Go6976

Protein kinase Ca/b inhibitor Go¨6976 promotes PC12 cell adhesion and spreading through membrane recruitment and activation of protein kinase Cd

Sung Youn Junga, O Bok Kima, Hyun Ki Kanga, Da Hyun Janga, Byung-Moo Mina,n, Frank H. Yub,n
aDepartment of Oral Biochemistry and Program in Cancer and Developmental Biology, Dental Research Institute, Seoul National University School of Dentistry, 28 Yeonkun-Dong, Chongno-Ku, Seoul 110-749, Republic of Korea
bProgram in Neurobiology, Seoul National University School of Dentistry, 28 Yeonkun-Dong, Chongno-Ku, Seoul 110-749, Republic of Korea

a r t i c l e i n f o r m a t i o n

Article Chronology: Received 21 July 2012 Received in revised form 2 October 2012
Accepted 3 October 2012 Available online 9 October 2012
Keywords:
PKCa/b inhibitor Go¨6976 Laminin
Cellular activity PKCd activation Small GTPases
a b s t r a c t

Go¨6976 is a nonglycosidic indolocarbazole compound widely used as a specific inhibitor of PKCa/b. In experiments probing for a role of PKCa in human laminin-2-integrin-mediated cell adhesion and spreading of PC12 cells, we observed unexpected enhancements of adhesion, spreading and stress fiber formation to 1 mM Go¨6976 with concomitant increase in membrane translocation of PKCd and autophosphorylation of focal adhesion kinase (FAK). Importantly, enhanced cellular behavior and membrane translocation of PKCd induced by Go¨6976 was retained in siRNA-transfected PC12 cells to knockdown PKCa expression. Go¨6976 also induced laminin-dependent cell adhesion in NIH/3T3 and CV-1 fibroblasts, suggesting of a mechanism that may be common to multiple cell-types. A specific inhibitor of PKCd, rottlerin, completely abrogated Go¨6976-dependent increase in PC12 cell adhesion to laminin as well as the activation of small GTPases, Rac1 and Cdc42, that are downstream of PKCd in adhesion receptor signaling. siRNA knockdown of Rac1 and Cdc42 expression inhibited cell spreading and lamellipodia formation in PC12 cells. Overall, these results suggest that Go¨6976 may stimulate membrane recruitment of PKCd through a mechanism that is independent of PKCa/b signaling. In addition, the activation of Rac1 and Cdc42 by human laminin-2-integrin-dependent activation of PKCd/
FAK signaling mediates cell spreading and lamellipodia formation in PC12 cells.
& 2012 Elsevier Inc. All rights reserved.

Introduction

Laminins are multifunctional basement membrane glycoproteins composed of a, b, and g polypeptide chains. The laminin a2 chain, a component of laminin-2 (a2b1g1), laminin-4 (a2b2g1), and laminin-12 (a2b1g3), is expressed in skeletal and cardiac

muscles, peripheral nerves, brain, and placenta [1]. Biological activities of laminin are mediated through binding to a subset of integrin family of cell adhesion receptors, a3b1, a6b1, a6b4, and a7b1 [2]. Integrin-mediated cell adhesion to laminin triggers signal transduction cascade, so-called ‘‘outside-in’’ signaling [3]
that induces activation of a number of pathways, including

nCorresponding authors.
E-mail addresses: [email protected] (B.-M. Min), [email protected] (F.H. Yu).

