Molecular and Cellular Neuroscience 19, 292–306 (2002)
Protein Kinase C � (PKC�) Is Required for ProteinTyrosine Phosphatase � (PTP�)-DependentNeurite Outgrowth
Jullia A. Rosdahl, Tracy L. Mourton, andSusann M. Brady-KalnayDepartment of Molecular Biology and Microbiology, Case Western Reserve University,School of Medicine, Cleveland, Ohio 44106-4960
Protein tyrosine phosphatase � (PTP�) is an adhesionmolecule in the immunoglobulin superfamily and is ex-pressed in the developing nervous system. We haveshown that PTP� can promote neurite outgrowth of reti-nal ganglion cells and it regulates neurite outgrowth me-diated by N-cadherin (S. M. Burden-Gulley and S. M.Brady-Kalnay, 1999, J. Cell Biol. 144, 1323–1336). We pre-viously demonstrated that PTP� binds to the scaffoldingprotein RACK1 in yeast and mammalian cells (T. Mourtonet al., 2001, J. Biol. Chem. 276, 14896–14901). RACK1 is areceptor for activated protein kinase C (PKC). In this ar-ticle, we demonstrate that PKC is involved in PTP�-de-pendent signaling. PTP�, RACK1, and PKC� exist in acomplex in cultured retinal cells and retinal tissue. Usingpharmacologic inhibition of PKC, we demonstrate thatPKC� is required for neurite outgrowth of retinal ganglioncells on a PTP� substrate. These results suggest thatPTP� signaling via RACK1 requires PKC� activity to pro-mote neurite outgrowth.
INTRODUCTION
Spatiotemporal patterning of the central nervous sys-tem is complex: trillions of neurons need to find theirsometimes distant targets. Through an intricate combi-nation of short- and long-range interactions, some at-tractive and some repulsive, an axon can find its way toits target tissue. The growing tip of the axon, called thegrowth cone, migrates along the surface of other neu-rons, on glial cells and on extracellular matrix. Celladhesion molecules (CAMs) mediate the cell–cell inter-actions required for migration and axonal guidance(reviewed in Tessier-Lavigne and Goodman, 1996). Yet,
the signaling cascades that convert these extracellularadhesive events into intracellular responses have not
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been well defined. A subgroup of CAMs consists ofreceptor protein tyrosine phosphatases (RPTPs) thattransduce signals via changes in tyrosine phosphoryla-tion.
The balance of protein tyrosine kinase (PTK) andprotein tyrosine phosphatase (PTP) activities controlthe level of tyrosine phosphorylation of proteins withina cell (reviewed in Hunter, 1998). Tyrosine phosphory-lation plays an important role in the growth and guid-ance of axons in the nervous system (reviewed in Desaiet al., 1997; Bixby, 2000; Stoker, 2001). For example, theEph PTKs and their ephrin ligands are axon guidancemolecules that play a crucial role in the spatiotemporalpatterning of the visual system (reviewed in Nakamoto,2000). RPTPs of the immunoglobulin (Ig) superfamilyhave also been implicated in axonal growth and guid-ance (reviewed in Brady-Kalnay, 2001). These CAM-like RPTPs are unique due to their dual functions ascell–cell adhesion molecules and tyrosine phosphatases(Brady-Kalnay, 2001), suggesting that they signal inresponse to adhesion. We recently demonstrated thatthe RPTP PTP� promotes neurite outgrowth in an invitro assay (Burden-Gulley and Brady-Kalnay, 1999). Arelated RPTP, PTP�, was also shown to promote neuriteoutgrowth from cerebellar neurons (Drosopoulos et al.,1999). PTP�, a member of the LAR subfamily of PTPs,promotes neurite outgrowth from forebrain and somecerebellar neurons (Wang and Bixby, 1999). In addition,a gradient of PTP� can act as a chemoattractant forembryonic forebrain neurons, which suggests that thisphosphatase may be a guidance molecule (Sun et al.,2000). Another LAR-like PTP, CRYP�, has been shown
doi:10.1006/mcnMCN
01.1071, available online at http://www.idealibrary.com on e.20
to regulate the interactions between neurons and glia inthe retina and to regulate retinal axon outgrowth (Haj et
al., 1999; Ledig et al., 1999a). Homophilic binding of theleech homolog of LAR (HmLAR2) may function in neu-ronal outgrowth as well as growth cone collapse andmutual repulsion (Gershon et al., 1998; Baker and Ma-cagno, 2000). In addition, CAM-like Drosophila RPTPsare required for proper neuronal guidance during de-velopment of the fly nervous system (Desai et al., 1996;Krueger et al., 1996; Desai et al., 1997). The regulation oftyrosine phosphorylation by RPTPs may control attrac-tion or repulsion of growth cones. This “steering” ofgrowth cones promotes extension along the appropriatepathways during development.
The neurite outgrowth assay provides a model forcontact-dependent growth and guidance, which isthought to be mediated by a series of adhesive eventsbetween the growth cone and molecules expressed onother cells or in matrices which form the “substrate” formigration. Neurite outgrowth is a frequently used as-say of adhesion molecule function because it not onlyrequires adhesion but also signaling to the cytoskeletonin order for a CAM to promote neurite outgrowth (re-viewed in Suter and Forscher, 2000). Only a limitednumber of CAMs are able to promote neurite out-growth. PTP� promotes neurite outgrowth of retinalganglion cells, which is blocked by anti-PTP� antibod-ies (Burden-Gulley and Brady-Kalnay, 1999). PTP� isexpressed at high levels in the retina and tectum duringthe developmental period in which RGC axons aregrowing to and forming connections with their CNStarget (Ledig et al., 1999b; Burden-Gulley and Brady-Kalnay, 1999). Thus, studying neurite outgrowth fromretina during this developmental period is an appropri-ate functional assay for PTP�.
