 17 (transmembrane domain), and a portion of exons 17 and 19 (cytoplasmic tail, 70 amino acids) (5). Of the 19 exons, 12 exons can be alternatively spliced (5). Most often, the alternative splicing occurs between exons 5 and 15 leading to an insertion in tandem of one or more variant exons (exon 6-exon 14;v1-v10) within the membrane-proximal region of the extracellular domain (5). For example, keratinocytes contain additional exons v3-v10, which are inserted into the CD44s transcripts (6-12). This isoform has been designated as CD44v3-10 (or Epican) (6-12). Various skin cancer cells and tissues express different CD44 variant (CD44v) isoforms (e.g. CD44v3 and CD44v10) in addition to CD44s and CD44v3-10 (Epican) (6-12). These CD44 isoforms have the same amino acid sequences at the two ends of the molecule but differ within the CD44 membrane-proximal region located at the external side of the membrane (5-12). Different CD44 isoforms are also further modified by extensive N- and O-glycosylations and glycosaminoglycan additions (13-15). Consequently, both post-translational modifications and/or alternative splicing within the CD44 molecule appear to determine the functional outcome of this important molecule.

The binding of HA to CD44 causes cells to adhere to extracellular matrix (ECM) components and has also been implicated in the stimulation of several different biological activities (16-19). The intracellular domain of CD44 binds to signaling proteins, such as RhoA- and Rac1-specific guanine nucleotide exchange factors (20-23), and cytoskeletal proteins including ankyrin (16-19) and the ERM proteins (ezrin, radixin, and moesin) (24). In addition, the cytoplasmic domain of CD44 is known to be tightly coupled with c-Src kinase, which promotes tyrosine phosphorylation of cortactin (a filamentous actin (F-actin)-associated protein) and cytoskeleton function resulting in specific structural changes in the plasma membrane and cell migration (18, 19, 61). These findings strongly suggest that the CD44 molecule provides a direct linkage between the ECM and the cytoskeleton.

RhoGTPases (small molecular weight GTPases (e.g. RhoA, Rac1, and Cdc42)) act as molecular switches that alternate between GTP- and GDP-bound states. The "activated" GTP-bound enzymes preferentially interact with downstream effector molecules and modulate the activities of the effectors (25). Previous work has indicated that HA also promotes the interaction between CD44 and several Rac1-specific guanine nucleotide exchange factors (e.g. Tiam1 (22) and Vav2 (23)) thereby up-regulating Rac1 signaling (22, 23, 26) and cytoskeleton-mediated cell functions (22-24). In keratinocytes, Rac1 has been shown to be co-localized with E-cadherin in cell-cell adhesion sites (27). Activation of Rho signaling is also involved in cytoskeleton-associated lamellipodia formation in keratinocytes (28).

Several enzymes have been identified as possible downstream targets for Rac1 signaling. One such enzyme is protein kinase N- (PKN) (also called PRK2), which belongs to a family of serine-threonine kinases known to interact with Rac1 in a GTP-dependent manner, and it shares a great deal of sequence homology with protein kinase C in the C-terminal region (29-31). The N-terminal region of PKN contains three homologous stretches of 70 amino acids (relatively rich in charged residues) and forms an antiparallel coiled-coil fold (ACC domain) (29). This ACC domain has been shown to interact with RhoGTPases such as RhoA and Rac1 (and to a lesser extent with Cdc42) (29-31). The C-terminal region contains the C2-like region, which functions as an auto-inhibitory domain (32). The ACC and the C2-like domains, together with the catalytic domain, are conserved among the PKN family members (29-32). In keratinocytes, RhoA-activated PKN has been found to be involved in Fyn/Src kinase-regulated cell-cell adhesion during Ca²⁺-induced differentiation (33). The question of whether HA/CD44-induced Rac1 activation and PKN-targeted downstream effectors are involved in regulating keratinocyte cell-cell adhesion and differentiation has not been clearly addressed.

HA binding to CD44-expressing cells also stimulates intracellular Ca²⁺ mobilization, which is a prerequisite for the onset of a variety of biological activities (34-36). Although the cellular and molecular mechanisms involved in HA/CD44-mediated Ca²⁺ signaling are currently not well understood, one of the likely pathways involves the phosphatidylinositol cascade that leads to Ca²⁺ release from intracellular stores (37). In response to different stimuli (including HA), phosphoinositide-specific phospholipase Cs (PLCs) hydrolyze phosphatidyl-4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate (IP₃) (35). IP₃, as a second messenger, binds to the IP₃ receptor and induces the release of Ca²⁺ from intracellular stores (34-37). At least four major PLC families exist, PLC-, PLC-, PLC-, and PLC-, and each family type has a number of subtypes (38, 39). One of the best characterized PLCs in keratinocytes is PLC-1, which is involved in inositol lipid signaling and keratinocyte differentiation (40, 41). Specifically, Xie and Bikle (40) have shown that blockage of PLC-1 expression with antisense PLC-1cDNA or down-regulation of PLC-1 activity with a specific inhibitor (41) can effectively impair Ca²⁺-induced keratinocyte differentiation. These findings suggest that intracellular PLC-1-mediated Ca²⁺ mobilization plays a critical role in regulating keratinocyte differentiation.

Although RacGTPase has been closely associated with remodeling of cortical actin (in particular, cortactin-cytoskeleton interaction) (28, 33, 42, 43), Ca²⁺ signaling (44), and cell-cell adhesion (33), Rac-specific effectors and their roles in regulating HA/CD44-mediated keratinocyte functions have not been identified or characterized. In this study we have investigated HA/CD44-mediated Rac1-PKN kinase signaling and its downstream effector function (PLC-1-mediated Ca²⁺ mobilization and cortactin-actin interaction) during human keratinocyte cell-cell adhesion and differentiation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture Model Systems—Normal human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM, Clonetics, San Diego, CA) as described previously (45). Briefly, keratinocytes were isolated from newborn human foreskins by trypsinization (0.25% trypsin, 4 °C, overnight), and primary cultures were established in KGM containing 0.07 mM calcium. After the first and second passages, keratinocytes were incubated with KGM containing 0.03 mM calcium and used in the experiments described below.

Antibodies and Reagents—Monoclonal rat anti-human CD44 antibody (clone, 020; isotype, IgG_2b; obtained from CMB-TECH, Inc., San Francisco) used in this study recognizes a common determinant of the CD44 class of glycoproteins including CD44s (the standard form), CD44 variant isoforms, and CD44_3-10 (Epican). Rabbit anti-phosphothreonine antibody and rabbit anti-phosphoserine antibody were obtained from Zymed Laboratories Inc.. Polyclonal mouse anti-involucrin and polyclonal mouse anti-transglutaminase were purchased from Covance Inc. (Princeton, NJ) and Neomarkers (Fremont, CA), respectively. Rabbit anti-PKN, rabbit anti-PLC1, mouse anti-RhoA, and mouse anti-Rac1 were obtained from Santa Cruz Biotechnology. Mouse anti-His and mouse anti-cortactin antibody (clone 4F11) were purchased from Invitrogen and Upstate Biotechnology, Inc. (Lake Placid, NY), respectively. Several other reagents including GST-tagged RhoA, GST-tagged Rac1, and inhibitors (e.g. U73122 [GenBank] , Xestospongin C, and BAPTA) were obtained from Calbiochem. Rooster comb hyaluronan (HA) and cytochalasin D were purchased from Sigma. High molecular weight HA polymers (10⁶ daltons) were purified by gel filtration column chromatography using Sephacryl S1000 column. The purity of high molecular weight HA polymers used in our experiments was further verified by anion exchange high performance liquid chromatography. No small HA fragments were detected in these preparations.

Method for Preparing His-tagged Dominant-negative Form (Containing ACC Domains) of PKN Kinase—The cDNA fragment encoding the ACC domains of PKN (amino acids 98-256) was generated by reverse transcription-PCR using two specific primers (5'-GGTGCAGCAGAAATTGGATGA-3' and 5'-CTTTGACGTGGACTTAGTGTTGGTG-3'). The PCR product digested with EcoRI and HindIII was purified with QIAquick PCR purification kit (Qiagen). The PKN-ACC fragment cDNA was subsequently cloned into pcDNA3.1/HisC vector that contains Xpress epitope to create His-tagged PKN-ACCcDNA. The inserted ACC domain sequence was confirmed by nucleotide sequencing analyses. This His-tagged PKN-ACC cDNA was then used for transient expression in keratinocytes as described below.