0014-4827/$ – see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2012.10.003

mitogen-activated kinases, Rho-family GTPases, phosphati- dylinositol 3-kinase, and PKC [4,5], thus controlling cell behaviors. Synthetic peptides or recombinant fragments containing bio- logically active motifs have been shown to partially mimic the effects of native laminin in various cellular functions, such as cell adhesion, spreading, migration, neurite outgrowth, and tumor metastasis [6–10], suggesting that laminin-derived-short peptide- modified biomaterials may have therapeutic potential in tissue regeneration applications.
PKC family of serine-threonine kinases plays key roles in many of the signaling pathways that control cellular function, including those mediated through integrin signaling. There are 12 isoforms of the PKC family that are grouped into three subfamilies based on their structure [11]: conventional PKC isozymes (PKCa, bI, bII, g) that are activated by diacylglycerol (DAG) and calcium; novel PKC isozymes (PKCd, e, Z, y, m) that are activated by DAG but not by calcium; atypical PKC isozymes (PKCz, i/l) that are unresponsive to either DAG or calcium. PKC activation by phorbol myristate acetate has been shown to induce the adhesion, spreading, and migration of cells [12]. Pharmacologic blockade of PKC inhibits cell adhesion and spreading as well as focal adhesion formation and focal adhesion kinase (FAK) phosphorylation [13,14]. In PC12 cells, we recently showed that the engagement of integrin a3b1 to a binding sequence within the human laminin a2 chain was sufficient to recruit PKCa and PKCd to the integrin receptor [6].
In preliminary experiments examining the role of PKCa in cell adhesion and spreading, we found that Go¨6976, a widely used, nonglycosidic indolocarbazole inhibitor of PKCa/b, had effects that could not be explained by inhibition of PKCa/b alone. Here, we show Go¨6976-dependent enhancement of cell adhesion and spreading does not require inhibition of PKCa isozyme. Further, Go¨6976 promotes membrane translocation of PKCd that activates downstream signaling elements Rac1 and Cdc42 small GTPases leading to increased PC12 cell adhesion and spreading activities in response to extracellular matrix protein, laminin.

Materials and methods

Cells

The PC12 cell line from transplantable rat pheochromocytoma was cultured in RPMI 1640 medium (BioWhittaker Cambrex, Walkersville, MD) containing 10% fetal bovine serum (FBS). The mouse embryo fibroblast cell line NIH/3T3 and normal African green monkey kidney fibroblast cell line CV-1, and human embryonic kidney epithelial cell line HEK293 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies, Grand Island, NY) containing 10% FBS. All cells were purchased from the American Type Culture Collection (Manassas, VA).

Construction, expression, and purification of human laminin a2 chain LG3 domain

Complementary DNA (cDNA) fragment corresponding to the C-terminal large globular (LG) 3 domain of laminin a2 chain was amplified by PCR using the full-length human laminin a2 chain cDNA template and subcloned into the mammalian expres- sion plasmid vector pRSET (Invitrogen, Carlsbad, CA) as described previously [6]. Recombinant LG (rLG) 3 protein was prepared as

described previously [15]. Expression of rLG3 protein was induced in Escherichia coli strain BL21 by 1 mM isopropyl-b-D-thiogalacto- pyranoside (Promega, Madison, WI) for 5 h at 37 1C and harvested by centrifugation. Cell pellets were suspended in urea-based lysis buffer (8 M urea, 10 mM Tris–HCl, pH 8.0, 100 mM NaH2PO4) containing 1 mM PMSF. Recombinant histidine 6-tagged LG3 proteins were purified using Ni2þ-nitrilotriacetic acid agarose (Qiagen, Valencia, CA) and dialyzed sequentially against 3, 2, 1, or 0.5 M urea, pH 3.0. Following the final dialysis against phosphate-buffered saline (PBS, pH 3.0) containing 1 mM PMSF, protein concentrations were determined using a Bradford reagent (BioRad, Hercules, CA).