The focus of this manuscript is PTP� which is widelyexpressed in the developing nervous system and devel-opmentally regulated in the visual system (Burden-Gulley and Brady-Kalnay, 1999; Ledig et al., 1999b). It isa member of the Ig superfamily of adhesion molecules.The extracellular segment of PTP� contains a MAMdomain, followed by an Ig domain and four fibronectintype III repeats (Gebbink et al., 1991). The extracellularportion of the protein is responsible for its adhesiveproperties and PTP� is known to bind homophilicallyvia its Ig domain (Brady-Kalnay and Tonks, 1994). TheMAM domain has also been shown to play a role incell–cell aggregation (Zondag et al., 1995). The intracel-lular segment of PTP� consists of a juxtamembraneregion followed by two phosphatase domains (Gebbinket al., 1991).
The juxtamembrane domain of PTP� has 20% homol-ogy with the conserved intracellular domain of thecadherins (Tonks et al., 1992). The cadherins are cal-
cium-dependent cell adhesion molecules. The intracel-lular domain of cadherins binds to the catenins whichin turn link this protein complex to the actin cytoskel-eton (Gumbiner, 1995; Aberle et al., 1996). We havepreviously shown that PTP� associates with the cad-herin–catenin complex (Brady-Kalnay et al., 1995, 1998;Hiscox and Jiang, 1998) and that PTP� is requiredfor N-cadherin-dependent neurite outgrowth (Burden-Gulley and Brady-Kalnay, 1999). In addition, PTP� alsopromotes neurite outgrowth of retinal ganglion cells(Burden-Gulley and Brady-Kalnay, 1999).
The precise signal transduction pathway utilized byPTP� to promote neurite outgrowth was not known.We previously performed a yeast two-hybrid assay toidentify interacting proteins and found that RACK1binds to the membrane proximal phosphatase domainof PTP� (Mourton et al., 2001). RACK1 is a scaffoldingprotein that was originally identified as a receptor foractivated C kinase (Ron et al., 1994). RACK1 is a mem-ber of the G� family of proteins distinguished by sevenpropeller-like protein-binding domains called WD40 re-peats (Garcia-Higuera et al., 1996). This cytoplasmicscaffolding protein binds the signaling molecules pro-tein kinase C (PKC), phospholipase-C�, the src tyrosinekinase, and cAMP phosphodiesterase-4 as well as the �subunit of integrin receptors and the � chain of theinterleukin-5 receptor (Disatnik et al., 1994; Chang et al.,1998; Yarwood et al., 1999; Liliental and Chang, 1998;Geijsen et al., 1999). RACK1 has also been shown to bindto the pleckstrin homology domains of dynamin,�-spectrin, RasGRF, and oxysterol binding protein (Ro-driguez et al., 1999). The interactions between RACK1with PKC and PLC� are mutually exclusive (Chang etal., 1998), as are the interactions between RACK1 withPTP� or src (Mourton et al., 2001). The interaction be-tween RACK1 and src is enhanced by PKC activationand tyrosine phosphorylation of the RACK1 protein(Chang et al., 2001). These studies suggest a modelwhere distinct complexes are formed via RACK1 inresponse to unique cellular signals.
RACK1 is a receptor for activated PKC (Ron et al.,1994). PKC(s) are a family of lipid-dependent serine/threonine kinases involved in many cellular processesincluding neurite outgrowth as well as growth, differ-entiation, secretion, apoptosis, and tumor development(reviewed in Gschwendt, 1999). Our goal was to deter-mine whether the PTP�/RACK1 complex associateswith PKC(s) as a part of a signaling pathway to regulatePTP�-dependent neurite outgrowth.
In this article, we demonstrate that PKC� is involvedin PTP�-dependent neurite outgrowth of retinal gan-glion cells. First, we show that most isoforms of PKC
293PKC� Regulates Neurite Outgrowth on PTP�
including PKC� are expressed in chick retinal cells.Second, we demonstrate that PTP�, RACK1, and PKC�exist in a complex in cultured retinal cells as well asretinal tissue. Third, using pharmacologic inhibition ofPKC, we demonstrate that PKC� activity is required forneurite outgrowth on a PTP� substrate. Together, theseresults suggest that PTP� signaling requires PKC� ac-tivity to promote neurite outgrowth of retinal ganglioncells.
RESULTS
PKC Isoforms Are Expressed in Chick Retinal Cells
PTP� interacts directly with the scaffolding proteinRACK1 in a yeast two-hybrid system and in a mamma-lian cell line (Mourton et al., 2001). RACK1 binds toactivated PKC (Ron et al., 1994). We investigatedwhether PKC interacts with PTP� and RACK1 in retinalcells. There are at least nine isoforms of PKC and mostare expressed in the retina and brain (Wood et al., 1997).Immunoblot analysis showed that all isoforms testedexcept for PKC� are expressed in embryonic day 8 (E8)chick retina and in E6 retinal neuroepithelial (RNE)cells (Fig. 1). The E8 retinas are used for the neuriteoutgrowth assays and E6 RNE cells are pluripotentneuroepithelial cells that can be cultured in vitro atdifferent densities. Most avian PKC isoforms migratedon SDS–PAGE at the same rate as their mammalianhomologue (rat brain extract) with the exception ofPKC�. Chick PKC� migrated slower than the strongestband in the mammalian positive control. There werealso several higher bands recognized by the PKC� an-tibody in both E8 retina and RNE cells. RACK1 wasexpressed in both retinal preparations (Fig. 1).
PTP�, RACK1, and PKC� Exist in a Complexin Retinal Cells
To examine whether PTP� associates with RACK1and PKC isoforms in chick retina, immunoprecipitationexperiments were performed using retina lysates (Fig.2). PTP� coimmunoprecipitates with RACK1 and the �isoform of PKC in both retina tissue and retinal cul-tures. The other classical PKC isoforms that are ex-pressed in retina were tested (� and �) but not detectedin a complex with PTP� in the retina (data not shown).To determine whether the interaction between PTP�with RACK1 and PKC� was dependent on cell contact(i.e., induced by PTP� homophilic binding), immuno-precipitation experiments were performed at two dif-
ferent cell densities to control the amount of cell–cellcontact (Fig. 2). At low cell density (approximately 1 �107 cells/100-mm dish) there is very little contact be-tween cells, whereas at high cell density (approximately4 � 107 cells/100-mm dish), the majority of cells are incontact with each other. The density experiments pro-vide a model for retinal cell–cell adhesion as the cellscontact other cells or axons. The immunoprecipitationexperiments were performed with two monoclonal an-tibodies generated against intracellular epitopes in thePTP� protein. The SK7 antibody binds to the juxtamem-brane domain of PTP� (Brady-Kalnay et al., 1998). The
FIG. 1. PKC isoforms are expressed in chick retina. Lysates madefrom embryonic day 8 neural retinas and embryonic day 6 retinalneuroepithelial cultures were separated by 10 or 12% SDS–PAGE,transferred to nitrocellulose, and probed with antibodies againstPKC�, �, �, �, �, �, and and RACK1. Rat brain lysate from Trans-duction Laboratories serves as the positive control.
