Atypical PKC (aPKC) plays a role in establishing cell polarity and has been indicated in neuronal differentiation and polarization, including neurite formation in rat pheochromocytoma PC12 cells, albeit by unclear mechanisms. Here, the role of the aPKC isoform, PKC iota (PKCι), in the early neuronal differentiation of PC12 cells , was investigated. NGF-treated PC12 cells with stably ex pressed exogenous wild-type PKCι showed decreased expression of a neuroendocrine marker, in creased expression of a neuronal marker and increased neurite formation. Stable expression of a kinase-inactive PKCι, but not constitutively active PKCι lacking a regulatory domain, had similar though less potent effects. Pharmacological inhibition of endogenous aPKC kinase activity in pa rental PC12 cells did not inhibit neurite formation, suggesting that some of the observed effects of PKCι expression on neuronal differentiation are kinase-independent. Interestingly, exogenous expression of wild-type and kinase-inactive PKCι had little effect on overall PKCι activity, but caused a decrease in PKC zeta (PKCζ) kinase activity, suggesting an interplay between the two isoforms that may underlie the observed results. Overall, these findings suggest that in PC12 and perhaps other neuroendocrine precursor cells, PKCι influences an early differentiation decision between the neuroendocrine (chromaffin) and sympathetic neuron cell lineages, potentially by affecting PKCζ function.
Protein kinase C (PKC) is a family of kinases that are involved in regulation of target proteins through the phosphorylation of their serine and/or threonine amino acid residues. PKCs are conserved among eukaryotes and play important roles in several signal transduction cascades. The PKC family consists of at least ten isozymes that are divided into three subfamilies based on their structure and activation mechanisms: conventional, novel and atypical [
There are two isoforms of aPKC, PKC iota (PKCι, named PKCλ in mice) and PKC zeta (PKCζ). aPKC is important for maintaining polarity in cells [
Toward further examining the roles of aPKC in early neuronal differentiation, we have employed the rat PC12 pheochromocytoma cell line as a model system. PC12 cells represent a cell type with both neuroendocrine and neuronal differentiation potential, as they both secrete catecholamines and upon NGF treatment, form neurites indicative of early neuronal differentiation [
PC12 and 293T cell lines were obtained from the American Type Culture Collection. PC12 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum, and 1% penicillin-streptomycin (100 U/ml and 10 μg/ml, respectively).
Plasmids directing expression of HA-tagged wild-type (WT) and constitutively active catalytic domain (CAT) and kinase-inactive (KI) mutants of PKCι [
PC12 cell lines grown on 60-mm culture dishes until confluent were rinsed with PBS and then lysed by incubating with 150 μl of lysis buffer (50 mM HEPES (pH 7.6), 250 mM NaCl, 0.5% Nonidet P-40, 0.5% Triton X-100, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na2VO3 and 2 μg/ml each of aprotinin, bestatin, and leupeptin) at 4˚C for 30 minutes. Lysed cells were scraped with a plastic scraper, resuspended by pipetting, collected, and were spun down in a refrigerated microcentrifuge for 15 minutes to remove all insoluble material. The supernatant was collected and a Bradford protein assay (Bio-Rad, Hercules, CA) was performed to determine protein concentrations. Equal amounts of protein for each well of a gel, ranging from 25 - 50 µg among the different blots performed, were mixed with an equivalent volume of 2× SDS buffer and were separated by SDS-PAGE. The separated proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane overnight at 30 volts for 16 hours and western blotting was then performed as described [
Mouse monoclonal antibodies: Anti-β1 integrin antibody from BD Biosciences (Franklin Lakes, NJ) and anti-α- tubulin from Sigma (St. Louis, MO) were used at a 1:1000 dilution in western blots. Rabbit polyclonal antibodies: Anti-HA antibody from Santa Cruz Biotechnology (Santa Cruz, CA) was used at a 1:200 dilution in western blots. Anti-PKCι, anti-PKCζ, anti-Tyrosine Hydroxylase and anti-MAP2 antibodies from Cell Signaling Technology (Danvers, MA), anti-PAR-6B from Sigma (St. Louis, MO) and anti-PAR-3 and anti-phosphoserine from EMD Millipore (Billerica, MA) were used at 1:1000 and 1:500 dilutions, respectively in western blots. Secondary antibodies were anti-mouse IgG-HRP and anti-rabbit IgG-HRP (Sigma, St. Louis, MO) used at a 1:2286 dilution.