Assays of cell adhesion, spreading, and stress fiber formation

The cell adhesion assay was described previously [16]. Briefly, 24-well culture plates (Nunc, Roskilde, Denmark) were coated with 5 mg/ml human placental laminin (Sigma-Aldrich, St. Louis, MO) or 25 mg/ml rLG3 proteins for 12 h at 4 1C, blocked with 1% heat-inactivated bovine serum albumin (BSA) in PBS for 1 h at 37 1C, and then washed with PBS. Trypsinized cells (2 ti 105 cells/
500 ml) were added to each plate and incubated at 37 1C in serum-free culture medium with and without PKC inhibitors, Go¨6976 (LC Laboratories, Woburn, MA; Calbiochem, San Diego, CA) and/or rottlerin (Calbiochem). After incubation period, unat- tached cells were removed by rinsing twice with PBS. Attached cells were fixed with 10% formalin for 15 min, stained with 0.5% crystal violet for 1 h, gently washed with distilled water three times, and dissolved with 2% SDS for 5 min. Absorbance was measured at 570 nm using a microplate reader (BioRad). To determine cell spreading, formalin-fixed and crystal violet- stained cell surface area was measured with Image-Pro plus software (Version 4.5; Media Cybernetics, Silver Spring, MD). To determine cell stress fiber formation, cells were attached to laminin or rLG3 for 3 h, fixed with formalin, permeabilized with 0.5% Triton X-100, blocked with BSA, incubated with rhodamin- conjugated phalloidin (1:300, Invitrogen) for 30 min at room temperature, and analyzed with confocal laser scanning microscope.

FAK phosphorylation assay and Western blotting

Cells were starved for 12 h by replacing RPMI 1640 medium with 0.1% FBS. Cells (2 ti 106 cells) were plated on 60-mm dishes
precoated with laminin (5 mg/ml) or rLG3 (25 mg/ml), and allowed to adhere for 15 min. Cells were washed with ice-cold PBS and lysed with 200 ml of RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF,
2mM Na3VO4, 1 mM glycerol phosphate) containing a protease inhibitor cocktail. RIPA lysates were denatured using SDS sample buffer and size-fractionated by SDS-PAGE and electroblotted. Immunoblots were probed with primary antibodies to FAK Tyr- 397 (Biosource, Camarillo, CA), FAK (Upstate Biotechnology, Lake Placid, NY), PKCa (Santa Cruz Biotechnology, Santa Cruz, CA), PKCb and PKCd (BD Biosciences, San Jose, CA), actin (Sigma- Aldrich), Rac1, or Cdc42 (Cell Biolabs, San Diego, CA). All blots were then incubated with appropriate anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (Cell Signaling, Danvers, MA). Signals were detected by ECL (iNtRON Biotechnology, Korea).

Cell fractionation

Membrane and cytosolic proteins were fractionated using plasma membrane protein extraction kit (BioVision, Mountain View, CA) following the recommended protocol. Briefly, cells were serum- starved for 12 h, trypsinized, and resuspended in serum-free medium containing 0.1% BSA. Cells were seeded (4 ti 106/100-mm dish) on laminin (5 mg/ml)- or rLG3 (25 mg/ml)-coated dishes and allowed to attach at 37 1C. Cells were lysed with the homogenize buffer of the kit (BioVision), and the cytosol fraction was collected after centrifugation at 10,000g for 30 min at 4 1C. Cell pellet was solubilized in RIPA buffer, and the soluble membrane fraction was recovered after centrifugation at 14,000g for 15 min at 4 1C. The protein concentration was determined using the Bradford reagent (BioRad), and analyzed by immunoblotting as described previously [6].
Rho GTPase assay

Rac1 and Cdc42 activation were determined by using the Rac1/
Cdc42 activation assay combo kit (Cell Biolabs) following the recommended protocol. Briefly, serum-starved cells were detached, resuspended in serum-free medium containing 0.1% BSA, and seeded on 100-mm dishes coated with laminin (5 mg/ml) or rLG3 (25 mg/ml) for indicated times at 37 1C. Cells were then lysed and rotated for 1 h at 4 1C with p21-activated protein kinase 1 (PAK1) p21-binding domain (PBD) Agarose beads which speci- fically bind the GTP-bound and activated forms of Rac1 and Cdc42 GTPases. The beads were washed extensively with the lysis buffer and, together with total lysates, boiled, and processed for immunoblotting.