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SK18 antibody binds to the membrane proximal cata-lytic domain of PTP� (Brady-Kalnay et al., 1998) whichalso contains the RACK1 binding site (Mourton et al.,2001). Both the full-length (200-kDa) and cleaved (100-kDa) forms of PTP� were immunoprecipitated by bothantibodies to PTP� (Fig. 2C).
When immunoprecipitates of PTP� were probed onimmunoblots with RACK1 antibodies, an associationwas detected in both E8 retina and E6 RNE cultures athigh cell density when the SK7 antibody was used (Fig.2A, lanes 11 and 12). Interestingly, the SK18 antibody,which immunoprecipitated PTP�, did not immunopre-cipitate RACK1 (Fig. 2A, lanes 13–15). The epitope ofSK18 antibody is the first phosphatase domain of PTP�(Brady-Kalnay et al., 1998) and the RACK1 binding sitein PTP� is also the first phosphatase domain (Mourtonet al., 2001). The PTP�/RACK1 complex could not beimmunoprecipitated using the SK18 antibody suggest-ing that the antibody binding site may overlap with the
RACK1 binding site on PTP�. In addition, PKC� wasalso detected by immunoblot in PTP� immunoprecipi-tates using the SK7 antibody in E8 retina and E6 RNEcultures at high cell density (Fig. 2B, lanes 5 and 6).PKC� was not detected in the SK18 immunoprecipitates(Fig. 2B, lanes 7–9), which also lack RACK1.
The association between PKC� and RACK1 in E6RNE cells was not affected by the degree of cell–cellcontact (Fig. 2A, lanes 7 and 8). RACK1 binds to acti-vated PKC (Ron et al., 1994). The RACK1/PKC� com-plex was observed in RNE cultures under all conditions(Fig. 2A, lanes 7 and 8). This result was not surprisingsince the retinal cell cultures were grown in the pres-ence of serum, which contains growth factors known tostimulate PKC. However, the association between PTP�with RACK1 (Fig. 2A, lanes 10 and 11) and PKC� (Fig.2B, lanes 4 and 5) in E6 RNE cells was dependent oncell–cell contact. Together, these results suggest thatPTP� exists in a signaling complex with RACK1 and
FIG. 2. RACK1 and PKC� coimmunoprecipitate with PTP� in E8 retina and in retinal neuroepithelial (RNE) cultures. (A) Lysates from E6 RNEcells at low (lanes 1, 4, 7, 10, 13) and high (lanes 2, 5, 8, 11, 14) density and from E8 retina (lanes 3, 6, 9, 12, 15) were immunoprecipitated withcontrol (lanes 1–3), RACK1 (lanes 4–6), PKC� (lanes 7–9), or PTP� (SK7, lanes 10–12; SK18, lanes 13–15) antibodies and were separated by 10%SDS–PAGE. Immunoblots were probed with RACK1 antibodies (A). RACK1 was detected in all RACK1 (A, lanes 4–6) and PKC� (A, lanes 7–9)immunoprecipitates. PTP� predominantly interacts with RACK1 in high-density RNE cultures (A, lane 11) and in E8 retina (A, lane 12), but notin low-density RNE cultures (A, lane 10). The interaction between PTP� and RACK1 is not preserved in immunoprecipitates using the SK18antibody (A, lanes 13–15). (B) Lysates from RNE cells at low (lanes 1, 4, 7) and high (lanes 2, 5, 8) density and from E8 retina (lanes 3, 6, 9) wereimmunoprecipitated with PKC� (lanes 1–3) or PTP� (SK7, lanes 4–6; SK18, lanes 7–9) antibodies. PKC� was detected in all PKC� immunopre-cipitates (B, lanes 1–3). PTP� predominantly interacts with PKC� in high-density RNE cultures (B, lane 5) and in E8 retina (B, lane 6), but notin low-density RNE cultures (B, lane 4). The complex between PTP� and PKC� is not immunoprecipitated by the SK18 antibody (B, lanes 7–9).(C) Lysates from RNE cells at low (lanes 1, 4) and high (lanes 2, 5) density and from E8 retina (lanes 3, 6) were immunoprecipitated with theSK7 (lanes 1–3) or SK18 (lanes 4–6) antibodies to PTP� and were separated by 6% SDS–PAGE. Immunoblots were probed with the PTP�antibody SK15. Both full-length and proteolytically processed forms of PTP� were detected in SK7 (C, lanes 1–3) and SK18 (C, lanes 4–6)immunoprecipitates.
295PKC� Regulates Neurite Outgrowth on PTP�
PKC� at high cell density. Most likely, RACK1 is me-diating the interaction between PTP� and PKC�.
PTP�, RACK1, and PKC� Are Presentin the Neurites and Growth Conesof Retinal Ganglion Cells
PTP� is expressed predominantly in retinal ganglioncells during retinal development and localized to theaxons and growth cones of retinal ganglion cells (Bur-den-Gulley and Brady-Kalnay, 1999). To determinewhether RACK1 and PKC� were also present in thesecells, we performed double-label immunocytochemis-try on retinal explants (Fig. 3). Neurites and growthcones growing on a laminin substrate were double la-beled for PTP� and RACK1 (Figs. 3A–3C) or PKC� andRACK1 (Figs. 3D–3F). All three proteins were present inthe neurites and growth cones of retinal ganglion cells.PTP� is expressed throughout the neurite and growthcone (Fig. 3A). RACK1 (Figs. 3B and 3E) and PKC� (Fig.3D) are localized predominantly to the central region ofthe growth cone as well as the shaft of the neurite.There is colocalization of both PTP� (Figs. 3A and 3B)and PKC� (Figs. 3D and 3E) with RACK1 in the shaft ofthe neurite as well as in the central region of the growthcone.
