L523S, an RNA-binding protein as a potential therapeutic target for lung cancer.

Approaches to vaccine-based immunotherapy of human cancer may ultimately require targets that are both tumour-specific and immunogenic. In order to generate specific antitumour immune responses to lung cancer, we have sought lung cancer-specific proteins that can be targeted for adjuvant vaccine therapy. By using a combination of cDNA subtraction and microarray analysis, we previously reported the identification of an RNA-binding protein within the KOC family, L523S, to be overexpressed in squamous cell cancers of the lung. We show here that L523S exhibits significant potential for vaccine immunotherapy of lung cancer. As an oncofetal protein, L523S is normally expressed in early embryonic tissues, yet it is re-expressed in a high percentage of nonsmall cell lung carcinoma. The specificity of L523S expression in lung cancer was demonstrated by both mRNA and protein measurements using real-time PCR, Western blot, and immunohistochemistry analyses. Furthermore, we show that immunological tolerance of L523S is naturally broken in lung cancer patients, as evidenced by detectable antibody responses to recombinant L523S protein in eight of 17 lung pleural effusions from lung cancer patients. Collectively, our studies suggest that L523S may be an important marker of malignant progression in human lung cancer, and further suggest that treatment approaches based on L523S as an immunogenic target are worthy of pursuit.

P504S immunohistochemical detection in 405 prostatic specimens including 376 18-gauge needle biopsies.

P504S is a recently described, prostate cancer-specific gene that encodes a protein involved in the beta-oxidation of branched chain fatty acids. A recent study has shown that immunohistochemical detection of P504S gene product is a sensitive and specific marker of prostatic carcinoma in formalin-fixed, paraffin-embedded tissues. We performed a detailed analysis of P504S protein expression in a large series of prostate and bladder specimens with special emphasis on staining in specific morphologic patterns of prostatic adenocarcinoma, posthormonal and radiation therapy cases, and invasive urothelial carcinoma. A total of 366 prostate needle core biopsies from 124 patients with prostate cancer, 10 biopsies from 2 patients without prostate cancer, 28 prostatectomy specimens (16 with specific morphologic patterns, 7 posthormonal therapy and 5 postradiation therapy specimens), 5 bladder specimens with invasive urothelial carcinoma, and a single transurethral resection specimen from a patient with hormonally treated prostate cancer and invasive urothelial carcinoma were stained with P504S monoclonal antibody at a 1:250 dilution using standard heat-induced epitope retrieval and avidin-biotin technique. Extent (0, no staining; 1+, 1-10% staining; 2+, 11-50% staining; 3+, > or =51% staining) and location (luminal, subluminal, and diffuse cytoplasmic) of immunoreactivity in carcinoma and benign tissues were recorded. A total of 153 of 186 biopsies (82%) with prostatic adenocarcinoma stained for P504S. Pseudohyperplastic, atrophic, ductal, and mucinous prostatic carcinomas stained similarly, as did cases treated with hormone or radiotherapy. In 81 of 377 (21%) foci of benign prostatic tissue there was staining that was almost always focal, faint, and noncircumferential. Seminal vesicles did not stain for P504S. Five of six (83%) specimens with invasive urothelial carcinoma had 2+ staining and one case had focal staining. We conclude that immunohistochemistry for P504S has potential utility in the diagnosis of prostate cancer, including those treated by hormones and radiation. Circumferential luminal to subluminal and diffuse cytoplasmic staining is the most specific staining pattern for prostatic carcinoma and is almost never associated with benign prostatic tissue. However, a negative P504S immunostain does not automatically rule out prostate cancer, as 18% of cases were negative. Additionally, occasional benign glands, high-grade prostatic intraepithelial neoplasia, atypical adenomatous hyperplasia, and urothelial carcinoma may express P504S. Therefore, we think that P504S is best used only in conjunction with strict light microscopic correlation and preferably with high molecular weight cytokeratin immunostaining.

Detection of mammaglobin in the sera of patients with breast cancer.

