Of the atypical PKCs, Par-4 and p62: recent understandings of the biology and pathology of a PB1-dominated complex.

The recent identification of a novel protein-protein interaction module, termed PB1, in critical signaling molecules such as p62 (also known as sequestosome1), the atypical PKCs, and Par-6, has unveiled the existence of a new set of signaling complexes, which can be central to several biological processes from development to cancer. In this review, we will discuss the most recent advances on the role that the different components of these complexes have in vivo and that are relevant to human disease. In particular, we will review what we are learning from new data from knockout mice, and the indications from human mutations on the real role of these proteins in the physiology and biology of human diseases. The role that PKCzeta, PKClambda/iota, and Par-4 have in lung and prostate cancer in vivo and in humans will be extensively covered in this article, as will the multifunctional role of p62 as a novel hub in cell signaling during cancer and inflammation, and the mechanistic details and controversial data published on its potential role in aggregate formation and signaling. All this published information is shedding new light on the proposed pathological implications of these PB1-regulators in disease and shows their important role in cell physiology.

The role of ubiquitin in neurotrophin receptor signalling and sorting.

NGF (nerve growth factor) binding to TrkA (tropomyosin receptor kinase A) induces dimerization, autophosphorylation and internalization of the receptor to signalling vesicles for delivery of differentiation signals. TrkA interacts with p75 receptor through the p62-TRAF-6 (tumour-necrosis-factor-receptor-associated factor 6) complex bridging the two receptors. The atypical protein kinase C is activated and recruited to the receptor complex as well. TrkA is Lys63-polyubiquitinated on Lys485 by the E3 (ubiquitin ligase), TRAF-6, and E2 (ubiquitin-conjugating enzyme), UbcH7. Inhibition of polyubiquitination has been observed to interrupt signalling and internalization. Furthermore, an absence of p62 prevents endosomal localization and signalling. Altogether, these findings reveal Lys63-linked polyubiquitin chains and the shuttling protein p62 co-ordinately regulate TrkA internalization, trafficking and sorting.

Nerve growth factor stimulates multisite tyrosine phosphorylation and activation of the atypical protein kinase C's via a src kinase pathway.

Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated differentiation of PC12 cells. In the present study, we report that PKC-iota becomes tyrosine phosphorylated in the membrane coincident with activation posttreatment with nerve growth factor. Tyrosine phosphorylation and activation of PKC-iota were inhibited in a dose-dependent manner by both PP2 and K252a, src and TrkA kinase inhibitors. Purified src was observed to phosphorylate and activate PKC-iota in vitro. In PC12 cells deficient in src kinase activity, both NGF-induced tyrosine phosphorylation and activation of PKC-iota were also diminished. Furthermore, we demonstrate activation of src by NGF along with formation of a signal complex including the TrkA receptor, src, and PKC-iota. Recruitment of PKC-iota into the complex was dependent on the tyrosine phosphorylation state of PKC-iota. The association of src and PKC-iota was constitutive but was enhanced by NGF treatment, with the src homology 3 domain interacting with a PXXP sequence within the regulatory domain of PKC-iota (amino acids 98 to 114). Altogether, these findings support a role for src in regulation of PKC-iota. Tyrosine 256, 271, and 325 were identified as major sites phosphorylated by src in the catalytic domain. Y256F and Y271F mutations did not alter src-induced activation of PKC-iota, whereas the Y325F mutation significantly reduced src-induced activation of PKC-iota. The functional relevance of these mutations was tested by determining the ability of each mutant to support TRAF6 activation of NF-kappaB, with significant impairment by the Y325F PKC-iota mutant. Moreover, when the Y352F mutant was expressed in PC12 cells, NGF's ability to promote survival in serum-free media was reduced. In summary, we have identified a novel mechanism for NGF-induced activation of atypical PKC involving tyrosine phosphorylation by c-Src.

Nerve growth factor stimulates the interaction of ZIP/p62 with atypical protein kinase C and targets endosomal localization: evidence for regulation of nerve growth factor-induced differentiation.