siRNAs for PKCa, Rac1 and Cdc42

siRNAs against rat PKCa, Rac1 and Cdc42, and a nonspecific control siRNA were purchased from Invitrogen. PC12 cells were plated at a density of 5 ti 105 cells/60-mm dish and transfected with 600 pmol of respective siRNA using Lipofectamine RNAi- MAX transfection reagent (Invitrogen) following manufacturer’s recommendations. After 48 h, the transfected cells were har- vested for analysis by immunoblot or were processed for cell adhesion and spreading determinations.
Statistical analysis

Statistical analysis of data was performed by analysis of variance (ANOVA) followed by pair-wise comparisons using a Scheffe’s adjustment. P values less than 0.05 were considered significant.

Results

PKCa/b inhibitor Go¨6976 promotes cell adhesion, spreading, and stress fiber formation

We previously identified that the RNIPPFEGCIWN motif within the human laminin a2 LG3 domain is crucial for the laminin binding to integrin a3b1 in PC12 cells [6]. The engagement of integrin a3b1 in turn signals cell adhesion, spreading, and migration through the membrane recruitment of PKCd and FAK

phosphorylation at Tyr-397. Among other proteins, PKCa has also been implicated as an important molecule in modulating integrin-mediated cell adhesion and spreading [13,17–19]. Gen- erally, experimental conditions that reduce PKCa activity nega- tively regulated cell adhesion and spreading. To determine the functional role of PKCa in laminin-induced PC12 cell adhesion and spreading, we used Go¨6976, an indolocarbazole kinase inhibitor specific for PKCa/b. In cell culture dishes coated with human placental laminin or rLG3 domain of human laminin a2 chain, addition of PC12 cell suspension in serum free medium leads to integrin-dependent cell adhesion and spreading [6]. At 10 nM Go¨6976, a concentration that is approximately 4-fold greater than the IC50 (2.3 nM) value for PKCa/b inhibition [20], there was no effect on cell adhesion and spreading compared to vehicle (Fig. 1A and B). Surprisingly, at a higher concentration that is used typically to block PKCa/b activity, 1 mM Go¨6976 treatment enhanced PC12 cell adhesion, spreading, and stress fiber formation on laminin and rLG3 (Fig. 1A and B). This Go¨6976-dependent increase in cell adhesion and spreading activity was consistently observed in multiple experiments and was also confirmed using different commercial sources of Go¨6976 (Calbiochem and LC Laboratories).
PKC activation plays a critical role in integrin-mediated pro- cesses and signaling in various cell types, including PC12 cells [13,21–23]. We have reported on the membrane recruitment of PKCd following laminin-dependent integrin engagement [6]. Indeed, pretreatment of PC12 cells with 1 mM Go¨6976 markedly increased PKCd in the membrane fraction compared with vehicle (Fig. 1C). The membrane translocation of PKCd from the cytosolic fraction occurred within 15 min of PC12 cell seeding on laminin or rLG3. Next, we examined whether enhanced membrane localization of PKCd by Go¨6976 treatment induces FAK phos- phorylation in cells adhering to laminin and rLG3. FAK phos- phorylation is necessary for integrin-mediated cell adhesion and spreading [24,25]. The levels of FAK phosphorylation at Tyr-397 were increased by Go¨6976 treatment in cells adhering to laminin and rLG3 (Fig. 1D). These results indicate that Go¨6976 treatment at 1 mM concentration positively regulates translocation of the PKCd from the cytosol to the membrane, leading to increased cell adhesion, spreading, stress fiber formation, and FAK phosphor- ylation at Tyr-397.