Cell Transfection—To establish a transient expression system, keratinocytes were transfected with various plasmid DNAs (e.g. His-tagged PKN-ACC domain cDNA or vector alone) using TransIT^R-Keratinocyte Transfection Reagent (Mirus, Madison, WI) according to those procedures described previously (46). Briefly, keratinocytes were plated at a density of 2 x 10⁶ cells per 100-mm dish and transfected with 25 µg/dish plasmid cDNA using TransIT^R-Keratinocyte Transfection Reagent. Transfected cells were grown in the culture medium containing G418 for at least 3 days. Various transfectants were then analyzed for their protein expression (e.g. PKN-ACC-related proteins) by immunoblot, PKN activity, and keratinocyte functional assays as described below.

Measurement of Rac1 Activation—Keratinocytes (5.0 x 10⁶ cells) were resuspended in a buffer containing 118 mM KCl, 5 mM NaCl, 0.4 mM CaCl₂,1mM EGTA, 1.2 mM magnesium acetate, 1.2 mM KH₂PO₄, 25 mM Tris-HCl (pH 7.4), 20 mg/ml bovine serum albumin. An aliquot of the cell suspension was added to the electroporation cuvette and incubated at 4 °C for 5-10 min followed by adding [³⁵S]GTPS (12.5 µCi). Subsequently, cells were electroporated at 25 microfarads and 2.0 kV/cm followed by incubating with 50 µg/ml HA (in the presence or absence of rat anti-CD44 antibody (50 µg/ml)) or without any HA treatment at 37 °C for 10 min. Subsequently, [³⁵S]GTPS-labeled cells were washed in PBS (pH 7.4) and solubilized in 1.0% Nonidet P-40 with 1 mM GTP, 25 mM magnesium acetate, and protease inhibitors in PBS (pH 7.4). Nonidet P-40-solubilized cells were then incubated with mouse anti-Rac1 IgG (5 µg/ml) plus goat anti-mouse-conjugated beads. The amount of [³⁵S]GTPS-Rac1 associated with anti-Rac1-conjugated immunobeads was measured by a gamma counter. The values expressed in Table I represent an average of triplicate determinations of five experiments with a standard deviation of less than 5%.

Untransfected keratinocytes (untreated or pretreated with the various inhibitors U73122 [GenBank] (1 µM), Xestospongin C (1 µM), BAPTA/AM (1 µM), cytochalasin D (20 µg/ml), and colchicine (1 x 10^-5 M) for 1 h at 37 °C) or keratinocytes transfected with PKN-ACCcDNA (or vector alone) were incubated with HA (50 µg/ml) at 37 °C for 10 min (or pretreated with anti-CD44 antibody followed by adding HA (50 µg/ml) or incubated with no HA). These cells were then immunoprecipitated with anti-PLC1 (or anti-cortactin) antibody followed by immunoblotting with anti-phosphoserine or anti-phosphothreonine, respectively. Subsequently, these blots were then developed using the ECL reagent according to the manufacturer's instructions. During these immunological analyses, an equal amount of cellular protein (50 µg/ml) immunoprecipitated with the antibody was applied to SDS-PAGE followed by immunoblot analyses.

In some experiments, keratinocytes (e.g. untransfected or transfected with His-tagged PKN-ACCcDNA or vector only) were incubated with HA (50 µg/ml) at 37 °C for 10 min (or pretreated with anti-CD44 antibody followed by adding HA (50 µg/ml) or incubated with no HA). These cells were then immunoprecipitated with rabbit anti-PKN IgG (or mouse anti-His IgG) followed by immunoblotting with mouse anti-Rac1 IgG/mouse anti-RhoA IgG or reblotted with anti-PKN IgG (or anti-His IgG) followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (or goat anti-rabbit IgG) (1:10,000 dilution). Cell lysates of keratinocytes (e.g. transfected with His-tagged PKNcDNA or vector only) were also immunoblotted with anti-PKN IgG and anti-His IgG, respectively. These blots were then developed using ECL reagent (Amersham Biosciences).

PKN-mediated Protein Phosphorylation Assay in Vitro—The PKN kinase reaction was carried out in 50 µl of the reaction mixture containing 40 mM Tris-HCl (pH 7.5), 2 mM EDTA, 1 mM dithiothreitol, 7 mM MgCl₂, 0.1% CHAPS, 0.1 µM calyculin A, 100 µM ATP, purified enzymes (e.g. 100 ng of PKN keratinocytes isolated from untransfected keratinocytes or keratinocytes transfected with His-tagged PKN-ACCcDNA or vector only) and 1 µg of PLC1 keratinocytes (or 1 µg of keratinocyte cortactin) in the presence of RhoA or Rac1 (incubated with 200 µM GDP or 200 µM GTP). After incubation for 30 min at 30 °C, the reaction mixtures were immunoprecipitated with anti-PLC1 beads (or anti-cortactin beads), boiled in SDS sample buffer, and subjected to SDS-PAGE. The protein bands associated with anti-PLC1 beads (or anti-cortactin beads) were revealed by anti-phosphoserine/anti-phosphothreonine-mediated immunoblot or anti-PLC1 (or anti-cortactin)-mediated immunoblot, respectively.

PLC1 Activity Assay—The procedures for analyzing the activity of unphosphorylated or phosphorylated PLC1 were similar to those described previously (47). Briefly, keratinocytes were solubilized in Nonidet P-40 buffer in the presence of protease inhibitors plus 1 µM NaF, 1 µM sodium orthovanadate, and 1 µM okadaic acid. Lysates were spun at 5000 x g, and the supernatants were collected. PLC1 was immunoprecipitated from the supernatants using rabbit anti-PLC1-conjugated beads. These PLC1-conjugated beads were then phosphorylated with PKN in the presence of GDP/GTP-bound RhoA or GDP/GTP-bound Rac1 or no PKN (as described above). Subsequently, unphosphorylated or RhoA/Rac1-PKN-phosphorylated PLC1 immunobeads were incubated with vesicles containing 100 µM phosphatidylethanolamine, 10 µM phosphatidylserine, 10 µM PIP₂, and 0.028 µM [³H]PIP₂ in PLC activity buffer (25 mM HEPES, 80 mM KCl, 3 mM EGTA, 0.5 mM dithiothreitol, pH 7.0) at 37 °C. In some cases, PLC1 isolated from keratinocytes (untransfected or transfected with PKN-ACCcDNA or vector alone), which were treated with HA (50 µg/ml) at 37 °C for 10 min (or pretreated with anti-CD44 antibody followed by adding HA (50 µg/ml) or incubated with no HA), was also incubated in this enzyme assay mixture. Reactions were carried out for 15 min and terminated by adding 10% trichloroacetic acid and 2 mg/ml bovine serum albumin. Precipitated proteins were removed by centrifugation. Radioactive [³H]IP₃ (liberated from [³H]PIP₂) in the trichloroacetic acid-soluble supernatant fraction was analyzed using scintillation counting. PIP₂ hydrolysis was linear for 15 min under the conditions used.

Double Immunofluorescence Staining—Keratinocytes (transfected with PKN-ACCcDNA or vector alone) were incubated with HA (50 µg/ml) at 37 °C for 36 h (or pretreated with anti-CD44 antibody followed by adding HA (50 µg/ml) or incubated with no HA) followed by fixing with 2% paraformaldehyde. Subsequently, these cells were stained with Texas Red-labeled rat anti-CD44 IgG. Texas Red-labeled cells were then rendered permeable by ethanol treatment followed by incubating with fluorescein (FITC)-conjugated immunoreagents (e.g. anti-PKN IgG, anti-PLC1 IgG, or anti-cortactin IgG). To detect nonspecific antibody binding, Texas Red-conjugated anti-CD44 or FITC-conjugated immunoreagent (e.g. anti-PKN IgG, anti-PLC1 IgG, and anti-cortactin IgG)-labeled cells were incubated with FITC-conjugated normal rabbit/normal mouse or Texas Red-conjugated normal rat IgG, respectively. No labeling was observed in such control samples. These fluorescein- and Texas Red-labeled samples were examined with a confocal laser scanning microscope.