Atypical protein kinase Cs zeta and lambda/iota play a functional role in the regulation of NGF-induced differentiation and survival of pheochromocytoma, PC12 cells [Coleman and Wooten, 1994; Wooten et al., 1999]. Here we demonstrate an NGF-dependent interaction of aPKC with its binding protein, ZIP/p62. Although, ZIP/p62 was not a PKC-iota substrate, the formation of a ZIP/p62-aPKC complex in PC12 cells by NGF occurred post activation of PKC-iota and was regulated by the tyrosine phosphorylation state of aPKC. Furthermore, NGF-dependent localization of ZIP/p62 was observed within vesicular structures, identified as late endosomes by colocalization with a Rab7 antibody. Both ZIP/p62 as well as PKC-iota colocalized with Rab7 upon NGF stimulation. Inhibition of the tyrosine phosphorylation state of PKC-iota did not prevent movement of ZIP/p62 to the endosomal compartment. These observations indicate that the subcellular localization of ZIP/p62 does not depend entirely upon activation of aPKC itself. Of functional importance, transfection of an antisense p62 construct into PC12 cells significantly diminished NGF-induced neurite outgrowth. Collectively, these findings demonstrate that ZIP/p62 acts as a shuttling protein involved in routing activated aPKC to an endosomal compartment and is required for mediating NGF's biological properties.

The atypical protein kinase C-interacting protein p62 is a scaffold for NF-kappaB activation by nerve growth factor.

Nerve growth factor (NGF) binding to both p75 and TrkA neurotrophin receptors activates the transcription factor nuclear factor kappaB (NF-kappaB). Here we show that the atypical protein kinase C-interacting protein, p62, which binds TRAF6, selectively interacts with TrkA but not p75. In contrast, TRAF6 interacts with p75 but not TrkA. We demonstrate the formation of a TRAF6-p62 complex that serves as a bridge linking both p75 and TrkA signaling. Of functional relevance, transfection of antisense p62-enhanced p75-mediated cell death and diminished NGF-induced differentiation occur through a mechanism involving inhibition of IKK activity. These findings reveal a new function for p62 as a common platform for communication of both p75-TRAF6 and TrkA signals. Moreover, we demonstrated that p62 serves as a scaffold for activation of the NF-kappaB pathway, which mediates NGF survival and differentiation responses.

Protein kinase C iota protects neural cells against apoptosis induced by amyloid beta-peptide.

Protein kinase C (PKC) isoforms are increasingly recognized as playing important roles in the regulation of neuronal plasticity and survival. Recent findings from studies of non-neuronal cells suggest that atypical isoforms of PKC can modulate apoptosis in various paradigms. Because increasing data support a role for neuronal apoptosis in the pathogenesis of Alzheimer's disease (AD), we tested the hypothesis that PKCiota (PKCiota) can modify vulnerability of neural cells to apoptosis induced by amyloid beta-peptide (ABP), a cytotoxic peptide linked to neuronal degeneration in AD. Overexpression of PKCiota increased the resistance of PC12 cells to apoptosis induced by ABP. Associated with the increased resistance to apoptosis were improved mitochondrial function and reduced activity of caspases. In addition, ABP-induced increases in levels of oxidative stress and intracellular calcium levels were attenuated in cells overexpressing PKCiota. These findings suggest that PKCiota prevents apoptosis induced by ABP by interrupting the cell death process at a very early step, thereby allowing the cells to maintain ion homeostasis and mitochondrial function.

Mapping of atypical protein kinase C within the nerve growth factor signaling cascade: relationship to differentiation and survival of PC12 cells.

The pathway by which atypical protein kinase C (aPKC) contributes to nerve growth factor (NGF) signaling is poorly understood. We previously reported that in PC12 cells NGF-induced activation of mitogen-activated protein kinase (MAPK) occurs independently of classical and nonclassical PKC isoforms, whereas aPKC isoforms were shown to be required for NGF-induced differentiation. NGF-induced activation of PKC-iota was observed to be dependent on phosphatidylinositol 3-kinase (PI3K) and led to coassociation of PKC-iota with Ras and Src. Expression of dominant negative mutants of either Src (DN2) or Ras (Asn-17) impaired activation of PKC-iota by NGF. At the level of Raf-1, neither PKC-iota nor PI3 kinase was required for activation; however, PKC-iota could weakly activate MEK. Inhibitors of PKC-iota activity and PI3K had no effect on NGF-induced MAPK or p38 activation but reduced NGF-stimulated c-Jun N-terminal kinase activity. Src, PI3K, and PKC-iota were likewise required for NGF-induced NF-kappaB activation and cell survival, whereas Ras was not required for either survival or NF-kappaB activation but was required for differentiation. IKK existed as a complex with PKC-iota, Src and IkappaB. Consistent with a role for Src in regulating NF-kappaB activation, an absence of Src activity impaired recruitment of PKC-iota into an IKK complex and markedly impaired NGF-induced translocation of p65/NF-kappaB to the nucleus. These findings reveal that in PC12 cells, aPKCs comprise a molecular switch to regulate differentiation and survival responses coupled downstream to NF-kappaB. On the basis of these findings, Src emerges as a critical upstream regulator of both PKC-iota and the NF-kappaB pathway.