Inhibition of PKC� Blocks PTP�-DependentNeurite Outgrowth
Purified PTP� is capable of promoting neurite out-growth from E8 retinal explants (Burden-Gulley andBrady-Kalnay, 1999). To determine whether PKCs wereinvolved in PTP�-dependent signaling to promote neu-rite outgrowth, PKC-specific inhibitors were used in theneurite outgrowth assay. The phorbol ester phorbol12-myristate 13-acetate (PMA) is a potent stimulator ofPKC activity upon short-term treatment (minutes).However, long-term treatment on the order of 24 hresults in inhibition of a broad spectrum of PKC iso-forms (Ballester and Rosen, 1985) including PKC�(Puceat et al., 1994; Chen and Wu, 1995). Retinal ex-plants were treated with PMA for the first 18–20 h afterplating, and then the PMA was washed out and re-placed with fresh medium. Under these conditions,PMA caused a significant decrease in neurite outgrowthon a PTP� substrate (Figs. 4A, 4B, and 5; Table 1). Thephotographs shown (Figs. 4A and 4B) are representa-tive of the median level of neurite outgrowth undercontrol and test conditions. The effect of PMA treatmentranged from no neurites to a few short neurites. Quan-titation of similar experiments indicated that overallneurite density was reduced by 94% and neurite lengths
FIG. 3. PTP�, RACK1, and PKC� are expressed in neurites and growth cones of retinal explants. Embryonic day 8 retinal explants werecultured on laminin overnight. The cells were fixed and double labeled with RACK1 antibodies (B, E) and PTP� antibodies (A) or PKC�antibodies (D) or secondary antibodies only (G–I). Phase contrast images are shown in C, F, and I. These proteins are present in neurites andgrowth cones of retinal ganglion cells. PTP� (A) is diffusely localized throughout the growth cone and neurite, whereas RACK1 (B, E) and PKC�(D) are localized predominantly in the central region of the growth cone as well as in the neurite.
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were reduced by 84% in cultures treated with PMAcompared to control cultures treated with DMSO.
To determine which PKC isoforms were required forPTP�-dependent outgrowth, isoform-specific PKC in-hibitors were added to neurite outgrowth assays.Go6976 specifically inhibits PKC� and PKC� isoforms.This inhibitor had no effect on neurite outgrowth on aPTP� substrate (Figs. 4C, 4D, and 5; Table 1). A slightincrease in neurite length was observed in the presenceof Go6976 (Fig. 5B), but this increase was not statisti-cally significant. Rottlerin specifically inhibits PKC�and treatment with this inhibitor resulted in a signifi-cant reduction in PTP�-dependent neurite outgrowth(Figs. 4E, 4F, and 5; Table 1). Quantitation of similarexperiments showed that overall average neurite den-sity was reduced by 82% and average neurite lengthswere reduced by 54% in rottlerin-treated cultures com-pared to the control treatment with DMSO. Together,these results suggest that the PKC signaling pathway isinvolved in PTP�-dependent neurite outgrowth andthe isoform mediating this effect is likely to be PKC�.
We wanted to test whether PKC� was specificallyinvolved in PTP�-dependent neurite outgrowth or if itwas required for neurite outgrowth on other substrates
as well. Therefore, control neurite outgrowth assaysperformed on two other adhesion molecules, lamininand N-cadherin, were treated with the PKC inhibitorsas described above. Laminin, an extracellular matrixmolecule that binds to integrin receptors, induces ro-bust outgrowth within 24 h of plating (Cohen et al.,1986). Cultures were treated with PMA for the first dayafter plating, and then the PMA-containing mediumwas washed out and replaced with normal medium.This washout procedure resulted in a slight overalldelay in neurite outgrowth for both experimental andcontrol cultures. After approximately 18 h of recoverytime, the cultures were photographed. Under these con-ditions, PMA-induced inhibition of PKC resulted in asmall decrease in length (10% decrease) and in density(28% decrease) in neurite outgrowth on laminin (Figs.6A, 6B, 8A, and 8B; Table 1). The isoform-specific PKCinhibitors Go6976 and rottlerin were also added to ret-inal neurite outgrowth cultures. Treatment with theseinhibitors had no effect on laminin-induced neurite out-growth (Figs. 6C, 6F, 8A, and 8B; Table 1). Therefore, weconcluded that PKC� is not involved in laminin-in-duced retinal neurite outgrowth.
The PKC inhibitors were also added to a neurite
FIG. 4. PKC inhibitors block neurite outgrowth on a PTP� substrate. Neural retina explants from E8 chick embryos were cultured on a PTP�substrate in the presence of DMSO (A, C, E) or 20 nM PMA (B), 7 nM Go6976 (D), or 0.5�M rottlerin (F). The explant tissue is on the left of eachpanel. The neurites in each dish were examined using dark-field optics at 3–4 days after plating. Long-term treatment with PMA is abroad-spectrum inhibitor of PKC and blocks neurite outgrowth on a PTP� substrate. Treatment with Go6976 (inhibits PKC� and �) has no effecton PTP�-dependent neurite outgrowth, whereas treatment with rottlerin (inhibits PKC�) blocks PTP�-dependent neurite outgrowth. Bar, 500 �m.