Measurement of Intracellular Ca²⁺ Mobilization—Keratinocytes (untreated or pretreated with the various inhibitors U73122 [GenBank] (1 µM), Xestospongin C (1 µM), BAPTA/AM (1 µM), cytochalasin D (20 µg/ml), and colchicine (1 x 10^-5 M) at 37 °C for 1 h or transfected with PKN-ACCcDNA or vector alone) were first incubated with 10 µM Fura-2/AM (Calbiochem) for 1 h at room temperature in a buffer solution containing 145 mM NaCl, 5 mM KCl, 0.1 mM MgCl₂, 5 mM glucose, and 15 mM HEPES (pH 7.3) in the presence or absence of 1 mM CaCl₂. Cells were subsequently washed three times with the same buffer. Cells (10⁷ cells/ml) (with or without rat anti-CD44 antibody (1 µg/ml)) resuspended in 0.1 M phosphate-buffered saline (pH 7.0) were incubated simultaneously with an equal volume of 0.1 M phosphate-buffered saline (pH 7.0) containing HA (50 µg/ml) (Sigma) into a 20-µl chamber alternately illuminated with 200-ms flashes of 340 and 380 nm every 10 ms (monitoring the emission wavelength of 510 nm) using a Dural wavelength fluorescence imaging system (Intracellular Imaging Inc., Cincinnati, OH). The concentration of intracellular Ca²⁺ was determined by the following equation: Ca²⁺ = K_d x ((R - R_min)/(R_max - R)) x F/B, where Ca²⁺ is intracellular Ca²⁺; K_d is the dissociation constant of Fura-2 for Ca²⁺; R is the ratio of the Fura-2 fluorescence excited at 340 divided by the fluorescence excited at 380 nm, R_min and R_max indicate minimal and maximal fluorescence ratios, respectively, obtained in ionomycin in the presence of 7 mM EGTA or 2.0 mM Ca²⁺; and F and B are the fluorescence voltage signals at 380 nm in 5.0 µM ionomycin in the presence of 7 mM EGTA and 2 mM Ca²⁺, respectively.

F-actin Cross-linking Assay—The procedures for conducting F-actin cross-linking experiments were the same as those described previously (48, 49) with some modifications. Unphosphorylated cortactin or PKN-phosphorylated cortactin (as described above) (50-100 nM) in 50 µl of TKM buffer (50 mM Tris-HCl (pH 7.4), 134 mM KCl, and 1 mM MgCl₂) was mixed with an equal volume of 8 µM of ¹²⁵I-labeled F-actin followed by a 30-min incubation at room temperature. Subsequently, the mixture was centrifuged at 25,000 x g for 10 min at room temperature. The supernatant was then collected, and the radioactivity in this fraction was counted. The decrease (or loss) of radioactivity in the supernatant fraction reflects F-actin precipitation because of the cross-linking reaction (48, 49). The F-actin cross-linking reaction in the presence of unphosphorylated cortactin (control) is designated as 100%. In some cases, unphosphorylated cortactin isolated from vector-transfected cells using His-conjugated Sepharose beads (control) is designated as 100%. Each assay was set up in triplicate and repeated at least three times. All data were analyzed statistically using the Student's t test, and statistical significance was set at p < 0.01.

Keratinocyte Cell-Cell Adhesion Assay—Keratinocytes (10⁷ cells/ml) (untreated or pretreated with the various inhibitors U73122 [GenBank] (1 µM), Xestospongin C (1 µM), BAPTA/AM (1 µM), cytochalasin D (20 µg/ml), and colchicine (1 x 10^-5 M) at 37 °C for 1 h or transfected with PKN-ACCcDNA or vector alone) grown in 0.03 mM Ca²⁺ were incubated with no HA, with HA (50 µg/ml), or pretreated with anti-CD44 antibody followed by adding HA (50 µg/ml) at 37 °C for various intervals (e.g. 24, 36, or 48 h). The degree of cell-cell adhesion was calculated as the cell-cell adhesion index (N_o - N_a)/N_o. Specifically, N_o represents the total number of cells (free cells plus aggregated cells) before HA treatment, and N_a is the number of free cells after HA treatment. The number of cell units was counted using a hemocytometer as described previously (50). The values expressed in Tables V, VI, VII represent an average of triplicate determinations of five experiments with a standard deviation of less than 5%.

View this table: [in this window] [in a new window]   TABLE VII Effect of PKN-ACC overexpression on HA-dependent keratinocyte cell-cell adhesion

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Eukaryotic cells contain a large number of protein serine/threonine kinases, which appear to play important roles in signal transduction required for various cell functions. One of the known downstream targets for the GTP-bound (activated) form of Rac1 is PKN kinase (also called PRK2 kinase) (29-31). By using a specific anti-PKN-mediated immunoblot technique, we have found significant levels of the PKN (molecular mass 120 kDa) are expressed in keratinocytes (Fig. 1A, lane 2). We believe that the PKN detected in keratinocytes revealed by anti-PKN-mediated immunoblot is specific because no protein is detected in these cells using preimmune rabbit IgG (Fig. 1A, lane 1).

View larger version (31K): [in this window] [in a new window]   FIG. 1. Characterization of PKN and CD44-PKN complex in human keratinocytes. A, detection of PKN in keratinocytes. Keratinocytes were solubilized by 1% Nonidet P-40 buffer followed by anti-PKN-mediated immunoblotting analyses. Lane 1, immunoblot of keratinocytes with preimmune serum; lane 2, immunoblot of keratinocytes with anti-PKN antibody. B, demonstration of RhoGTPase binding properties of PKN. PKN (isolated from keratinocytes) was incubated with GDP- or GTP-loaded forms of either Rac1- or RhoAGST-conjugated beads. Proteins associated with Rac1/RhoA-GST beads were then analyzed by immunoblotting with anti-PKN as described under "Materials and Methods." Lane 1, PKN associated with GDP-bound RhoA beads; lane 2, PKN associated with GTP-bound RhoA beads; lane 3, PKN associated with GDP-bound Rac1 beads; lane 4, PKN associated with GTP-bound Rac1 beads. C, analysis of the CD44-PKN complex in keratinocytes. Keratinocytes (untreated or treated with HA) were solubilized by 1% Nonidet P-40 buffer and immunoprecipitated (IP) with anti-CD44 antibody followed by immunoblotting using anti-PKN antibody (a) or anti-CD44 antibody (b), respectively. Lane 1, detection of PKN in the complex isolated from untreated keratinocytes by anti-CD44-mediated immunoprecipitation followed by immunoblotting with anti-PKN (a) or reblotting with anti-CD44 (b) as a loading control. Lane 2, detection of PKN in the complex isolated from keratinocytes (treated with HA) by anti-CD44-mediated immunoprecipitation followed by immunoblotting with anti-PKN (a) or reblotting with anti-CD44 (b) as a loading control.

In addition, we have addressed the question of whether there is an interaction between CD44 and PKN in human keratinocytes. To this end we first carried out anti-CD44-mediated immunoprecipitation followed by anti-PKN immunoblot (Fig. 1C, a) or anti-CD44 immunoblot (Fig. 1C, b), respectively, using untreated keratinocytes. Our results indicate that a low level of PKN (Fig. 1C, a, lane 1) is present in the anti-CD44-immunoprecipitated materials (Fig. 1C, b, lane 1). Subsequently, we have determined that HA treatment causes the recruitment of a significant amount of PKN (Fig. 1C, a, lane 2) into the CD44-PKN complex (Fig. 1C, b, lane 2). These findings clearly establish that CD44 and PKN are closely associated with each other in vivo, particularly following HA treatment of the keratinocytes.