Purification and characterization of a novel protamine kinase in HL60 cells.

A protamine kinase from HL60 cells was purified to near homogeneity by DEAE-Sephacel, protamine-agarose, Hydroxylapatite, and S-200 chromatography. It was purified by 75.8-fold through four chromatographic steps, and 0.67% of total activity was recovered. The purified enzyme had an apparent molecular mass of 120 kDa and was activated by Mg(2+) or Mn(2+), but inhibited by Ca(2+). Neither phospholipid nor phorbol ester significantly affected the enzyme activity. Staurosporine was the most potent inhibitor of the enzyme among the protein kinase inhibitors tested, K(252a), H(7), heparin, and staurosporine. The purified protamine kinase exhibited a maximum velocity of 5,000 pmol/min/mg and K(m) of 1.3 mM for protamine sulfate as a substrate. Myelin basic protein and protamine sulfate served as the best substrates for the protamine kinase among those tested. The activity of the protamine kinase remained unchanged upon treatment with PMA, retinoic acid, dimethyl sulfoxide, or 1,25 dihydroxy vitamin D(3) for 15 min, while treatment with a differentiating agent, 1,25 dihydroxy vitamin D(3), for one week increased its activity. These results suggest that protamine kinase in HL60 cells is involved in the late stage of the macrophage-monocytic differentiation pathway and may play a role in maintenance of the differentiation after HL60 cells are committed.

Function for NF-kB in neuronal survival: regulation by atypical protein kinase C.

Activation of the transcription factor nuclear factor kappa B (NF-kB) has been intensely studied in the past several years due to its role as an inducible regulator of inflammation, apoptosis, transformation, and oncogenesis. Recently, increasing evidence supports a role for NF-kB in regulation of anti-apoptotic gene expression and promotion of cell survival (May and Ghosh [1999] Science 284:272-273). Studies in the past 5 years have provided evidence that NF-kB regulates neuronal survival as well. Moreover, atypical protein kinase (aPKC) has been shown to play a novel role in modulating the NF-kB pathway. In this review, I focus on neurons and the factors that contribute to regulation of NF-kB via aPKC.

Identification of Src as a novel atypical protein kinase C-interacting protein.

Atypical protein kinase C-zeta (PKC-zeta) participates in nerve growth factor (NGF) signaling and is required for NGF-induced differentiation of PC12 cells. The biological activity of PKC-zeta is likely mediated by interaction of PKC-zeta with specific proteins. Affinity column chromatography employing the PKC-zeta regulatory domain coupled to glutathione-agarose was used to search for proteins that bound PKC-zeta. Two proteins (59/60 kDa) were recovered from NGF-stimulated PC12 cell lysates that bound the matrix. Western blot analysis of pooled column fractions identified these proteins as tubulin and src, respectively. Using purified preparations of src and tubulin, PKC-zeta was shown to interact with both proteins using blot overlay. To demonstrate a functional interaction in vivo, PC12 cells expressing a temperature-sensitive v-src were shifted to the permissive temperature (37 degrees C), followed by immunoprecipitation. At the permissive temperature where src was active, PKC-zeta was tyrosine phosphorylated and coassociated with src in vivo; by comparison, at the nonpermissive temperature (40 degrees C) PKC-zeta was not tyrosine phosphorylated. Taken together, these findings support a novel role for the interaction of src and atypical PKC in vivo, which is dependent upon the activity of src and the tyrosine phosphorylation state of PKC-zeta.

Overexpression of atypical PKC in PC12 cells enhances NGF-responsiveness and survival through an NF-kappaB dependent pathway.

Removal of atypical PKC blocks NGF-induced differentiation of PC12 cells.1 We now examine the consequences that overexpression of atypical PKCs had upon NGF responses. PC12 cells were stably transfected with either PKC-iota or PKC-zeta. Overexpression of atypical PKCs markedly enhanced NGF- induced neurite outgrowth as well as enhanced NGF-stimulated JNK kinase. Cotransfection of HA-JNK1 along with increasing concentrations of PKC-iota, resulted in dose-dependent phosphorylation of GST c-Jun (1 - 79). NGF treatment of PC12 cells resulted in activation of NF-kappaB. In comparison, overexpression of atypical PKC-iota was by itself sufficient to activate NF-kappaB and shift the kinetics of NGF-induced kappaB activity. Furthermore, transfection of full-length antisense PKC-iota blocked basal and NGF-stimulated NF-kappaB. Differentiated and undifferentiated PC12 cells overexpressing atypical PKC-iota were protected from serum deprivation-induced cell death. Collectively, these findings demonstrate that atypical PKC-iota lies in a pathway that regulates NF-kappaB and contributes to both neurotrophin-mediated differentiation and survival signaling.