Go¨6976-dependent membrane translocation of PKCd in PC12 cells transfected with PKCa siRNA

Go¨6976 is structurally similar to ATP and is widely used as isozyme-specific competitive inhibitor of PKCa and PKCb with IC50 in the low nanomolar range [20]. At 10 nM Go¨6976, PKCa activity is expected to be largely inhibited and at 1 mM, PKCa is expected to be completely inactive. Because increased cell adhesion and spreading of PC12 cells in Fig. 1 occurred only in response to 1 mM Go¨6976 treatment, it suggested that the Go¨6976 effect on cell adhesion activity may not result from a direct pharmacological inhibition of PKCa/b. We utilized knock- down approach with siRNA specific for PKCa to test this hypothesis. PC12 cells did not express PKCb as determined by immunoblot using PKCb-specific monoclonal antibody (clone 36/
PKCb; BD Biosciences) (Fig. 2A). In PC12 cells transfected with PKCa siRNA, but not control siRNA, resulted in pronounced diminution of PKCa protein expression compared to vehicle

Fig. 1 – High concentration of PKCa/b inhibitor Go¨6976 promotes cell adhesion, spreading, and stress fiber formation through the membrane localization of PKCd and FAK phosphorylation at Tyr-397. (A) Increased cell adhesion (left panel) and spreading (right panel) to laminin and rLG3 in PC12 cells pretreated with 10 nM and 1 lM Go¨6976 for 15 min and then cultured on laminin- or rLG3-coated plates for 1 h (left panel) or 3 h (right panel). Data are expressed as a percentage of the value of cells pretreated without Go¨6976 (mean7S.D., n ¼ 3). ti po0.01. (B) Rhodamine-phalloidin staining and actin stress fiber formation in PC12 cells pretreated with and without Go¨6976. PC12 cells were seeded for 3 h in serum-free medium on glass slide chambers coated with laminin or rLG3. Data are percentage of cells with actin stress fibers and expressed as mean7S.D. (n ¼ 3). Bar, 50 lm. ti po0.01. (C) Immunoblots of PKCd from cytosolic and membrane fractions of cells pretreated with Go¨6976. PC12 cells pretreated with
1 lM Go¨6976 or vehicle for 15 min were seeded on laminin or rLG3-coated dishes for the indicated times. Immunoblots were stripped and reprobed with actin antibody. (D) Pretreatment with 1 lM Go¨6976 increased laminin- and rLG3-induced FAK phosphorylation at Tyr-397. PC12 cells were pretreated with or without Go¨6976 for 15 min and seeded on laminin- or
rLG3-coated plates for 15 min in serum-free medium.

(Fig. 2B). Despite the reduced levels of PKCa, 1 mM Go¨6976 significantly increased cell adhesion and spreading of the PKCa siRNA-transfected PC12 cells on both laminin and rLG3 (Fig. 2C). The lower Go¨6976 concentration (10 nM) was not different from vehicle treatment. Qualitatively similar enhancements of Go¨6976-mediated cell adhesion and spreading to laminin and rLG3 were found in control siRNA-transfected (data not shown) and untransfected cells (Fig. 1A). In addition, the membrane translocation of PKCd was significantly increased following 1 mM Go¨6976 treatment to PKCa siRNA-transfected PC12 cells that were seeded on laminin or rLG3 (Fig. 2D). Evidently, the high concentration of Go¨6976 enhances laminin-dependent cell adhe- sion and spreading and the membrane recruitment of PKCd through a mechanism that does not involve PKCa.

Inhibition of PKCd blocks Go¨6976-dependent enhancement of cell adhesion activity