PLC1 Serves as a New Cellular Substrate for Rac1-dependent PKN Kinase and Regulates Ca²⁺ Signaling in Keratinocytes—HA-CD44 interaction has been shown to be tightly coupled with intracellular Ca²⁺ mobilization pathways in many different cell types (34-36). In these signaling events, PLC1 initially hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP₂) into IP₃ which leads to Ca²⁺ release from intracellular stores (37-39). In searching for a possible linkage between HA/CD44-mediated Rac1 signaling, PKN kinase, and intracellular Ca²⁺ regulation, we have demonstrated that PKN isolated from keratinocytes is capable of phosphorylating PLC1 at threonine residues (as detected by anti-PLC1-mediated immunoprecipitation followed by anti-phosphothreonine-mediated immunoblot) (Fig. 2A, a) but not serine residues (as detected by anti-PLC1-mediated immunoprecipitation followed by anti-phosphoserine-mediated immunoblot) (Fig. 2A, b). These PKN-mediated PLC1 phosphorylation experiments were carried out in the presence of RhoA (either unactivated RhoA (GDP-bound RhoA) (Fig. 2A, a, lane 1, and A, b, lane 1)or activated RhoA (GTP-bound RhoA) (Fig. 2A, a, lane 2, and A, b, lane 2)) or Rac1 (either unactivated Rac1 (GDP-bound Rac1) (Fig. 2A, a, lane 3, and A, b, lane 3) or activated Rac1 (GTP-bound Rac1) (Fig. 2A, a, lane 4 and A, b, lane 4)). Specifically, the level of PKN-mediated threonine phosphorylation (but not serine) of PLC1 is significantly up-regulated if activated Rac1 (GTP-bound Rac1) is present (Fig. 2A, a, lane 4, and A, b, lane 4). A much lesser up-regulation of PKN-mediated PLC1 phosphorylation is detected in the presence of GDP-bound Rac1 or GDP/GTP-bound RhoA (Fig. 2A, a, lanes 1-3, and A, b, lanes 1-3). These findings indicate that PLC1 serves as a cellular substrate for Rac1 (and to a lesser extent RhoA)-dependent kinases such as PKN in vitro.

View larger version (41K): [in this window] [in a new window]   FIG. 2. Detection of PLC1 phosphorylation by PKN in vitro (A) and in vivo (B). A, the PKN kinase reaction was carried out in the reaction mixture containing ATP and purified PKN and PLC1 in the presence of RhoA or Rac1 (incubated with GDP or GTP). After incubation at 30 °C for 30 min, the reaction mixtures were immunoprecipitated (IP) with anti-PLC1 beads, boiled in SDS sample buffer, and subjected to SDS-PAGE. The protein bands associated with anti-PLC1 beads were revealed by anti-phosphothreonine/anti-phosphoserine-mediated immunoblot or anti-PLC1-mediated immunoblot, respectively, as described under "Materials and Methods." Lane 1, PLC1 phosphorylation by GDP-RhoA-treated PKN using anti-phosphothreonine (a)/anti-phosphoserine (b)-mediated immunoblot or anti-PLC1-mediated immunoblot (c). Lane 2, PLC1 phosphorylation by GTP-RhoA-treated PKN using anti-phosphothreonine (a)/anti-phosphoserine (b)-mediated immunoblot or anti-PLC1-mediated immunoblot (c). Lane 3, PLC1 phosphorylation by GDP-Rac1-treated PKN using anti-phosphothreonine (a)/anti-phosphoserine (b)-mediated immunoblot or anti-PLC1-mediated immunoblot (c). Lane 4, PLC1 phosphorylation by GTP-Rac1-treated PKN using anti-phosphothreonine (a)/anti-phosphoserine (b)-mediated immunoblot or anti-PLC1-mediated immunoblot (c). B, keratinocytes treated with HA (or pretreated with anti-CD44 plus HA or no HA) were solubilized by Nonidet P-40 and immunoprecipitated with anti-PLC antibody followed by immunoblotting with anti-phosphothreonine/anti-phosphoserine, respectively. Lane 1, detection of PLC1 phosphorylation by anti-PLC-mediated immunoprecipitation followed by immunoblotting with anti-phosphothreonine (a)/anti-phosphoserine (b) or (reblotting with anti-PLC1 as a loading control (c)) in keratinocytes treated with no HA. Lane 2, detection of PLC1 phosphorylation by anti-PLC-mediated immunoprecipitation followed by immunoblotting with anti-phosphothreonine (a)/anti-phosphoserine (b) or (reblotting with anti-PLC1 as a loading control (c)) in keratinocytes treated with HA. Lane 3, detection of PLC1 phosphorylation by anti-PLC-mediated immunoprecipitation followed by immunoblotting with anti-phosphothreonine (a)/anti-phosphoserine (b) or (reblotting with anti-PLC1 as a loading control (c)) in keratinocytes pretreated with anti-CD44 followed by adding HA treatment.

Next, we examined the potential impact of PKN-mediated phosphorylation on the regulation of PLC1 activity (measured by PLC1-mediated IP₃ production). Our results indicate that the level of IP₃ production is slightly elevated using PLC1 phosphorylated with GDP-RhoA or GTP-RhoA-activated PKN (Table II). PLC1 activity is greatly stimulated using PLC1 treated with GTP-bound Rac1-activated PKN (Table II) (but not PKN in the presence of GDP-bound Rac1 (Table II)). The amount of IP₃ production induced by PLC1 activated by GTPRac1-PKN is also significantly higher than PLC1-activated by GDP-RhoA-PKN or GTP-RhoA-PKN (Table II). In control experiments, we found that unphosphorylated PLC1 (in the absence of PKN) (Table II) can induce only a basal level or very minimal amounts of IP₃ production. Therefore, we conclude that Rac1-activated (and to a lesser extent RhoA-activated) PKN phosphorylation of PLC1 is directly involved in the activation of PLC1 activity. Moreover, we have determined that the stimulation of PLC1 activity occurs immediately after HA addition to CD44-expressing keratinocytes (Table III). Preincubation with anti-CD44 antibody blocks PLC1 activation in keratinocytes treated with HA (Table III) indicating that HA induces PLC1 activity in a CD44-dependent manner in keratinocytes.

View this table: [in this window] [in a new window]   TABLE II Measurement of PLC1 activity, effects of Rac1/RhoA-PKN phosphorylation on PLC1 activity

View this table: [in this window] [in a new window]   TABLE III Measurement of PLC1 activity, role of HA and CD44 in regulating PLC1 activity

View larger version (31K): [in this window] [in a new window]   FIG. 3. Measurement of Ca2+ mobilization in keratinocytes. A, intracellular Ca2+ mobilization was measured by a fluorescence spectrophotometer using cells loaded with 10 µM Fura-2/AM as described under "Materials and Methods." Subsequently, Fura-2-labeled cells (preincubated with 0.1 mM EGTA) were treated with HA (indicated by arrowhead) (A), pretreated with anti-CD44 antibody followed by HA treatment (indicated by arrowhead) (B), pretreated with U-73122 followed by HA treatment (indicated by arrowhead) (C), pretreated with Xestospongin C (Xesto. C) followed by HA treatment (indicated by arrowhead) (D), or pretreated with BAPTA followed by HA treatment (indicated by arrowhead) (E).