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outgrowth assay on a N-cadherin substrate, a calcium-dependent cell–cell adhesion molecule that binds via ahomophilic mechanism (Takeichi, 1988) and is a potentstimulator of neurite outgrowth (Bixby and Zhang,1990). When cultures plated on a N-cadherin substratewere treated with PMA, neurite outgrowth was unaf-
fected by broad-spectrum PKC inhibition (Figs. 7A, 7B,8C, and 8D; Table 1). In addition, treatment with theisoform-specific PKC inhibitors resulted in a slight re-duction in neurite outgrowth (Figs. 7C–7F, 8C, and 8D;Table 1). Inhibition of PKC� and PKC� with Go6976caused a small reduction (19% decrease) in neurite
TABLE 1
Statistical Analysis of Neurite Outgrowth Assays with PKC Inhibitors
Substrate Treatment (n) Length (�m) � SEM P Density (pixels) � SEM P
FIG. 5. Quantitation of the effects of PKC inhibition on PTP�-dependent neurite outgrowth. E8 chick retina explants cultured on a PTP� substratewere treated with DMSO (black bars) or a PKC inhibitor (PMA, gray bars; Go6976, striped bars; rottlerin, hatched bars). Treatment with PMA(broad-spectrum inhibitor) and with rottlerin (PKC� inhibitor) caused a statistically significant (asterisk) reduction in neurite density (A) and length(B), but treatment with Go6976 (PKC� and � inhibitor) had no effect on neurite density or length. For statistical analysis, see Table 1.
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length and a slight reduction in neurite density that wasnot significant. Rottlerin, the PKC� inhibitor, causedsmall reductions in both overall neurite density (36%decrease) and neurite length (27% decrease) comparedto DMSO-treated controls. These results suggest that aPKC� signaling pathway may play a minor role inN-cadherin-mediated neurite outgrowth. Together, theresults of the PKC inhibitor studies clearly indicate arequirement for PKC� activity in PTP�-dependent neu-rite outgrowth.
DISCUSSION
PTP� is capable of promoting neurite outgrowth ofretinal ganglion cell neurons (Burden-Gulley andBrady-Kalnay, 1999). The ability of PTP� to bind toRACK1 in yeast and mammalian cells suggests thatPTP�-dependent signaling may involve RACK1 (Mour-ton et al., 2001) and perhaps the other RACK1 interact-ing proteins such as the PKCs. To determine whetherPTP� forms a signaling complex with RACK1 andPKCs, we examined whether PTP� interacts with PKCsand RACK1 in E8 chick retina using immunoprecipita-tion. Immunoprecipitates of PTP� contained RACK1and PKC�. A complex of PTP�, RACK1, and PKC� wasalso observed in E6 RNE cells and the interaction was
dependent upon cell–cell contact. The density depen-dence of the complex formation suggests that cell–cellbinding stimulates the complex to form which may beinvolved in the propagation of signals. A workingmodel of neurite outgrowth is that PTP� on the culturedish (which mimics PTP� on the neuronal cell surface)binds to PTP� on the growth cone and this bindingstimulates the formation of the PTP�–RACK1–PKC�signaling complex (see Fig. 9).
To determine whether PKCs, which bind to RACK1in their activated state, are involved in PTP�-dependentsignaling, we examined the ability of various PKC in-hibitors to affect neurite outgrowth on a PTP� sub-strate. Long-term treatment with the phorbol esterPMA inhibits a broad spectrum of PKC isoforms andcaused a large reduction in neurite outgrowth on aPTP� substrate. To determine which isoform of PKCwas mediating this effect, isoform-specific inhibitorswere added to neurite outgrowth assays. Inhibition ofthe classical PKC isoforms � and � with Go6976 had noeffect on PTP�-dependent neurite outgrowth. How-ever, treatment with rottlerin, which inhibits PKC� re-sulted in a large reduction in neurite outgrowth. To-gether, these results suggest that PKC� activity may beregulated by PTP� signaling to promote neurite out-growth.
A role for PKC in neurite outgrowth has been previ-
FIG. 6. Long-term PMA treatment has a subtle effect on laminin-induced neurite outgrowth. Neural retina explants from E8 chick embryoswere cultured on a laminin substrate in the presence of DMSO (A, C, E) or 20 nM PMA (B), 7 nM Go6976 (D), or 0.5 �M rottlerin (F). The explantsare on the left of each panel. The neurites in each dish were examined using dark-field optics at 42 h (A, B) or 24 h (C–F) after plating. Long-termtreatment with PMA is a broad-spectrum inhibitor of PKC and causes a small reduction in neurite outgrowth on a laminin substrate. Treatmentwith Go6976 (inhibits PKC� and �) or rottlerin (inhibits PKC�) has no effect on Laminin-induced neurite outgrowth. Bar, 750 �m.
299PKC� Regulates Neurite Outgrowth on PTP�
ously examined. Several groups have used phorbol es-ters as well as nonspecific protein kinase and PKC-specific inhibitors to study the role of PKC. They haveexamined both neuronal-like cell lines and primaryneuronal cultures with seemingly contradictory results,depending on cell type, culture conditions, the stage ofdevelopment used, and other variables. Treatment withphorbol ester inhibited neurite outgrowth from mouseneuroblastoma cells (Ishii et al., 1978; Herrick-Davis etal., 1991), chick sensory ganglia (Ishii, 1978), and ratsympathetic ganglia (Campenot et al., 1991). Con-versely, phorbol ester treatment stimulated neurite for-mation from human neuroblastoma cells (Spinelli et al.,1982), chick ciliary ganglia (Bixby, 1989), and PC12 cells(O’Driscoll et al., 1995). Interestingly, neurite outgrowthfrom rat cerebellar neurons is inhibited by high doses ofphorbol esters, whereas it is stimulated by low doses(Cambray-Deakin et al., 1990). Very recently, Kabir andcolleagues (2001) have reported that stimulation of PKCby phorbol ester treatment of aplysia neurons resultedin the advance of microtubules into the peripheral re-gion of the growth cone.
In our study, we examined the role of PKCs in neuriteoutgrowth of embryonic chick retinal cells cultured onthe adhesion molecules PTP�, laminin, and N-cadherin.We observed a large reduction in neurite outgrowth ona PTP� substrate with prolonged treatment of PMA,compared to a small reduction on a laminin substrateand no effect on N-cadherin-dependent neurite out-growth. Heacock and Agranoff (1997) observed that
protein kinase inhibitors including prolonged phorbolester treatment and PKC-specific inhibitors were capa-ble of inhibiting neurite outgrowth of regeneratingadult goldfish retinal cells. Using PKC isoform-specificinhibitors, we found that inhibition of PKC� mimickedthe effect observed with the broad-spectrum inhibitionof PKC on a PTP� substrate. A role for PKC� in neuriteoutgrowth appears to be unique to PTP�-dependentneurite outgrowth, since this specific inhibitor had noeffect on a laminin substrate and only a subtle effect ona N-cadherin substrate.