Cells grown to confluence in 100-mm culture dishes were rinsed with PBS and lysed as described for western blot lysate preparation. For each HA immunoprecipitation, 25 µl of monoclonal HA-agarose beads (Sigma, St. Louis, MO) was used. Beads were washed 3 times with PBS and once with lysis buffer, resuspended in lysis buffer and incubated with 500 µg of lysate for 3 hours with rotation at 4˚C. Immune complexes were washed four times with lysis buffer and eluted by incubating with 30 μl of 2× SDS gel loading buffer, which was then heated to 55˚C for 3 minutes. 10 μl of the eluted immunoprecipitates was loaded on one SDS page gel (for HA western blotting), whereas the remaining 20 μl was loaded onto another gel (for PAR-6B and PAR-3 western blotting). Proteins were separated by SDS-PAGE and visualized by Western blotting. Quantification of bands was performed from scans of the western blots using Image J (version 1.42q). PAR-3 immunoprecipitations were performed similarly except that lysates were supplemented with 0.5% sodium deoxycholate and incubated with 1.5 µl of PAR-3 antibody for 1 hour. 20 µl of Protein G Sepharose 4 Fast Flow (GE Healthcare Biosciences, Pittsburgh, PA) was added incubated for another hour. Immune complexes were washed as above, except that 40 µl of 2× SDS gel loading buffer was used, which were then divided into 2 aliquots to be loaded into different lanes.
Wells from a 6-well plate were covered with 0.5 ml of a 20 µg/ml solution of laminin (Sigma), which was allowed to dry. Each cell line was plated in triplicate wells at an initial density of 1 × 104 cells per well and allowed to attach for 24 hours. Each cell line was then incubated for a period of 48 hours in serum-free DMEM that was supplemented with 100 ng/ml of NGF (nerve growth factor, Becton Dickenson). Images of these cell lines at 48 hours post-treatment with NGF were captured using a Zeiss Axiovert S100 inverted microscope equipped with a Nikon digital camera with NIS-elements F 2.20 digital-imaging software. In all analyses, neurites that were counted were neuron-like extensions of the cell membrane that were equal to or longer than twice the diameter of the cell. Student’s t-tests were performed to determine statistical significances with respect to control cells. To analyze neurite outgrowth after PMA treatment, cells were treated as previously described, except they were allowed to adhere for 3 - 5 hours and then treated with phorbol 12-myristate-13-acetate (PMA, 1 µM) for two days prior to NGF treatment. The PMA was removed, and the cells were then treated with NGF (100 ng/ml) in serum-free media and imaged after 48 hours, as previously. To analyze the effects of PKC inhibitors on neurite outgrowth, parental PC12 cells were treated as previously described, except media containing serum was used during the NGF incubation. The PKC inhibitor, Gö6983 (200 nM), was added to certain wells at the same time as NGF, and the cells were imaged after 48 hours, as previously.
PC12 cell lines grown to near-confluence on laminin-coated 100-mm culture dishes were incubated with NGF (100 ng/ml) in serum-free media for 1 day and subjected to either PKCι or PKCζ immunoprecipitation. Briefly, cells were lysed as described above and 500 µg of each lysate was incubated at 4˚C with 2 µg of either antiPKCι or PKCζ antibody for 1 hour with rotation. Immune complexes were captured by incubating with 20 µl of pre-rinsed Protein G Sepharose 4 Fast Flow (GE Healthcare Biosciences, Pittsburgh, PA) for 1 hour at 4˚C with rotation. The Sepharose beads with immunoprecipitated PKCι or PKCζ were then washed four times with lysis buffer and incubated with 25 µl of PKC reaction mixture from the PepTag Non-Radioactive Protein Kinase C Assay (Promega, Madison WI). The assay was carried out as directed by the manufacturer, except that 20 µl of the reaction was separated on a 0.6% agarose gel to resolve the fluorescent phosphorylated and unphosphorylated PKC substrates. Images were captured on a FOTO/Analyst Investigator gel documentation workstation (Fotodyne, Hartland, WI) and band intensities were quantified using Image J software (version 1.47). After the reaction mixture was removed and separated, the beads and the remaining 5 µl of reaction mixture were incubated with 30 μl of 2× SDS gel loading buffer, which was then heated to 55˚C for 3 minutes, separated by SDSPAGE, and subjected to PKCζ western blotting.