Atypical PKC zeta is activated by ceramide, resulting in coactivation of NF-kappaB/JNK kinase and cell survival.

Both protein kinase C (PKC) and ceramide play a critical role in cell signaling, but the relationship between PKC and ceramide is unclear. Low concentrations of ceramide were observed to transiently stimulate PKC zeta activity in vitro and in vivo, whereas high doses of ceramide lead to inhibition of PKC zeta. Inhibition of activity was accompanied by enhanced binding of the negative regulator, Par4 to PKC zeta. Treatment of PC12 cells with low doses of ceramide promoted survival in serum-free media and activation of nuclear factor-KB, whereas higher doses (>2.5 microM) resulted in cell death. Overexpression of either aPKC isoform, PKC zeta or iota, resulted in enhanced survival of PC12 cells at high doses of ceramide and in ceramide-stimulated Jun N-terminal kinase (JNK), without any apparent effect on mitogen-activated kinase. These findings support a role for ceramide-induced PKC zeta activity in the control of cell survival signaling via a pathway that also activates JNK kinase.

Delta-protein kinase C phosphorylation parallels inhibition of nerve growth factor-induced differentiation independent of changes in Trk A and MAP kinase signalling in PC12 cells.

We investigated the ability of bryostatin 1 to block nerve growth factor (NGF)-induced differentiation of pheochromocytoma PC12 cells and to effect expression of protein kinase C (PKC) isoforms. Compared with phorbol myristate acetate (PMA), a likewise potent activator of PKC, high doses of bryostatin (> 200 nM) failed to down-regulate delta-PKC, as with zeta-PKC, whereas, alpha-PKC was completely down-regulated. Two forms of delta-PKC were expressed in PC12 cells, a phosphorylated 78.000 M(r) species and a de-phosphorylated 76.000 M(r) form. High-dose bryostatin treatment resulted in a 4.5-fold increase in phosphorylated delta-PKC and a 2.5-fold increase in phosphotyrosine. Inhibition of tyrosine kinase activity, with either herbimycin or genistein, prior to addition of bryostatin abrogated protection from down-regulation and led to simultaneous increases in ubiquitinated 110.000 M(r)-delta-PKC. Similarly, pre-treatment of cells with N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal, an inhibitor of the proteasome pathway, prior to low-dose treatment with bryostatin resulted in a dose-dependent accumulation of delta-PKC and inhibition of down-regulation. Protection of delta-PKC from down-regulation by high-dose bryostatin requires a counter-balance between protein tyrosine kinase and phosphatase systems. High doses of bryostatin blocked NGF-induced neurite outgrowth without altering Y-490 TrK A phosphorylation or an alteration in pp44/42 mitogen-activated protein kinase. Our findings suggest that the phosphorylation state of delta-PKC may regulate its ability to participate in signal coupling and modulation of cell growth and differentiation pathways. Moreover, these data reveal the existence of a signalling pathway independent of MAP kinase that affects NGF differentiation in a negative fashion.

Transport of protein kinase C isoforms to the nucleus of PC12 cells by nerve growth factor: association of atypical zeta-PKC with the nuclear matrix.

In an effort to understand the role of protein kinase C (PKC) in nerve growth factor-induced differentiation, we studied the expression of PKC using isoform-specific antibodies. Western blot analysis on whole cell lysates showed that alpha,beta,gamma,epsilon,zeta, iota/lambda and mu were expressed in PC12 cells, except for theta which was absent. In nuclei obtained from control PC12 cells, small amounts of delta, epsilon, iota/lambda and zeta were detected. A computer-assisted search algorithm was used to search for the presence of bipartite nuclear targeting motifs. In classical PKC isoforms alpha,beta,gamma, two bipartite motifs were present, while atypical iota/lambda and zeta-PKC displayed one motif, whereas novel PKC isoforms did not exhibit any bipartite motif structure. Treatment of cells with differentiating doses of nerve growth factor (NGF) resulted in changes of differential magnitude for all of the nuclear PKC isoforms in response to NGF. However, little change in gamma-PKC was observed in response to NGF. This analysis indicated that other factors may contribute to transport of PKC into the nucleus, in addition to the bipartite motif itself. Atypical zeta-PKC is required for NGF-induced neurite outgrowth of PC12 cells (Coleman and Wooten: J Mol Neurosci 5:39-57, 1994). Increases in nuclear zeta-PKC were NGF dose-dependant with a concomitant decrease in cytoplasmic immunoreactivity. The localization of zeta-PKC was investigated by means of immunoelectron microscopy which revealed the localization of this isoform within the inner nuclear matrix bound to chromatin. Taken together, these findings suggest that zeta-PKC may be involved in the regulation of nuclear processes.