A role for PKC has been previously shown for neu-rons growing on laminin (Bixby, 1989) and N-cadherin(Bixby and Jhabvala, 1990) in other cell types. In ciliaryganglion neurons, short-term phorbol ester treatment(6–8 h) resulted in a potentiation of neurite outgrowthon suboptimal laminin concentrations (Bixby, 1989) andon a N-cadherin substrate (Bixby and Jhabvala, 1990).Additionally, the broad-spectrum PKC inhibitor H7 in-hibited neurite outgrowth on laminin (Bixby, 1989).Treatment with H7 resulted in potentiation of the initialgrowth response on N-cadherin (8 h) but inhibition ofneurite outgrowth by 16 h (Bixby and Jhabvala, 1990).Together with our results from retinal ganglion cells, itappears that the PKC pathway is regulated by specificadhesion molecules, perhaps in a PKC isoform-specificmanner. No previous data are available from the stud-ies mentioned above regarding the PKC isoforms in-volved in laminin or N-cadherin-dependent neuriteoutgrowth, but our study suggests that under these
FIG. 7. PKC inhibitors have little effect on N-cadherin-dependent neurite outgrowth. Neural retina explants from E8 chick embryos werecultured on a N-cadherin substrate in the presence of DMSO (A, C, E) or 20 nM PMA (B), 7 nM Go6976 (D), or 0.5 �M rottlerin (F). The explantsare on the left of each panel. The neurites in each dish were examined using dark-field optics at 48 h after plating. Long-term treatment withPMA is a broad-spectrum inhibitor of PKC and has no effect on N-cadherin-dependent neurite outgrowth. Treatment with Go6976 (inhibits PKC� and �) and rottlerin (inhibits PKC�) caused a slight reduction in N-cadherin-dependent neurite outgrowth. Bar, 750 �m.
300 Rosdahl, Mourton, and Brady-Kalnay
conditions, PKC� activity is not required for retinalganglion cell neurite outgrowth on laminin or N-cad-herin.
Our neurite outgrowth results suggest a role forPKC� in retinal ganglion cell outgrowth on PTP�. Os-borne and colleagues (1992, 1994) have examined thelocalization of several PKC isoforms in the retinas ofseveral species including chick. PKC�, PKC�, and PKCwere expressed in the inner segments of the photore-ceptor cells of adult chick retina. Interestingly, closeinspection of the photomicrographs suggests that lowlevels of PKC� were also localized to the ganglion cell
layer (Osborne et al., 1994). Our studies were performedin embryonic chick retina. Our immunocytochemicaldata confirm that PTP�, RACK1, and PKC� are allcoexpressed in retinal ganglion cells (Fig. 3). The PKCfamily plays a number of roles in the retina includingdopamine release, modulation of glutamate receptorsand GABAC receptor function, involvement in retinalischemia, cytoskeletal regulation, and inositol phos-phate signaling (Wood et al., 1997). We provide evi-dence that PKC� is involved in PTP�-dependent neu-rite outgrowth of retinal ganglion cells.
Both N-cadherin and PTP� are homophilic binding
FIG. 8. Quantitation of the effects of PKC inhibition on laminin and N-cadherin-dependent neurite outgrowth. E8 chick retina explants culturedon a laminin (A, B) or N-cadherin (C, D) substrate were treated with DMSO (black bars) or a PKC inhibitor (PMA, gray bars; Go6976, stripedbars; rottlerin, hatched bars) for 24 or 42 h. Treatment with PMA (broad-spectrum inhibitor) caused a small but statistically significant reductionin laminin-induced neurite density (A) and length (B). Treatment with Go6976 (PKC� and � inhibitor) and rottlerin (inhibits PKC�) had no effecton laminin-induced neurite density (A) or length (B). Treatment with PMA had no effect on neurite density (C) or length (D) on N-cadherin.Treatment with rottlerin caused a statistically significant decrease (asterisk) in both neurite density (C) and length (D), and treatment withGo6876 caused a statistically significant decrease (asterisk) in neurite length (D). For statistical analysis, see Table 1.
301PKC� Regulates Neurite Outgrowth on PTP�
proteins (Takeichi, 1988; Brady-Kalnay et al., 1993).These proteins bind to themselves in trans; i.e., PTP�binds to PTP� on two apposing cells. PTP� and N-cadherin are known to bind to one another in cis (withinthe plane of the membrane) via their cytoplasmic do-mains (Brady-Kalnay et al., 1995, 1998). We have previ-ously shown that PTP� is required for N-cadherin-mediated neurite outgrowth (Burden-Gulley andBrady-Kalnay, 1999). We have further demonstratedthat the catalytic activity of PTP� is required for N-cadherin-dependent neurite outgrowth (Burden-Gulleyand Brady-Kalnay, 1999). N-cadherin homophilic bind-ing may generate a specific signal within the neuronalcell body, axon, or growth cone (Fig. 9, illustrated assignal X). The N-cadherin/PTP� cis interaction mayinvolve another downstream signaling pathway (Fig. 9,illustrated as signal Y). We have no evidence to datethat N-cadherin homophilic binding activates PTP� cat-alytic activity or PTP�-dependent signaling cascadesthat involve RACK1. We would argue from our datathat the N-cadherin homophilic binding signal does notrequire PKC�. Based upon our results, we hypothesizethat PTP� homophilic binding generates signals thatare likely to involve RACK1 and PKC� (Fig. 9). PTP�may regulate PKC� via its interaction with RACK1.Since PTP� coimmunoprecipates with RACK1 in di-
verse cell types (chick retina, shown here and MvLucells, a mink lung cell line, Mourton et al., 2001), theinteraction between PTP� and RACK1 may be a com-mon mechanism for membrane localization of RACK1and signaling complex formation.