PC12 cell lines stably expressing exogenous HA-tagged versions of either wild-type (WT) PKCι or kinase-inactive (KI) and constitutively active catalytic domain (CAT) mutants of PKCι were created. To test for proper expression of these proteins, an HA western blot was performed (
the PKCι in these cell lines, a western blot was performed using a PKCι antibody that detects both endogenous and exogenous PKCι (
The PC12 cell lines were analyzed for levels of neuroendocrine and neuronal markers. Decreased levels of tyrosine hydroxylase, the rate-limiting enzyme for catecholamine production and marker for the catecholaminergic phenotype [
To determine whether PKCι WT affects the ability of PC12 cells to form neurites upon NGF exposure [
Because differences in the NGF-mediated neurite outgrowth were observed with the PKCι WT and PKCι KI cell lines, the effect of expression of these proteins on neuronal and catecholaminergic markers was readdressed, focusing on changes that occur after NGF exposure. Thus, the cell lines were treated with NGF for 5 days and western blots were performed (
maintenance [
Previous studies have reported that aPKC is necessary for neurite outgrowth in PC12 cells [
To further assess the role of aPKC function in neuritogenesis, neurite outgrowth assays were also performed on the parental PC12 cells in the presence of a PKC inhibitor (
inhibited by the 200 nM Gö6983 treatment (
Since aPKC kinase activity was seemingly dispensable for increased neurite and neuronal differentiation, we sought to identify non-kinase functions of aPKC that may play a role in some of the observed phenomena. aPKC proteins are known to participate in multiprotein complexes and can influence the activities and localization of the constituents of these complexes. One important member of such multiprotein aPKC complexes is the protein, PAR-6 [
In order to determine whether expression of exogenous PKCι WT and KI have an effect on the kinase activity of endogenous PKCι that may underlie some of the observed effects on neuronal differentiation, a nonradioactive PKC assay was utilized. Both endogenous and exogenous PKCι were immunoprecipitated from our set of PC12 cell lines (which were grown on laminin and treated with NGF) and PKC kinase activity in the immune complexes was determined, as has been done by others [
In order to see whether expression of PKCι affected the kinase activity of the other aPKC isoform, PKCζ, in these cells, the experiment was repeated, except PKCζ was immunoprecipitated from the cells and assayed as above. Surprisingly, the PKC kinase activity of immunoprecipitated PKCζ was greatly diminished in both PKCι WT and PKCι KI cell lines (Figures 7(c) and (d)), with PKCι WT showing the greatest effect. Since the decrease in overall PKCζ activity mirrors the cell lines that had increased pro-neuronal effects, it is likely that this decrease in PKCζ activity plays a role in the increase in neurite formation and neuronal characteristics observed with the PKCι WT and KI cell lines.
In this paper, the role of PKCι in PC12 differentiation was investigated by expressing wild-type PKCι and kinase-inactive and constitutively active (catalytic domain) mutants of PKCι in PC12 cells. Expression of wildtype PKCι and kinase-inactive PKCι suppressed neuroendocrine characteristics of PC12 cells and promoted neuronal differentiation in a manner that partly did not depend on PKCι kinase activity. However, expression of these proteins did negatively affect PKCζ kinase activity in NGF-treated cells. The present findings suggest that expression of PKCι can influence the choice of differentiation of PC12 cells between the neuroendocrine (chromaffin) and sympathetic neuron lineages, potentially through effects on PKCζ.
It is curious that in our assays with NGF present, PKCι KI showed a somewhat similar ability as PKCι WT to increase neuronal characteristics in the cells in which it was expressed. These results cannot be attributed to gross overexpression of these proteins since there was a lack of increase in overall PKCι levels in these cell lines (in
It is notable that in the absence of NGF treatment, only the PKCι WT cell line showed a decrease of neuroendocrine function, as assayed by tyrosine hydroxylase levels. Also, although in NGF-treated PC12 cells expression of PKCι KI led to a more neuronal phenotype, it was much less efficient than PKCι WT at down-regulating neuroendocrine characteristics (i.e., tyrosine hydroxylase, as seen in
Since aPKC activities in neuronal differentiation may rely on its ability to interact with key effectors of cytoskeleton dynamics and morphogenesis, we attempted to determine whether there was some difference in these interactions that could account for our observations, focusing on subsets of the PKCι/PAR-6/PAR-3/Cdc42 complexes [
The aPKC/PAR-6 complex has been reported to bind to the activated form of Cdc42 [
It is intriguing that expression of PKCι CAT, which is constitutively active and did not bind to PAR-6 or PAR-3, did have any effect in the assays used here. It was noted that levels of PKCι CAT were lower than PKCι WT and KI, however it was expected that its constitutively active status would compensate for its lower abundance, which did not occur. It is likely that PAR-6 and PAR-3 binding plays a central role in aPKC functions in cell determination, such that expression of the aPKC catalytic domain is insufficient for its neuronal-promoting functions. Along those lines, it is interesting that the greatest pro-neuronal effects in the majority of assays here were observed with the PKCι WT cell line, which did have greatest amount of expression. This may suggest that the endogenous PKCι protein is somehow different from the exogenous protein in some manner (although overall cellular PKCι kinase activity is unaffected) such that levels of the exogenous protein are the key factor mediating these results. We do not preclude the notion that the HA epitope tag on the C-terminal renders the PKCι protein defective in some way that does not impinge on overall PKCι kinase activity. Given that the C-terminus of aPKC has been shown to have key interactions that affect its function [
The supposition that the exogenous PKCι WT and KI are defective in some manner does not provide a simple explanation for the negative effect of PKCι WT and KI on PKCζ kinase activity. A search of the literature did not find any reported instances of PKCι expression affecting PKCζ function. Moreover, the two isoforms are considered functionally equivalent, except for a reported inhibitor that affects PKCι and not PKCζ [
We thank Dr. Jae-Won Soh (Inha University, Korea) for generously providing vectors directing expression of HA-tagged wild-type and mutant forms of both PKC iota. We also thank Dr. Benjamin Weeks (Adelphi University) for discussion and for guiding us in the use of PC12 cells and neurite outgrowth assays.
Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health [award number R15CA121992].