Nucleolin is a protein kinase C-zeta substrate. Connection between cell surface signaling and nucleus in PC12 cells.

We have previously shown that protein kinase C (PKC)-zeta is activated and required for nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma PC12 cells (Wooten, M. W., Zhou, G., Seibenhener, M. L., and Coleman, E. S. (1994) Cell Growth & Diff. 5, 395-403; Coleman, E. S., and Wooten, M. W. (1994) J. Mol. Neurosci. 5, 39-57). Here we report the characterization and identification of a 106-kDa nuclear protein as a specific substrate of PKC-zeta. NGF treatment of PC12 cells resulted in translocation of PKC-zeta and coincident phosphorylation of a protein that was localized within the nucleoplasm of nuclei isolated from PC12 cells. Addition of PKC-zeta pseudosubstrate peptide in vitro or myristoylated peptide in vivo diminished phosphorylation of pp106 in a dose-dependent fashion. Likewise, addition of purified PKC-zeta, but neither PKC-alpha nor delta, to nuclear extracts resulted in an incremental increase in the phosphorylation of pp106. Expression of dominant-negative PKC-zeta inhibited NGF-induced phosphorylation of pp106, by comparison overexpression of PKC-zeta enhanced basal phosphorylation without a noticeable effect upon NGF-induced effects. Amino acid sequence analysis of four peptides derived from purified pp106 revealed that this protein was homologous to nucleolin. Using an in vitro reconstitution system, purified nucleolin was likewise shown to be phosphorylated by purified PKC-zeta. The staining intensity of both enzyme and substrate in the nucleus increased upon treatment with NGF. In vivo labeling with 32Pi and stimulation of PC12 cells with NGF followed by immunoprecipitation with anti-nucleolin antibody corroborated the in vitro approach documenting enhanced phosphorylation of nucleolin by NGF treatment. Taken together, the findings presented herein document that nucleolin is a target of PKC-zeta that serves to relay NGF signals from cell surface to nucleus in PC12 cells.

Modulation of zeta-protein kinase C by cyclic AMP in PC12 cells occurs through phosphorylation by protein kinase A.

Although cyclic AMP (cAMP) has been reported to cross talk with the protein kinase C (PKC) system, effects of elevated intracellular cAMP on the activities of specific PKC isoforms have not been studied. We report findings from a permeabilized cell assay that was used to examine changes in the activity of the atypical PKC isoforms brought about by exposure of PC12 cells to agents that elevate intracellular cAMP. We found that increases in intracellular cAMP led to rapid stimulation of atypical PKC activity, 40-70% above control, for a sustained period of time, a response that occurred independent of the phorbol 12-myristate 13-acetate (PMA)-sensitive PKC isoforms. Changes in intracellular cAMP levels resulted in a dose-dependent redistribution of zeta-PKC to the cytoplasm with a concomitant increase in the phosphorylation state of the enzyme. Incubation of purified zeta-PKC with increasing concentrations of PKA likewise caused a twofold increase in the phosphorylation state of zeta-PKC. In contrast to the positive effect that PKA-mediated phosphorylation had on the activity of zeta-PKC, the enzyme displayed reduced binding to ras when phosphorylated. Taken together, these findings are consistent with the hypothesis that protein phosphorylation of PKC acts as a positive effector of its enzyme activity and may serve as a negative modulator for interaction with other proteins.

Atypical zeta-protein kinase c displays a unique developmental expression pattern in rat brain.