Current procedures for the diagnosis of breast cancer are cumbersome and invasive, making detection of this disease difficult. A rapid screening test for early detection of breast cancer would allow for better management of this deadly disease. In this report, we show that, with the exception of the skin, mammaglobin mRNA is specifically expressed in mammary tissue and commonly overexpressed in breast cancer. Mammaglobin is not expressed in other types of cancer including colon, lung, ovarian, and prostate cancer. Breast-specific expression of mammaglobin protein was shown using immunohistochemical methods. Mammaglobin is secreted from both established breast cancer cell lines and primary breast carcinoma cells cultured in vitro. Using a monoclonal antibody-based assay for monitoring the presence of mammaglobin in serum, elevated levels of mammaglobin were detected in sera of patients with breast cancer, but not in healthy women. Thus, mammaglobin, which is overexpressed and secreted from breast carcinoma cells, is detectable in sera of patients with breast cancer and may provide a rapid screening test for the diagnosis and management of breast cancer.

L552S, an alternatively spliced isoform of XAGE-1, is over-expressed in lung adenocarcinoma.

Using a combination of cDNA subtraction and microarray analysis, we report here the identification and characterization of L552S, an over-expressed, alternatively spliced isoform of XAGE-1 in lung adenocarcinoma. Real-time RT-PCR analysis shows that L552S is expressed at levels greater than 10-fold in 12 of 25 lung adenocarcinoma tumors compared with the highest expression level found in all normal tissues tested. L552S is expressed in both early and late stages of lung adenocarcinoma, but it was not detected in large cell carcinoma, small cell carcinoma, or atypical lung neuroendocrine carcinoid. The full-length cDNA for L552S comprises 770 bp and encodes a polypeptide of 160 amino acids. C-terminal 94 amino acids of L552S are identical to a cancer testis antigen, XAGE-1, found in Ewing's sarcoma. Genomic sequence analysis has revealed that L552S and XAGE-1 are alternatively spliced isoforms, and expression of both L552S and XAGE-1 isoforms are present in lung adenocarcinoma. Immunohistochemistry analysis using affinity purified L552S polyclonal antibodies demonstrated specific nuclear staining in 10 of 12 lung adenocarcinoma samples. Furthermore, antibody responses to recombinant L552S protein were observed in seven of 17 lung pleural effusion fluids of lung cancer patients. These results strongly imply that L552S protein is immunogenic and suggest that it might have use as a vaccine target for lung cancer.

A p53 homologue and a novel serine proteinase inhibitor are over-expressed in lung squamous cell carcinoma.

LSCC is a common type of lung cancer and accounts for approximately 30% of all lung cancers. We have used a combination of subtraction and cDNA microarray technology to identify genes preferentially over-expressed in LSCC. Here we report extensive molecular characterization of two novel full-length cDNA sequences, L530S and L531S. Although L530S and L531S were found to be differentially over-expressed in LSCC, the expression profiles for these two genes were not identical. L530S expression was specifically elevated in LSCC whereas L531S transcript was up regulated in both LSCC and head and neck squamous cell carcinoma samples. L530S is a homologue of p53, and L531S belongs to a new member of serine proteinase inhibitors with significant homology to SCCA1 and SCCA2. Furthermore, L531S protein was found to be expressed in lung cancers by IHC analysis. The distinct as well as similar expression profiles exhibited by L530S and L531S suggest that each gene may play a unique role for tumorgenesis of LSCC. Identification of these genes not only allows us to further explore their diagnostic and therapeutic potentials for LSCC, but also provides us with additional tools and reagents for understanding the biology behind LSCC, and differentiating LSCC from other types of lung cancer at the molecular level.

P504S: a new molecular marker for the detection of prostate carcinoma.