View larger version (52K): [in this window] [in a new window]   FIG. 4. Detection of cortactin phosphorylation by PKN in vitro (A) and in vivo (B). A, the PKN kinase reaction was carried out in the reaction mixture containing ATP, purified PKN, and cortactin in the presence of RhoA or Rac1 (incubated with GDP or GTP). After incubation for various times (e.g. 0, 10, 20, 30, 60, and 120 min) at 30 °C, the reaction mixtures were immunoprecipitated (IP) with anti-cortactin beads and boiled in SDS sample buffer and subjected to SDS-PAGE. The protein bands associated with anti-cortactin beads were revealed by anti-phosphoserine/anti-phosphothreonine-mediated immunoblot or anti-cortactin-mediated immunoblot, respectively, as described under "Materials and Methods." Lane 1, cortactin phosphorylation by GDP-RhoA-treated PKN using anti-phosphothreonine (a)/anti-phosphoserine (b)-mediated immunoblot or anti-cortactin-mediated immunoblot (c). Lane 2, cortactin phosphorylation by GTP-RhoA-treated PKN using anti-phosphothreonine (a)/anti-phosphoserine (b)-mediated immunoblot or anti-cortactin-mediated immunoblot (c). Lane 3, cortactin phosphorylation by GDP-Rac1-treated PKN using anti-phosphothreonine (a)/anti-phosphoserine (b)-mediated immunoblot or anti-cortactin-mediated immunoblot (c). Lane 4, cortactin phosphorylation by GTP-Rac1-treated PKN using anti-phosphothreonine (a)/anti-phosphoserine (b)-mediated immunoblot or anti-cortactin-mediated immunoblot (c). B, keratinocytes treated with HA (or pretreated with anti-CD44 plus HA or no HA) were solubilized by Nonidet P-40 and immunoprecipitated with anti-cortactin antibody followed by immunoblotting with anti-phosphothreonine/anti-phosphoserine, respectively. Lane 1, detection of cortactin phosphorylation by anti-cortactin-mediated immunoprecipitation followed by immunoblotting with anti-phosphothreonine (a)/anti-phosphoserine (b) or (reblotting with anti-cortactin as a loading control) (c) in keratinocytes treated with no HA. Lane 2, detection of cortactin phosphorylation by anti-cortactin-mediated immunoprecipitation followed by immunoblotting with anti-phosphothreonine (a)/anti-phosphoserine (b) or (reblotting with anti-cortactin as a loading control) (c) in keratinocytes treated with HA. Lane 3, detection of cortactin phosphorylation by anti-cortactin-mediated immunoprecipitation followed by immunoblotting with anti-phosphothreonine (a)/anti-phosphoserine (b) or (reblotting with anti-cortactin as a loading control) (c) in keratinocytes pretreated with anti-CD44 followed by HA treatment.

Cortactin is known to be an actin-binding protein (48, 49). Our data indicate that unphosphorylated cortactin (in the absence of PKN) (Fig. 5a) or cortactin with low level phosphorylation by PKN, in the presence of unactivated RhoA (GDP-bound RhoA) (Fig. 5b), activated RhoA (GTP-bound RhoA) (Fig. 5c), or unactivated Rac1 (GDP-bound Rac1) (Fig. 5d), is capable of cross-linking the actin filaments into bundles in vitro. However, serine/threonine phosphorylation of cortactin by Rac1 (GTP-bound form)-activated PKN down-regulates its ability to cross-link filamentous actin (Fig. 5e). These results are consistent with previous findings suggesting cortactin (phosphorylated versus unphosphorylated forms) plays an important role as an F-actin modulator required for cytoskeleton reorganization.

View larger version (15K): [in this window] [in a new window]   FIG. 5. Measurement of the F-actin cross-linking activity of cortactin. Purified cortactin treated with PKN (in the presence of GDP- or GTP-bound form of Rac1/RhoA) was subjected to F-actin cross-linking analysis. Specifically, unphosphorylated cortactin (in the absence of PKN) (a), cortactin phosphorylated by PKN in the presence of unactivated RhoA (GDP-bound RhoA) (b), activated RhoA (GTP-bound RhoA) (c), unactivated Rac1 (GDP-bound Rac1) (d), or activated Rac1 (GTP-bound Rac1) (e) was incubated with a TKM buffer (50 mM Tris-HCl (pH 7.4), 134 mM KCl, and 1 mM MgCl2). Unphosphorylated or phosphorylated cortactin was then mixed with 125I-labeled F-actin followed by a 30-min incubation at room temperature. Subsequently, the mixture was centrifuged at 25,000 x g for 10 min at room temperature. The supernatant was then collected, and the radioactivity in this fraction was counted. The decrease (or loss) of radioactivity in the supernatant fraction reflects F-actin precipitation because of the cross-linking reaction. The F-actin cross-linking reaction in the presence of unphosphorylated cortactin (in the absence of PKN) (control) is designated as 100%. Each assay was set up in triplicate and repeated at least three times. All data were analyzed statistically using the Student's t test, and statistical significance was set at p < 0.01.

View larger version (39K): [in this window] [in a new window]   FIG. 6. Interaction between the PKN-ACC fragment and Rac1/RhoA in vitro and detection of PKN-Rac1-RhoA complex in PKN-ACC cDNA-transfected/vector-transfected cells. A, illustration of PKN full-length (a) and His-tagged PKN-ACCcDNA construct (b). B, characterization of the binding interaction between His-tagged PKN-ACC and GST Rac1/GST-RhoA beads in vitro. Lane 1, anti-His-mediated immunoblot of PKN-ACC fragment associated with GDP-bound RhoA-beads. Lane 2, anti-His-mediated immunoblot of PKN-ACC fragment associated with GTP-bound RhoA-beads. Lane 3, anti-His-mediated immunoblot of PKN-ACC fragment associated with GDP-bound Rac1-beads. Lane 4, anti-His-mediated immunoblot of PKN-ACC fragment associated with GTP-bound Rac1-beads. C, analyses of PKN-ACC-RhoA-Rac1 complex formation in keratinocyte transfectants. Keratinocytes (transfected with His-tagged PKN-ACCcDNA or vector alone) were solubilized by 1% Nonidet P-40 buffer. Cell lysates were then used for anti-His-mediated immunoprecipitation followed by immunoblotting with anti-Rac1/RhoA antibody or anti-His antibody, respectively. as described under "Materials and Methods." a, Anti-Rac1-mediated immunoblot of anti-His-mediated immunoprecipitated materials isolated from keratinocytes transfected with vector alone (lane 1) or with His-tagged PKN-ACCcDNA (lane 2). b, anti-RhoA-mediated immunoblot of anti-His-mediated immunoprecipitated materials isolated from keratinocytes transfected with vector alone (lane 1) or with His-tagged PKN-ACCcDNA (lane 2). c, anti-His-mediated immunoblot of anti-His-mediated immunoprecipitated materials isolated from keratinocytes transfected with vector alone (lane 1) or with His-tagged PKN-ACCcDNA (lane 2). D, analyses of PKN expression (I) and PKN-ACC-RhoA-Rac1 complex formation (II) in keratinocyte transfectants. Keratinocytes transfected with His-tagged PKN-ACCcDNA or vector alone (untreated, treated with HA, or pretreated with anti-CD44 followed by HA addition) were solubilized by 1% Nonidet P-40 buffer. Cell lysates were then used for anti-PKN-mediated immunoblot or anti-PKN-mediated immunoprecipitation followed by immunoblotting with anti-Rac1/RhoA antibody or anti-PKN antibody, respectively, as described under "Materials and Methods." (I), anti-PKN-mediated immunoblot of the cell lysate isolated from keratinocytes transfected with vector alone (untreated (lane 1), treated with HA (lane 2) or pretreated with anti-CD44 followed by HA addition (lane 3)) or His-tagged PKN-ACCcDNA (untreated (lane 4), treated with HA (lane 5), or pretreated with anti-CD44 followed by HA addition (lane 6)). (II), a, anti-Rac1-mediated immunoblot of anti-PKN-mediated immunoprecipitated materials isolated from keratinocytes transfected with vector alone (untreated (lane 1), treated with HA (lane 2), or pretreated with anti-CD44 followed by HA addition (lane 3)) or PKN-ACCcDNA (untreated (lane 4), treated with HA (lane 5), or pretreated with anti-CD44 followed by HA addition (lane 6)). (II), b, anti-RhoA-mediated immunoblot of anti-PKN-mediated immunoprecipitated materials isolated from keratinocytes transfected with vector alone (untreated (lane 1), treated with HA (lane 2), or pretreated with anti-CD44 followed by HA addition (lane 3)) or PKN-ACCcDNA (untreated (lane 4), treated with HA (lane 5), or pretreated with anti-CD44 followed by HA addition (lane 6)). (II), c, anti-PKN-mediated immunoblot of anti-PKN-mediated immunoprecipitated materials isolated from keratinocytes transfected with vector alone (untreated (lane 1), treated with HA (lane 2), or pretreated with anti-CD44 followed by HA addition (lane 3)) or PKN-ACCcDNA (untreated (lane 4), treated with HA (lane 5), or pretreated with anti-CD44 followed by HA addition (lane 6)).