A model for PTP�-dependent signaling is beginningto emerge. PKC is activated at the cell membrane bydiacylyglycerol and phospholipid (reviewed in Ronand Kazanietz, 1999). Activated PKC binds to RACK1,translocates to the plasma membrane, and phosphory-lates key substrates. Several PKC enzymatic substratesare necessary for neurite outgrowth, including GAP-43,CAP-23, and MARCKS (Aigner and Caroni, 1993; Meiriet al., 1998; Laux et al., 2000; Frey et al., 2000). PTP�could regulate PKC by several possible mechanisms.First, PTP� may serve to localize RACK1 and PKC (viaits RACK1 association) to a particular site in the plasmamembrane where cytoskeletal changes will occur topromote neurite outgrowth. A change in subcellularlocalization may affect substrate availability of PKC andtherefore its downstream signaling. It is also possiblethat PKC is an enzymatic substrate of PTP�, since PKC�is phosphorylated on tyrosine residues (Gschwendt,1999). In addition, since src can alter the association ofPTP� with RACK1 (Mourton et al., 2001), PTP� mayaffect the phosphotyrosine levels of PKC� indirectly by
FIG. 9. A model of PTP� function in neurite outgrowth. PTP� on the surface of migrating axons binds to PTP� on the surface of neighboringaxons and other cells. PTP�–PTP� binding initiates a signaling cascade that includes recruitment of RACK1 and PKC�. PTP�/PTP�-dependentsignaling is likely to be distinct from N-cadherin/N-cadherin-dependent signaling (signal X) although PTP� catalytic activity is required forN-cadherin-dependent neurite outgrowth (signal Y).
302 Rosdahl, Mourton, and Brady-Kalnay
displacing src from RACK1. RACK1 itself can be ty-rosine phosphorylated and the interaction betweenRACK1 and src is mediated by phosphotyrosines in thesixth WD repeat (Chang et al., 2001). RACK1 could be asubstrate for PTP� or PTP� could modulate protein–protein interactions between PKC, src, and RACK1.
The RPTPs are unique molecules combining adhesionand signaling functions. The signaling functions ofthese molecules have been difficult to study due to thehigh degree of in vitro promiscuity. The present studyhas demonstrated that PTP� exists in a signaling com-plex with the scaffolding protein RACK1 and PKC�. Wehave determined that the PKC� isoform is required forneurite outgrowth on a PTP� substrate in chick retinalganglion cells. In conclusion these results suggest thatPKC� activity is required for PTP�-dependent signaltransduction to promote neurite outgrowth.
EXPERIMENTAL METHODS
Materials
Fertilized eggs were purchased from SPAFAS (Pres-ton, CT). RPMI 1640 medium, laminin, and l-glutaminewere obtained from Gibco-BRL (Grand Island, NY).Fetal bovine serum was purchased from Summit Bio-technology (Ft. Collins, CO). Black nitrocellulose filterswere purchased from Vanguard International, Inc.(Neptune, NJ). The trypsin/EDTA solution used to dis-sociate the retina was purchased from Mediatech Cell-gro (Herndon, VA). Chemical inhibitors PMA, rottlerin,and Go6976 were purchased from Calbiochem (San Di-ego, CA). Antibodies were obtained from the followingsources: PKC�, �, �, and and RACK1 monoclonalantibodies, from Transduction Laboratories (Lexington,KY); PKC�, �, and � polyclonal antibodies, from SantaCruz Biotechnology, Inc. (Santa Cruz, CA). ProteinA–Sepharose was purchased from Pharmacia Biotech(Piscataway, NJ) and goat anti-mouse IgG (or IgM)–Sepharose were purchased from Zymed (South SanFrancisco, CA). Flurophore-conjugated secondary anti-bodies and SlowFadeLight were purchased from Mo-lecular Probes (Eugene, OR). Tween 20 was obtainedfrom Fisher (Pittsburgh, PA). All other reagents wereobtained from Sigma (St. Louis, MO).
Methods
Retinal neuroepithelial cultures. Retinal neuroepi-thelial cultures were prepared from E6 retinas using theprocedure described in BurdenGulley and Brady-Kal-
nay (1999). Briefly, retinas were incubated in 0.25%trypsin and 0.1% EDTA for 20 min at 37°C. The retinaswere dissociated by trituration and resuspended inRPMI 1640 with 10% tryptose phosphate broth, 4% fetalbovine serum, 1% chick serum, 2 mM l-glutamine, 2units/ml penicillin, 2 �g/ml streptomycin, 5 ng/mlamphotericin. The cells were cultured for 2–4 days at37°C in 95% air/5% CO2.
Analysis of PTP�, PKC isoforms, and RACK1 inretina. E8 retinas were dissected and transferred tolysis buffer (20 mM Tris, pH 7.6, 1% Triton X-100, 5 mMEDTA, 1 mM benzamidine, 200 �M phenyl arsine ox-ide, 1 mM vanadate, 0.1 mM ammonium molybdate,and 2 �l/ml protease inhibitor cocktail). After homog-enization, the tissue lysates were incubated on ice for 30min. Triton-insoluble material was removed by centrif-ugation at 14,000 rpm, and the lysate was incubated insample buffer at 95°C and separated by 10% or 12%SDS–PAGE. Proteins were transferred to nitrocellulosemembrane and immunoblotted as described (Brady-Kalnay et al., 1993).
Retinal neuroepithelial cultures were scraped in coldlysis buffer (20 mM Tris, pH 7.6, 1% Triton X-100, 1 mMbenzamidine, 200 �M phenyl arsine oxide, 1 mM van-adate, 0.1 mM ammonium molybdate, and 2 �l/mlprotease inhibitor cocktail) and incubated on ice for 30min. Lysate preparation proceeded as described abovefor E8 retinal lysates.