The ability to diagnose prostate carcinoma would be improved by the detection of a tumor-associated antigen. P504S, a cytoplasmic protein, was recently identified by cDNA library subtraction in conjunction with high throughput microarray screening from prostate carcinoma. The aim of this study was to establish the pattern of expression of P504S in prostate carcinoma and benign prostatic tissue. A total of 207 cases, including 137 cases of prostate carcinoma and 70 cases of benign prostate, from prostatectomies (n = 77), prostate needle biopsies (n = 112), and transurethral prostate resections (n = 18) were examined by immunocytochemistry for P504S. P504S showed strong cytoplasmic granular staining in 100% of prostate carcinomas regardless of Gleason scores and diffuse (>75% of tumor) staining in 92% of cases. In contrast, 171 of 194 (88%) of benign prostates, including 56 of 67 (84%) benign prostate cases and 115 of 127 (91%) cases of benign glands adjacent to cancers were negative for P504S. The remainders of benign prostates were focally and weakly positive for P504S. The staining pattern of these normal glands was different and easily distinguishable from that observed in prostate carcinoma. Expression of P504S was not found in basal cell hyperplasia, urothelial cells/metaplasia and small atrophic glands that may mimic prostate carcinoma. Our findings indicate that P504S is a highly sensitive and specific positive marker for prostate carcinoma.

MEK kinase 1 gene disruption alters cell migration and c-Jun NH2-terminal kinase regulation but does not cause a measurable defect in NF-kappa B activation.

MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase that, in addition to regulating the c-Jun NH(2)-terminal kinase (JNK) pathway, is involved in the control of cell motility. MEKK1(-/-) mice are defective in eyelid closure, a TGFalpha-directed process involving the migration of epithelial cells. MEKK1 expression in epithelial cells stimulates lamellipodia formation, a process required for cell movement. In addition, mouse embryo fibroblasts derived from MEKK1(-/-) mice are inhibited in their migration relative to MEKK1(+/+) fibroblasts. MEKK1 is required for JNK but not NF-kappaB activation in response to virus infection, microtubule disruption, and stimulation of embryonic stem cells with lysophosphatidic acid. MEKK1 is not required for TNFalpha or IL-1 regulation of JNK or NF-kappaB activation in macrophages or fibroblasts. Thus, MEKK1 senses microtubule integrity, contributes to the regulation of fibroblast and epithelial cell migration, and is required for activation of JNK but not NF-kappaB in response to selected stress stimuli.

Live cell fluorescence imaging of T cell MEKK2: redistribution and activation in response to antigen stimulation of the T cell receptor.

T cell activation requires engagement of the T cell receptor (TCR) at the interface of conjugates formed with antigen-presenting cells. TCR engagement is accompanied by a redistribution of specific signaling molecules to the cytoplasmic region of the TCR complex. In this study, immunocytochemistry and live cell fluorescence imaging demonstrate that T cell MEK kinase 2 (MEKK2) is translocated to the T cell/antigen-presenting cell interface in response to antigen activation. MEKK2 translocation occurs more rapidly as the antigen concentration is increased. Biochemical activation of MEKK2 follows TCR stimulation, and expression of a dominant-negative MEKK2 inhibits TCR-mediated conjugate stabilization and ERK and p38 MAP kinase phosphorylation. Live cell fluorescence imaging thus enables characterization of signal transducers that are dynamically translocated following TCR engagement.

Regulation of the MAPK family members: role of subcellular localization and architectural organization.

The members of the mitogen-activated protein kinase (MAPK) family are regulated by a diverse array of extracellular cues ranging from cytokines, growth factors and neuropeptides, which activate cell surface receptors, to stresses such as cold, heat, osmolarity changes and irradiation. The MAPK pathways control genetic expression by modifying transcription factor activity and cue important cell fate decisions including survival, proliferation, and programmed cell death (apoptosis). One interesting feature of the MAPK pathways is that the components are evolutionarily conserved from yeast to human, and many of the pathways are similarly organized and regulated. Unlike previously imagined, architectural organization or the multimeric organization of signaling proteins into complexes which are localized to distinct subcellular regions is an important mechanism that influences the regulation of these pathways. In addition, extracellular stimuli can induce relocalization of specific signal transduction proteins. The formation of multimeric signaling complexes, as well as the dynamic movement of signaling proteins, contribute to determine signaling specificity and efficacy. This review describes what is currently known about the subcellular localization of MAPK pathway signaling proteins and the relocalization that occurs during events associated with activation of the MAPK family members.