Furthermore, we have demonstrated that threonine (but not serine) phosphorylation of PLC1 (Fig. 7, a-c, lane 2) is greatly enhanced in vector-transfected keratinocytes treated with HA as compared with those observed in untreated vector-transfected cells (Fig. 7, a-c, lane 1) or pretreated with anti-CD44 antibody followed by HA treatment (Fig. 7, a-c, lane 3). Both PLC1 activity (measured by the production of IP₃) and intracellular Ca²⁺ mobilization are also up-regulated in vector-transfected keratinocytes treated with HA (Table IV). In contrast, the level of PLC1 phosphorylation, PLC1-mediated IP₃ production, and Ca²⁺ mobilization is relatively low in untreated vector-transfected keratinocytes (Table IV) or pretreated with anti-CD44 antibody plus HA (data not shown). However, overexpression of the PKN-ACC fragment by transfecting keratinocytes with PKN-ACCcDNA not only significantly reduces the ability of PLC1 to respond to HA/CD44-stimulated phosphorylation (Fig. 7, a-c, lanes 4-6) but also down-regulates enzymatic activity (measured by PLC1-mediated IP₃ production) and Ca²⁺ mobilization (Table IV). Therefore, we believe that the ACC fragment of PKN (by interfering endogenous Rac1 association with PKN) (Fig. 6D (II), a, lanes 4-6) functions as a potent dominant-negative mutant that effectively inhibits HA/CD44-induced Rac1-PKN activation and PLC1 phosphorylation required for Ca²⁺ signaling.

View larger version (62K): [in this window] [in a new window]   FIG. 7. Detection of PLC1/cortactin phosphorylation in keratinocyte transfectants. a, anti-phosphothreonine-mediated immunoblot of anti-PLC1-mediated immunoprecipitated materials isolated from keratinocytes transfected with vector alone (untreated (lane 1), treated with HA (lane 2), or pretreated with anti-CD44 followed by HA addition (lane 3)) or PKN-ACCcDNA (untreated (lane 4), treated with HA (lane 5), or pretreated with anti-CD44 followed by HA addition (lane 6)). b, anti-phosphoserine-mediated immunoblot of anti-PLC1-mediated immunoprecipitated materials isolated from keratinocytes transfected with vector alone (untreated (lane 1), treated with HA (lane 2), or pretreated with anti-CD44 followed by HA addition (lane 3)) or PKN-ACCcDNA (untreated (lane 4), treated with HA (lane 5), or pretreated with anti-CD44 followed by HA addition (lane 6)). c, anti-PLC1-mediated immunoblot of anti-PLC1-mediated immunoprecipitated materials isolated from keratinocytes transfected with vector alone (untreated (lane 1), treated with HA (lane 2), or pretreated with anti-CD44 followed by HA addition (lane 3)) or PKN-ACCcDNA (untreated (lane 4), treated with HA (lane 5), or pretreated with anti-CD44 followed by HA addition (lane 6)). d, anti-phosphothreonine-mediated immunoblot of anti-cortactin-mediated immunoprecipitated materials isolated from keratinocytes transfected with vector alone (untreated (lane 1), treated with HA (lane 2), or pretreated with anti-CD44 followed by HA addition (lane 3)) or PKN-ACCcDNA (untreated (lane 4), treated with HA (lane 5), or pretreated with anti-CD44 followed by HA addition (lane 6)). e, anti-phosphoserine-mediated immunoblot of anti-cortactin-mediated immunoprecipitated materials isolated from keratinocytes transfected with vector alone (untreated (lane 1), treated with HA (lane 2), or pretreated with anti-CD44 followed by HA addition (lane 3)) or PKN-ACCcDNA (untreated (lane 4), treated with HA (lane 5), or pretreated with anti-CD44 followed by HA addition (lane 6)). f, anti-PLC1-mediated immunoblot of anti-cortactin-mediated immunoprecipitated materials isolated from keratinocytes transfected with vector alone (untreated (lane 1), treated with HA (lane 2), or pretreated with anti-CD44 followed by HA addition (lane 3)) or PKN-ACCcDNA (untreated (lane 4), treated with HA (lane 5), or pretreated with anti-CD44 followed by HA addition (lane 6)). g, anti-His-mediated immunoblot of the cell lysate isolated from keratinocytes transfected with vector alone (untreated (lane 1), treated with HA (lane 2), or pretreated with anti-CD44 followed by HA addition (lane 3)) or His-tagged PKN-ACCcDNA (untreated (lane 4), treated with HA (lane 5), or pretreated with anti-CD44 followed by HA addition (lane 6)).

View this table: [in this window] [in a new window]   TABLE IV Effect of PKN-ACC overexpression on HA-dependent keratinocyte signaling

By using double immunofluorescence staining and confocal microscopic analyses, we have determined that CD44 is expressed on the cell surface of vector-transfected keratinocytes (Fig. 8A, a) and that PKN kinase (Fig. 8A, c) is diffusely distributed in both the cell membrane and the cytoplasm of untreated vector-transfected keratinocytes. A low level of CD44-PKN co-localization is detected in these keratinocytes in the absence of HA (Fig. 8A, e). We have also observed that both PLC1 (Fig. 8B, c) and cortactin (Fig. 8C, c) are distributed in the cytosol and not complexed with CD44 (Fig. 8, B, a, and C, a) in the plasma membrane in untreated vector-transfected keratinocytes. Very little CD44 co-localization with PLC1 (Fig. 8B, e) and cortactin (Fig. 8C, e) was observed in these untreated cells. Upon HA addition, keratinocyte cell-cell adhesion occurs (Fig. 8A, b, d, and f) and there is a dramatic recruitment of PKN (Fig. 8A, d) from the cytosol to CD44-containing plasma membranes (Fig. 8A, b and f) in vector-transfected keratinocytes. These results demonstrate that HA induces a close association between CD44 and PKN in the plasma membrane of vector-transfected keratinocytes. Both PLC1 (Fig. 8B, d) and cortactin (Fig. 8C, d) are also cocolocalized with CD44 (Fig. 8, B, b, d, and f, and C, b, d, and f) in the plasma membranes and cell-cell adhesion junction in vector-transfected keratinocytes treated with HA. In contrast, we have noted that overexpression of PKN-ACC by transfecting keratinocytes with PKN-ACCcDNA significantly reduces HA-mediated cell-cell adhesion (Fig. 8, D-F; Tables V, VI, VII) and PKN (Fig. 8D, c and d) co-localization with CD44 (Fig. 8, D, a and b, and D, e and f). Similarly, the interaction between CD44 (Fig. 8, E, a and b, and F, a and b) and PLC1 (Fig. 8E, c and d)/cortactin (Fig. 8F, c and d) at the cell-cell adhesion site (Fig. 8, E, e and f, and F, e and f) is greatly inhibited in these transfectants in the absence or the presence of HA. Treatment of keratinocytes with rat anti-CD44 antibody (but not normal rat IgG) or various agents such as U73122 [GenBank] (a PLC inhibitor), Xestospongin C (an IP₃ receptor blocker), BAPTA (a membrane-permeable Ca²⁺chelator), or cytochalasin D (a microfilament disrupting agent known to prevent actin polymerization) effectively inhibits HA/CD44-induced cell-cell adhesion (Tables V, VI, VII). Transfection of keratinocytes with PKN-ACCcDNA also readily impairs HA-dependent cell-cell adhesion of keratinocytes (Tables V, VI, VII). These observations strongly suggest that CD44-mediated PKN activation together with PLC1-regulated Ca²⁺ signaling and cytoskeleton function are involved in HA-dependent cell-cell adhesion in keratinocytes.