We employed rottlerin, a specific pharmacological inhibitor of
PKCd activity (IC50 ¼ 3–6 mM), to assess whether the PKCd signaling is required for the increased cell adhesion activity induced by the high concentration of Go¨6976. PC12 cells seeded on rLG3-coated dishes showed increased cell adhesion over the
3h time course incubation (Fig. 3A). Treatment with 1 mM Go¨6976 increased cell adhesion compared to the vehicle treat- ment at all-time points, and additive treatment with 1 mM Go¨6976 and 5 mM rottlerin completely blocked cell adhesion activity to a similar level of 5 mM rottlerin treatment alone. We have shown previously that rottlerin block of PKCd reduces PC12 cell spreading on laminin- and rLG3-coated dishes [6]. These results suggest that high concentration of Go¨6976 induces membrane recruitment and signaling through the PKCd pathway to enhance cell adhesion activity. Qualitatively similar results were obtained in CV-1 (African green monkey kidney) and NIH/
3T3 (mouse embryonic) fibroblasts seeded on rLG3 where the increased cell adhesion to 1 mM Go¨6976 after 2 h of seeding was blocked by co-incubation with 5 mM rottlerin (Fig. 3B and C). This result suggests that the enhancement of laminin-dependent cell adhesion activity induced by the high concentration of Go¨6976 is not a phenomenon limited to PC12 cells and may occur in other cell types.

PKCd-downstream role of small GTPases, Rac1 and Cdc42, in cell spreading and lamellipodia formation

Our results provide additional support for the involvement of PKCd in the early stages of cells adhesion and spreading.

Fig. 2 – High concentration Go¨6976 promotes cell adhesion and spreading in PKCa siRNA-transfected cells through
the membrane localization of PKCd. (A) Immunoblot
of PKCb from PC12 cell lysate. Whole cell lysates from PC12, HEK293, and rat brain were immunoblotted and probed
with PKCb-specific monoclonal antibody and stripped and reprobed with actin antibody. (B) Immunoblot
of PC12 cell lysates prepared 48 h following transfection with control siRNA or PKCa siRNA and probed with
anti-PKCa antibody, and stripped and reprobed with actin antibody. (C) Increased cell adhesion (left panel) and spreading (right panel) to laminin and rLG3 in PKCa siRNA- transfected cells pretreated with high concentration Go¨6976. Forty-eight hours following transfection with
control siRNA or PKCa siRNA, PC12 cells were pretreated with
10nM and 1 lM Go¨6976 for 15 min and then cultured on laminin- or rLG3-coated plates for 1 h (left panel) or 3 h (right panel) in serum-free medium. Data are expressed as percentage of the value for cells pretreated vehicle (mean7S.D., n ¼ 3). tipo0.01; titipo0.05. (D) High concentration of Go¨6976 recruits PKCd to membrane fraction in laminin- and LG3-mediated adhesion and spreading of PKCa knockdown PC12 cells. The assay conditions were the same as described in C, except that cells were cultured on laminin- or rLG3-coated plates for 15 min.

Fig. 3 – Go¨6976-dependent enhancement of cell adhesion activity is blocked by inhibition of PKCd in PC12, NIH/3T3
and CV-1 cells. (A) Time course of cell adhesion to rLG3-coated plates in PC12 cells treated with and without Go¨6976 and rottlerin. PC12 cells were pretreated with 1 lM Go¨6976, 5 lM rottlerin, and both for 15 min and then cultured on rLG3- coated plates for 7, 15, 30, 60, 120, and 180 min in serum-free medium. Data are expressed as mean7S.D. (n ¼ 3). (B and C) Cell adhesion to rLG3-coated plates in CV-1 (B) and NIH/3T3 (C) cells treated with Go¨6976, rottlerin, and both. The assay conditions were the same as described in (A), except that cells were cultured for 120 min on rLG3-coated plates. Data are expressed as the mean7S.D. (n ¼ 3). ti po0.01; titi po0.05.