Analysis of PTP�, RACK1, and PKC� in neurites.E8 retinal explant cultures growing on laminin were fixedwith 4% paraformaldehyde, 0.01% glutaraldehyde inPEM buffer (80 mM Pipes, 5 mM EGTA, 1 mM MgCl2, 3%sucrose) for 1 h and then rinsed with phosphate-bufferedsaline (PBS) and incubated in block buffer (20% goat se-rum, 1% phosphate buffered saline (BSA), 1% saponin inPBS). Cultures were incubated in primary antibodies di-luted in block buffer overnight at 4°C. Cultures wererinsed extensively with TNT buffer (0.1 M Tris, 0.15 MNaCl, 0.05% Tween 20) and then incubated in secondary,fluorophore-conjugated antibodies diluted in block bufferfor 90 min at room temperature. After extensive rinsingwith TNT buffer, the cultures were coverslipped withSlowFadeLight antifading reagent and photographed at100� on an inverted fluorescent microscope (Carl Zeiss,Inc.). Images were acquired using MetaMorph software(Universal Imaging Corp.).
Immunoprecipitations. Antibodies to PTP� or RACK1were incubated with protein A–Sepharose or goat anti-mouse IgG (or IgM) conjugated to Sepharose for 2 h atroom temperature and then washed three times with PBS(9.5 mM phosphate, 137 mM NaCl, pH 7.5) before addi-tion to cell lysates. Purified monoclonal antibodies were
303PKC� Regulates Neurite Outgrowth on PTP�
used at 0.6 mg of IgG per milliliter of beads, ascites fluidwas used at 1 mg of IgG per milliliter of beads, andpolyclonal serum was used at 3 mg of IgG per milliliter ofbeads. Immunoprecipitates were prepared from 250 �g ofthe Triton-soluble lysate of cells. The immunoprecipitateswere incubated overnight at 4°C on a rocker, centrifugedat 3000g for 1 min. The supernatant was removed and thebeads were separated then washed four times in lysisbuffer and the bound material was eluted by addition of100 �l of 2� sample buffer and heated for 5 min at 95°C.One-fifth of the immunoprecipitate (20 �l) was loaded perlane of the gel, and the proteins were separated by 10%(for analysis of RACK1 and PKC) and 6% (for analysis ofPTP�) SDS–PAGE and transferred to nitrocellulose forimmunoblotting.
Neurite outgrowth assays. PTP� was purified fromadult rat brains as described (Burden-Gulley andBrady-Kalnay, 1999). N-cadherin was purified from em-bryonic chick brains as described (Bixby and Zhang,1990). Briefly, 35-mm tissue culture dishes were coatedwith nitrocellulose in methanol (Lagenaur and Lem-mon, 1987) and allowed to dry. Protein (2–4 �g) wasspread across the center of the dishes, and they wereincubated 20–30 min at room temperature. Remainingbinding sites on the nitrocellulose were blocked with2% BSA in calcium-, magnesium-free Hanks’ bufferedsaline (CMF), and the dishes were rinsed with RPMI1640 medium.
Embryonic day 8 (stage 32.5–33 according to Ham-burger and Hamilton, 1951) chick eyes were dissected andthe retina was flattened with the photoreceptor side downonto black nitrocellulose filters (0.45 �m pore size) thathad previously been incubated in 0.1% concanavalin A toaid in attachment of the retina to the filter. The filter wasthen cut into 350-�m-wide strips perpendicular to theoptic fissure using a McIlwain tissue chopper. Strips wereinverted onto substrate-coated culture dishes so that theganglion cell layer was directly adjacent to the substra-tum. Cells were cultured in RPMI 1640 medium with 10%fetal bovine serum, 2% chick serum, 2 mM l-glutamine, 2units/ml penicillin, 2 �g/ml streptomycin, 5 ng/ml am-photericin. For pharmacologic inhibition studies, PKCchemical inhibitors (final concentrations 7 nM Go6976, 0.5�M rottlerin, or 20 nM PMA) were added to the culturesand they were incubated at 37°C in 95% air/5% CO2. Forthe PMA experiments, after 18–20 h the medium wasexchanged for normal medium without inhibitors. For theGo6976 and rottlerin experiments, the inhibitors remainedin the culture for the entire culture period. Treatment withrottlerin at the IC50 (3–6 �M) was toxic to the neurons;however, treatment with 0.5 �M rottlerin was not cyto-toxic. Go6976 was used at its IC50 concentration. Cultures
were examined at approximately 24 and 42 h after platingfor laminin and 48 h N-cadherin substrates and at approx-imately 72 and 96 h for cultures on a PTP� substrate.Neurite outgrowth from each explant was photographedon a Nikon inverted microscope.
We used an established method for quantitation ofneurite outgrowth in control and perturbation condi-tions (Drazba and Lemmon, 1990). Measurement ofboth length and density provides two distinct means toassess the overall growth (density is, in part, a measureof length also since it takes into account all neurites onthe dish). All measurements were done at intervals thatallowed us to examine the most robust neurite out-growth on each substrate. To quantify the neurite out-growth, the 35-mm negatives were scanned and thedigitized images were analyzed using the Metamorphimage analysis program (Universal Imaging Corp.,West Chester, PA). Lengths of the five longest neuritesper explant were measured perpendicular to the ex-plant tissue. To measure the number of neurites perexplant, the region of neurite outgrowth was outlined,the neurites were highlighted using the threshold func-tion, and the total number of highlighted pixels wascalculated. The neurite length and density measure-ments were analyzed by Fisher’s PLSD, Scheffe, andStudent’s t test (Statview 4.51, Abacus Concepts, Inc.).The data from all like experiments were combined andgraphed (SigmaPlot 2000, SPSS Inc.).
ACKNOWLEDGMENTS
The authors acknowledge the expert advice and technical assis-tance of Sonya Ensslen and Dr. Susan Burden-Gulley. We also thankDr. Burden-Gulley for her critical reading of the manuscript. Thecontributions of Dr. Vance Lemmon are also greatly appreciated. Thiswork was supported by the National Institutes of Health Grant 1RO1-EY12251 to S.B.-K. J.R. was also supported by the Visual SciencesTraining Grant (T32-EY07157). Additional support was provided bythe Visual Sciences Research Center Core Grant from the NationalEye Institute (PO-EY11373).
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