View larger version (59K): [in this window] [in a new window]   FIG. 8. Immunofluorescence staining of CD44, PKN, PLC1, and cortactin in keratinocyte transfectants. Keratinocytes transfected with vector alone or PKN-ACCcDNA were fixed by 2% paraformaldehyde. Subsequently, cells were rendered permeable by ethanol treatment and stained with various immunoreagents as described under "Materials and Methods." A, Texas Red-labeled anti-CD44 (red color) (a), FITC-labeled anti-PKN (green color)(c), and co-localization of Texas Red anti-CD44 and FITC-PKN (e) (an overlay image of a and c) in vector-transfected keratinocytes treated with no HA. Texas Red-labeled anti-CD44 (red color) (b), FITC-labeled anti-PKN (green color) (d), and colocalization of Texas Red anti-CD44 and FITC-PKN (f) (an overlay image of b and d) in vector-transfected keratinocytes treated with HA. B, Texas Red-labeled anti-CD44 (red color) (a), FITC-labeled anti-PLC1 (green color)(c), and co-localization of Texas Red anti-CD44 and FITC-PLC1(e) (an overlay image of a and c) in vector-transfected keratinocytes treated with no HA. Texas Red-labeled anti-CD44 (red color) (b), FITC-labeled anti-PLC1 (green color) (d), and co-localization of Texas-red-anti-CD44 and FITC-PLC1 (f) (an overlay image of b and d) in vector-transfected keratinocytes treated with HA. C, Texas Red-labeled anti-CD44 (red color) (a), FITC-labeled anti-cortactin (green color) (c), and co-localization of Texas Red anti-CD44 and FITC-cortactin (e) (an overlay image of a and c) in vector-transfected keratinocytes treated with no HA. Texas Red-labeled anti-CD44 (red color)(b), FITC-labeled anti-cortactin (green color) (d), and co-localization of Texas Red anti-CD44 and FITC-cortactin (f) (an overlay image of b and d) in vector-transfected keratinocytes treated with HA. D, Texas Red-labeled anti-CD44 (red color) (a), FITC-labeled anti-PKN (green color) (c), and co-localization of Texas Red anti-CD44 and FITC-PKN (e) (an overlay image of a and c) in PKN-ACCcDNA-transfected keratinocytes treated with no HA. Texas red-labeled anti-CD44 (red color) (b), FITC-labeled anti-PKN (green color) (d), and co-localization of Texas Red anti-CD44 and FITC-PKN (f) (an overlay image of b and d) in PKN-ACCcDNA-transfected keratinocytes treated with HA. E, Texas Red-labeled anti-CD44 (red color) (a), FITC-labeled anti-PLC1 (green color) (c), and co-localization of Texas Red anti-CD44 and FITC-PLC1 (e) (an overlay image of a and c) in PKN-ACCcDNA-transfected keratinocytes treated with no HA; Texas Red-labeled anti-CD44 (red color) (b), FITC-labeled anti-PLC1 (green color)(d), and co-localization of Texas-red-anti-CD44 and FITC-PLC1(f) (an overlay image of b and d) in PKN-ACCcDNA-transfected keratinocytes treated with HA. F, Texas Red-labeled anti-CD44 (red color) (a), FITC-labeled anti-cortactin (green color) (c), and co-localization of Texas Red-anti-CD44 and FITC-cortactin (e) (an overlay image of a and c) in PKN-ACCcDNA-transfected keratinocytes treated with no HA. Texas Red-labeled anti-CD44 (red color) (b), FITC-labeled anti-cortactin (green color) (d), and co-localization of Texas Red anti-CD44 and FITC-cortactin (f) (an overlay image of (b and d) in PKN-ACCcDNA-transfected keratinocytes treated with HA.

View larger version (62K): [in this window] [in a new window]   FIG. 9. Expression of differentiation markers (involucrin and transglutaminase) in untransfected keratinocytes and PKN-ACCcDNA/vector-transfected keratinocytes. Keratinocytes (untreated or pretreated with various agents (e.g. anti-CD44 IgG, U-73122, Xestospongin C (Xesto. C), BAPTA, or cytochalasin D (Cyto. D) (20 µg/ml)) for 1 h or transfected with PKN-ACCcDNA (or vector alone)) were incubated with HA (50 µg/ml) at 37 °C for 36 h. Subsequently, cells were solubilized by 1% Nonidet P-40 buffer. Cell lysates were then used for anti-involucrin or anti-transglutaminase-mediated immunoblot. A, anti-involucrin (a) or anti-transglutaminase-mediated (b) immunoblot of cell lysate isolated from keratinocytes treated with no drug in the absence (lane 1) or the presence of HA (lane 2); from keratinocytes treated with anti-CD44 IgG in the absence (lane 3) or the presence of HA (lane 4); from keratinocytes treated with U-73122 in the absence (lane 5) or the presence of HA (lane 6); from keratinocytes treated with Xestospongin C in the absence (lane 7) or the presence of HA (lane 8), from keratinocytes treated with BAPTA in the absence (lane 9) or the presence of HA (lane 10), or from keratinocytes treated with cytochalasin D in the absence (lane 11) or the presence of HA (lane 12). B, anti-involucrin (a) or anti-transglutaminase-mediated (b) immunoblot of cell lysate isolated from vector-transfected keratinocytes (in the absence (lane 1) or the presence of HA (lane 2)) or PKN-ACCcDNA-transfected keratinocytes (in the absence (lane 3) or the presence of HA (lane 4)).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Previous studies by Bikle and co-workers (40) have shown that a multiphasic increase in intracellular Ca²⁺ concentration following the addition of exogenous 1-2 mM Ca²⁺ to keratinocytes is required for Ca²⁺-induced keratinocyte differentiation. Inhibition of intracellular Ca²⁺ mobilization by treatment with the Ca²⁺ chelator BAPTA prevents Ca²⁺-induced differentiation (41). These findings suggest that intracellular Ca²⁺ mobilization plays a critical role in regulating keratinocyte differentiation (40, 41). The binding of HA to CD44 increases intracellular Ca²⁺ levels in many cell types including keratinocytes (Fig. 3). The mechanism by which keratinocytes respond to HA/CD44-induced Ca²⁺ signaling is not fully established. PLC1 is known to generate 2-s messengers from the hydrolysis of phosphatidylinositol 4,5-bisphosphate, diacylglycerol, and IP₃. Diacylglycerol is an activator of protein kinase C, and IP₃ interacts with intracellular membrane receptors (e.g. IP₃ receptors) leading to an increased release of stored Ca²⁺ ions (37-39). Most important, targeted deletion of PLC1 causes embryonic lethality in mice (55). These findings suggest that PLC1 plays a critical role in Ca²⁺ signaling and developmental processes. In fact, keratinocytes are known to possess functionally active inositol lipid signaling systems, one of which involves PLC1 activation (40). During growth factor signaling, PLC1 binds to tyrosine autophosphorylation sites of the receptor-linked tyrosine kinases leading to tyrosine phosphorylation and stimulation of PLC1 activity (56). Because tyrosine phosphorylation of PLC1 and PLC1 activity (measured by PLC1-mediated IP₃ production) do not correlate well in certain case(s) (38), we were prompted to explore additional mechanisms including post-translational modifications of PLC1 by certain serine/threonine kinase(s), such as the PKN family of kinases, during HA/CD44-mediated activation in keratinocytes.

PKN belongs to a family of serine/threonine kinase that displays high sequence homology to that of the protein kinase C family members (29). There are at least three different isoforms of PKN (PKN PAK-1/PRK-1, PKN, and PKN/PRK2) detected in mammals (29). PKN isoforms appear to be involved in a variety of cellular functions (29-31). PKN is a serine-threonine kinase known to interact with Rac1 in a GTP-dependent manner and shares a great deal of homology to protein kinase C in the C-terminal region (29). PKN also has a unique regulatory region containing ACC domains that bind to the small GTPases (29-31). In a previous study PKN has been shown to be involved in Fyn kinase activity and the cell-cell adhesion in mouse keratinocytes treated with exogenously added Ca²⁺ (33). By using a PKN-specific antibody, we have confirmed the presence of PKN in human keratinocytes (Fig. 1). Furthermore, we have demonstrated that PKN (by binding to activated Rac1) is capable of inducing marked threonine phosphorylation of PLC1 (isolated from keratinocytes) in vitro (Fig. 2). The ability of PKN to phosphorylate PLC1 in the presence of unactivated Rac1 appears to be greatly reduced (Fig. 2). These results clearly indicate that PKN acts as one of the downstream effectors of Rac1 signaling and utilizes PLC1 as one of its cellular targets. Both Rac1 signaling (Table I) and PLC phosphorylation in vivo (Fig. 2) can be stimulated by HA and blocked by cells pretreated with anti-CD44 antibody during HA stimulation (Table I and Fig. 2). Thus, it appears that Rac1-activated PKN-mediated phosphorylation of PLC1 is closely coupled with HA-mediated CD44 activation in keratinocytes. Most important, PKN-mediated phosphorylation of PLC1 up-regulates PLC1 activity (measured by PLC1-mediated IP₃ production) (Table II) required for Ca²⁺ signaling.