The expression of atypical zeta-protein kinase C (PKC) was examined during prenatal and postnatal rat brain development. Immunoblot as well as transcript analysis revealed a dramatic increase in expression at 2-3 days post-birth, which declined thereafter and remained at levels observed in the adult brain. The expression of zeta-PKC precedes that of the other PKC isoforms in developing rat brain. Subcellular fractionation of pup and adult brain documented distribution between all three distinct fractions (A,B,C), including the low speed pellet composed of nuclei. In adult brain, the kinase was enriched in the A fraction of the sucrose gradient. Specific substrate proteins of zeta-PKC were characterized in each of the subcellular fractions from both pup and adult brain. Four predominant proteins pp76, pp60-doublet, pp54 and pp45 were identified as zeta-PKC endogenous substrates. All four proteins were phosphorylated on serine residues, while the pp60-doublet was also phosphorylated on tyrosine. The pp60-doublet was the most predominant substrate, specifically enriched in the A fraction of a sucrose gradient of adult brain and immunoprecipitated by monoclonal antibody to pp60c-src.

A role for zeta protein kinase C in nerve growth factor-induced differentiation of PC12 cells.

Studies were undertaken to compare the signal-induced redistribution of conventional protein kinase C (cPKC) to nonclassical protein kinase C (nPKC) family members in response to phorbol 12-myristate 13-acetate (PMA) or nerve growth factor (NGF) treatment of PC12 cells. cPKC-alpha and -beta and nPKC-delta and -epsilon were predominantly cytoplasmic, whereas PKC-zeta displayed approximately equal distribution between the cytoplasm and membrane fraction. Treatment of PC12 cells with PMA induced rapid translocation of both c- and nPKC isoforms to the membrane fraction, although the kinetics varied between isoforms with epsilon being most sensitive, followed by delta > zeta > alpha. Both PKC-epsilon and delta translocated in the presence of minute concentrations of PMA, whereas cPKC was less sensitive, and PKC-zeta was least sensitive. NGF treatment, on the other hand, induced translocation of cPKCs and delta and epsilon nPKC, albeit with differential magnitude, whereas PKC-zeta was found predominantly in the cytoplasm. Chronic treatment of PC12 cells with PMA (1 microM) caused a rapid disappearance of alpha, beta, delta, and epsilon PKC isoforms, whereas the expression of PKC-zeta was unaltered over 4 days. NGF induced an increase in cytoplasmic PKC-zeta in control, or PMA down-regulated PC12 cells. Moreover, the increase in cytoplasmic PKC-zeta was blocked by pretreatment with sphingosine (2.5 microM). Furthermore, PKC-zeta was activated by NGF in PMA down-regulated PC12 cells, as determined by the extent of epsilon-peptide phosphorylation using a permeabilized cell assay. In addition, the zeta-pseudosubstrate peptide inhibited NGF-induced activation of PKC-zeta.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve growth factor-induced differentiation of PC12 cells employs the PMA-insensitive protein kinase C-zeta isoform.

To elucidate the role of protein kinase C (PKC) in nerve growth factor (NGF)-induced differentiation, PMA downregulation of pheochromocytoma (PC12) cells was undertaken. Prolonged treatment (2 d) of PC12 cells with PMA (1 microM) resulted in depleting the cells of alpha, beta, delta, and epsilon-PKC isoforms, but had no effect on the expression of the atypical PKC isoform zeta. PC12 cells, which expressed only PKC zeta, were evaluated for their responses to NGF. Removal of the PMA-sensitive PKC isoforms enhanced the ability of NGF to promote neurite extension. Both the percentage cells with neurites and length of neurites were increased in the PMA-treated cells, whereas no effect was observed on the number of neurites per cell or branching of individual neurites. In addition, PMA downregulation resulted in an increase in the incorporation of 3H-thymidine without any significant effect on the expression of c-fos. Addition of NGF to PC12 cells depleted of the PMA-sensitive PKC isoforms resulted in the activation of PKC zeta (Wooten et al., 1994). To test whether the transient activation of PKC zeta is a necessary component of the neuritogenetic pathway, antisense oligonucleotide strategy was utilized to remove this particular PKC isoform. The addition of a 20-bp antisense oligonucleotide directed against the 5' coding sequence of PKC zeta attenuated NGF-induced neurite outgrowth in PC12 cells lacking PMA-sensitive PKC isoforms. Sense oligonucleotide directed at the same site was without effect on NGF responses. These data indicate that PKC zeta comprises a portion of the NGF pathway and underscores the importance of this isoform in neuronal differentiation. Moreover, these findings demonstrate that the PMA-insensitive pathway, which was previously characterized as PKC-independent, and the neurite induction pathway are synonymous and mediated by PKC zeta.