Signaling downstream of PKCd from the focal adhesion sites is not well understood in laminin-integrin-mediated cell spreading. Small GTPase Rac1 is downstream of FAK [26], and FAK- dependent activation of Rac1 promotes formation of membrane ruffles and lamellipodia as well as regulating cell migration [14,27]. Cdc42, similar to Rac1, is another member of the Rho GTPase family that has a role in neuronal lamellipodia formation [28]. We examined the expression and activation of Rac1 and Cdc42 by pull-down assays in PC12 cells seeded on laminin and rLG3 using the PAK1 PBD Agarose beads which specifically bind to the active (GTP-bound) state of Rac1 or Cdc42 proteins. The levels of activated Rac1 increased slightly and that of the activated Cdc42 increased markedly in cells seeded on laminin and rLG3 compared to cells in suspension (Fig. 4A and B). Next, we treated the cells with 5 mM rottlerin to examine whether PKCd signaling is involved in the activation of the small GTPases. There were clear reductions in the levels of active Cdc42 in

Fig. 4 – Involvement of small GTPases, Rac1 and Cdc42, in cell spreading, lamellipodia formation induced by laminin and rLG3. (A and B) Immunoblot analysis of active Rac1 and Cdc42 in PC12 cells pretreated with rottlerin. PC12 cells were pretreated with 5 lM rottlerin for 15 min and then cultured on laminin- or rLG3-coated dishes or kept suspended in rottlerin-containing medium for the indicated times. The cells were lysed and incubated with PAK1 PBD Agarose beads for 1 h at 4 1C. Active Rac1 (A) and Cdc42 (B) proteins were determined by immunoblot analysis using anti-Rac1 and anti-Cdc42 antibodies. (C) Immunoblot analysis of the expression of Rac1 and Cdc42 in Rac1 siRNA-, Cdc42 siRNA-, and Rac1 siRNA plus Cdc42 siRNA-transfected cells. (D) Inhibition of cell spreading on laminin- and rLG3-coated culture slides in Rac1 siRNA-, Cdc42 siRNA-, and Rac1 siRNA plus Cdc42 siRNA-transfected PC12 cells seeded for 3 h. Results are expressed as a percentage from the value of control siRNA- transfected cells (mean7S.D., n ¼ 3). ti po0.01.

rottlerin-pretreated cells that were seeded on laminin and rLG3 (Fig. 4B). The decreased levels of active Rac1 in rottlerin-treated cells were also evident in cells seeded on laminin for 30 min, and in cells seeded on rLG3 for 1 h (Fig. 4A). In rottlerin-treated cells seeded on laminin for 1 h, the small level of active Rac1 pulled- down was not different compared to the vehicle treated cells (data not shown). These results suggest that PKCd signaling pathway is involved in the activation of Rac1 and Cdc42 down- stream of laminin-integrin and rLG3-integrin engagement.
To investigate the role of Rac1 and Cdc42 in cell spreading, we used siRNA experiments to reduce the expression of Rac1 and Cdc42. Compared to the control siRNA-transfected cells, Rac1 and Cdc42 expressions were markedly reduced in the respective siRNA-transfected cells (Fig. 4C). Suppression of Rac1 expression inhibited cell spreading and lamellipodia formation in cells seeded on laminin and rLG3, but the suppression of Cdc42 expression alone did not (Fig. 4D). Interestingly, blocking expres- sion of both Rac1 and Cdc42 synergistically reduced cell spread- ing and lamellipodia formation in cells seeded on laminin and rLG3 compared to suppression of Rac1 expression alone (Fig. 4D), suggesting that Rac1 may be activated by Cdc42 during cell spreading and lamellipodia formation. Collectively, these results
indicate that in cell adhesion to laminin and rLG3, integrin engagement activates Rac1 and Cdc42 in a PKCd-dependent manner, leading to cell spreading and lamellipodia formation.

Discussion

The present study investigated the link between PKC and cellular behavior such as cell adhesion, spreading, and stress fiber formation during the early stage cell adhesion signaling of cultured PC12 cells on laminin-2. We have recently reported that human laminin-2 which has a central role in peripheral myelinogenesis [1] promotes outside-in integrin signaling in PC12 cells through a3b1 integrin in PC12 cells [6]. PKC activation is known to be involved in signaling pathways of several integrin-mediated processes, such as cell adhesion, spreading, migration, and focal adhesion assembly [11,12,22]. The activa- tion of PKC results from the phospholipase C-mediated increase in intracellular DAG levels following the ligand engagement of integrin b1 [29]. Furthermore, PKC activation in response to cell adhesion is accompanied by a change in subcellular localization [14,27] mediated by specific interactions with binding proteins,