The ACC domain of PKN has been shown to be involved in the regulation of GTP-dependent PKN function (29-31). In Fig. 6, we have detected that the ACC domain is closely associated with Rac1 as a complex. Transfection of keratinocytes with the ACC domain cDNA of PKN (Fig. 6) effectively competes for endogenous activated Rac1 binding to PKN (Fig. 6), inhibits the ability of PKN to phosphorylate PLC1 (Fig. 7), blocks HA/CD44-mediated Ca²⁺ signaling (Table IV), and down-regulates keratinocyte cell-cell adhesion and differentiation (Figs. 8 and 9; Tables V, VI, VII). These results strongly suggest that the ACC domain of PKN is a potent inhibitor of PKN-mediated PLC1 phosphorylation and Ca²⁺ signaling required for HA/CD44-mediated keratinocyte function. Our observations are consistent with previous findings suggesting that small GTPases (e.g. Rac1) are important for Ca²⁺ sensitization in hamster muscle resistance arteries (58). By using dominant-negative and dominant-active forms of Rac1, Hong-Geller and Cerione (44) have also suggested that these small GTPases regulate signal transduction at the level of PLC1 and IP₃ production.

The cytoskeletal protein cortactin contains a repeat domain and a C-terminal SH3 domain resembling neufectin, a F-actin-associated protein (59). In fact, cortactin is considered to be an actin-binding protein (48, 49). Cortactin not only serves as a cellular substrate for c-Src and Fyn but also localizes with actin at cell adhesion sites during Ca²⁺-induced keratinocyte differentiation (60). Tyrosine phosphorylation of cortactin is greatly reduced in keratinocytes derived from Fyn knock-out mice, indicating Fyn kinase may be directly involved in cortactin phosphorylation (60). Our previous study demonstrated that HA activates Src kinase-mediated cortactin phosphorylation and modulates cortactin-F-actin binding in tumor cells (61). In this study, we have determined that cortactin is a cellular substrate for Rac1-activated PKN kinase in keratinocytes (Fig. 4). Serine/threonine phosphorylation of cortactin by Rac1 (and to a lesser extent RhoA)-activated PKN alters its ability to interact with filamentous actin (Fig. 5). In addition, we have confirmed that cortactin phosphorylation by the PKN kinase (isolated from untransfected or vector-transfected keratinocytes in the presence of HA treatment) significantly down-regulates its ability to cross-link filamentous actin (Table IV). In contrast, the reduction of cortactin phosphorylation (isolated from keratinocytes transfected with PKN-ACCcDNA with or without HA treatment) is sufficient to promote cross-linking of actin filaments into bundles in vitro (Table IV). These results are consistent with previous findings suggesting cortactin plays an important role as a regulator for F-actin-based cytoskeleton function.

To elucidate further the CD44 and PKN interaction with cortactin and PLC1 in vivo, we have used immunocytochemical staining and confocal microscopy to monitor morphological changes and the intracellular distribution of various signaling related proteins (e.g. CD44, endogenous PKN, PLC1, and cortactin) in keratinocytes transfected with PKN-ACCcDNA or vector alone. Our results indicate that that HA promotes co-localization of cortactin and PLC1 with CD44 and PKN at the plasma membrane region and cell-cell adhesion sites in vector-transfected keratinocytes (Fig. 8). In contrast, overexpression of the dominant-negative form of PKN (PKN-ACC fragment) by transfecting keratinocytes with PKN-ACCcDNA effectively inhibits HA-mediated recruitment of cortactin, PLC1, or PKN kinase to CD44 (Fig. 8) at the cellular membranes and impairs HA-induced cell-cell adhesion (Fig. 8; Tables V, VI, VII). These findings demonstrate that the ability of cortactin/PLC1 to be recruited into CD44-associated membrane region and cell-cell adhesion sites is tightly coupled with an active form of PKN kinase in keratinocytes during HA signaling. Treatment of keratinocytes with various inhibitors (e.g. the PLC inhibitor (U73122 [GenBank] ), the IP₃ receptor blocker (Xestospongin C), the Ca²⁺ chelator (BAPTA), or the microfilament inhibitor (cytochalasin D)) also displays marked inhibitory effects on HA/CD44-induced cell-cell adhesion (Tables V, VI, VII). These data also strongly suggest that CD44-mediated PKN activation is required for PLC1-regulated Ca²⁺ signaling and cytoskeleton function during HA-dependent cell-cell adhesion in keratinocytes.

The early events of keratinocyte differentiation (shortly after HA/CD44-induced Ca²⁺ signaling) are accompanied by the expression of keratinocyte differentiation markers including involucrin (precursor for the cornified envelope). Within 24-48 h after HA binding to CD44, transglutaminase is also expressed and activated (Fig. 9). One of the mechanisms by which intracellular Ca²⁺ triggers differentiation during HA-CD44 interaction may involve the Ca²⁺-dependent activation of protein kinase C isoforms (e.g. protein kinase C, -, -, -, and - isoforms) (62), which regulate various signal cascades and serve as regulators of keratinocyte differentiation-dependent gene expression (e.g. involucrin and/or transglutaminase gene expression) (63-67). HA/CD44-induced intracellular Ca²⁺ may also be required for activating Ca²⁺-dependent enzymes such as transglutaminase. In fact, Ca²⁺-activated transglutaminase has been shown to catalyze the isopeptide bond between peptide-bound glutamine and lysine resulting in the cross-linking of involucrin and other proteins into the insoluble cornified envelope to establish barrier function in the skin (38, 68-70). In addition, HA/CD44-induced Ca²⁺ mobilization may contribute to cytoskeleton reorganization and cell-cell adhesion for the maintenance of epidermal integrity. The fact that the dominant-negative form of PKN (PKN-ACC domains) and various inhibitors (e.g. PLC inhibitor, IP₃ receptor blocker, Ca²⁺ chelator, and cytoskeleton inhibitor) can prevent HA- and CD44-mediated keratinocyte differentiation (Fig. 9) suggests that PLC1 activation, Ca²⁺ signaling, and cortactin-cytoskeleton function regulated by Rac1-dependent PKN play important roles in HA/CD44-induced keratinocyte differentiation.

These results taken together allow us to propose the following signaling model: HA-CD44 interaction (Fig. 10, step 1) with Rac1-dependent PKN kinase activation (step 2) promotes phosphorylation of PLC1 phosphorylation (step 3a) and cortactin (step 3b), leading to the onset of PLC1-mediated IP₃ production/Ca²⁺ signaling (step 4a) and cortactin-mediated cytoskeleton function (step 4b) required for keratinocyte functions (e.g. cell-cell adhesion and differentiation) (step 5) (Fig. 10). We believe that the successful identification of these HA/CD44-mediated signaling pathways has provided valuable new insight toward a better understanding of keratinocyte functions and normal epidermal development.

View larger version (40K): [in this window] [in a new window]   FIG. 10. A proposed model for the interaction between HA/CD44-mediated Rac1-PKN activation and PLC1-regulated Ca2+ signaling/cortactin-cytoskeleton binding during keratinocyte cell-cell adhesion and differentiation. HA-CD44 interaction (step 1) with Rac1-dependent PKN kinase activation (step 2) promotes phosphorylation of PLC1 phosphorylation (step 3a) and cortactin (step 3b), leading to the onset of PLC1-mediated IP3 production/Ca2+ signaling (step 4a) and cortactin-mediated cytoskeleton function (step 4b) required for keratinocyte functions (e.g. cell-cell adhesion and differentiation) (step 5).

	FOOTNOTES

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.

¹ The abbreviations used are: ECM, extracellular matrix; HA, hyaluronan; ACC, antiparallel coiled-coil; FITC, fluorescein isothiocyanate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; IP₃, inositol 1,4,5-trisphosphate; PLC, phospholipases C; PIP₂, phosphatidylinositol 4,5-bisphosphate; GST, glutathione S-transferase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; PKN, protein kinase N-; GTPS, guanosine 5'-3-O-(thio)triphosphate; F-actin, filamentous actin.

	ACKNOWLEDGMENTS

 
We thank Dr. Gerard J. Bourguignon for assistance in the preparation of this paper.

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