such as the receptors for activated C kinases 1 [30]. The receptors

for activated C kinases 1 binds the activated PKC isoforms, and this complex associates with the integrin b1 cytoplasmic domain, leading to integrin clustering and increased cell adhesion and spreading [31].
In this study, we found that the treatment with the specific PKCa/b blocker Go¨6976 (1 mM) increased cell adhesion, spread- ing, and FAK phosphorylation at Tyr-397 through the transloca- tion of the PKCd from the cytosol to the membrane fraction. Inhibition of PKCa leads to attenuated focal adhesion formation and decreased stress fibers in rat embryo fibroblasts [19], and attenuated cell spreading in vascular smooth muscle [32]. Unexpectedly, the Go¨6976-mediated increase in cell adhesion and spreading and the membrane translocation of PKCd persisted in siRNA knockdown of PKCa in PC12 cells. Together with the fact that PC12 cells do not express the PKCb isoforms, our results suggest that the Go¨6976 effects may occur through a mechanism that is independent of PKCa/b. Indeed, Go¨6976 has recently been shown to inhibit tyrosine kinases associated with neutrophin receptors, trk A and trk B, Janus kinase 2 and FLT3 tyrosine kinases, and guanylyl cyclase-A and -B [33–35]. Whether these signaling members are part of the mechanism by which PKCa/b inhibitor Go¨6976 treatment induces activation and membrane translocation of PKCd in response to laminin-2-integrin engage- ment adhesion signaling in PC12 cells remains to be elucidated.
Currently, the mechanisms by which the small GTPases, Rac1 and Cdc42, regulate integrin-mediated cell adhesion, and spread- ing are poorly understood. In this study we found that PKCd regulated the activations of Rac1 and Cdc42 in rLG3 and laminin- 2-mediated cell adhesion, indicating that the interaction of a3b1 integrin with PKCd directly or indirectly activates Rac1 and Cdc42, and thereby promotes cell spreading. Although it is possible that high Go¨6976 may also directly activate Rac1 and Cdc42, in addition to its effects through PKCd, we did not test this idea in the present study. Moreover, FAK is known to be involved in Rac1 activation and targeting through the tyrosine phosphor- ylation of bp21-activated kinase-interacting exchange factor [36], indicating that FAK-bp21-activated kinase-interacting exchange factor-Rac1 pathway may be important for the promo- tion of cell spreading. Our results demonstrated that cell adhe- sion to rLG3 and laminin activated the small GTPases, Rac1 and Cdc42, and further, both Rac1 and Cdc42 siRNA-knockdown PC12 cells showed synergistically impaired cell spreading and lamellipodia formation compared to Rac1 siRNA-treated cells. Since Cdc42 siRNA-treatment alone did not inhibit cell spreading and lamellipodia formation, Cdc42 activation in PC12 cells may occur through Rac1 signaling pathway, similar to the activation of Cdc42 in NIE-115 neuroblastoma cells that induces formation of lamellipodia through the STEF/Tiam1-Rac1 pathway [28]. Therefore, it is possible that not only Rac1 but also Cdc42 may be positive regulators of formation of lamellipodia in PC12 cells.
As shown here, Go¨6976 induced human laminin-2-integrin- mediated cell adhesion and spreading and FAK phosphorylation through a PKCa/b-independent recruitment of PKCd in PC12 cells. Our results suggest that the notion of Go¨6976 being a specific and selective inhibitor of PKCa/b warrants caution. In addition, our results also showed that the activation of Rac1 and Cdc42 by the a3b1 integrin and PKCd/FAK signaling pathways mediates cell spreading and lamellipodia formation in PC12 cells.
Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea Government (MEST) (2010-0014662) (to B.-M. M).

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