MEK kinase 1 (MEKK1) transduces c-Jun NH2-terminal kinase activation in response to changes in the microtubule cytoskeleton.

Cell shape change and the restructuring of the cytoskeleton are important regulatory responses that influence the growth, differentiation, and commitment to apoptosis of different cell types. MEK kinase 1 (MEKK1) activates the c-Jun NH2-terminal kinase (JNK) pathway in response to exposure of cells to microtubule toxins, including taxol. MEKK1 expression is elevated 3-fold in mitosis and microtubule toxin-treated cells accumulated at G2/M of the cell cycle. Targeted disruption of MEKK1 expression in embryonic stem cells resulted in the loss of JNK activation and increased apoptosis in response to taxol. Targeted disruption of the MEK kinase 2 gene had no effect on activation of the JNK pathway in response to microtubule toxins demonstrating a specific role of MEKK1 in this response. Cytochalasin D-mediated disruption of actin fibers activates JNK and stimulates apoptosis similarly in MEKK1(-/-) and wild type cells. The results show that MEKK1 is required for JNK activation in response to microtubule but not actin fiber toxins in embryonic stem cells. MEKK1 activation can protect cells from apoptosis in response to change in the integrity of the microtubule cytoskeleton.

Signal transduction pathways regulated by arsenate and arsenite.

Arsenate and arsenite activate c-Jun N-terminal kinase (JNK), however, the mechanism by which this occurs is not known. By expressing inhibitory mutant small GTP-binding proteins, p21-activated kinase (PAK) and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinases (MEKKs), we have identified specific proteins that are involved in arsenate- and arsenite-mediated activation of JNK. We observe a distinct difference between arsenate and arsenite signaling, which demonstrates that arsenate and arsenite are capable of activating unique proteins. Both arsenate and arsenite activation of JNK requires Rac and Rho. Neither arsenate nor arsenite signaling was inhibited by a dominant-negative mutant of Cdc42 or Ras. Arsenite stimulation of JNK requires PAK, whereas arsenate-mediated activation of JNK was unaffected by inhibitory mutant PAK. Of the four MEKKs tested, only MEKK3 and MEKK4 are involved in arsenate-mediated activation of JNK. In contrast, arsenite-mediated JNK activation requires MEKK2, MEKK3 and MEKK4. These results better define the mechanisms by which arsenate and arsenite activate JNK and demonstrate differences in the regulation of signal transduction pathways by these inorganic arsenic species.

Role of MEKK1 in cell survival and activation of JNK and ERK pathways defined by targeted gene disruption.

Targeted disruption of the gene encoding MEK kinase 1 (MEKK1), a mitogen-activated protein kinase (MAPK) kinase kinase, defined its function in the regulation of MAPK pathways and cell survival. MEKK1(-/-) embryonic stem cells from mice had lost or altered responses of the c-Jun amino-terminal kinase (JNK) to microtubule disruption and cold stress but activated JNK normally in response to heat shock, anisomycin, and ultraviolet irradiation. Activation of JNK was lost and that of extracellular signal-regulated protein kinase (ERK) was diminished in response to hyperosmolarity and serum factors in MEKK1(-/-) cells. Loss of MEKK1 expression resulted in a greater apoptotic response of cells to hyperosmolarity and microtubule disruption. When activated by specific stresses that alter cell shape and the cytoskeleton, MEKK1 signals to protect cells from apoptosis.

The TAO of MEKK.

Cloning and characterization of MEKK1 in 1993 revealed that in addition to Raf there were other pathways activated by extracellular stimuli that were responsible for ERK activation. Since then, three additional MEKK family members have been cloned adding even further diversity to the regulation of MAPK pathways. The MEKK family members are regulated by a diverse array of extracellular stimuli ranging from growth factors to DNA damaging stimuli and so are important for the cell to sense exposure to various environmental stimuli. One important aspect of MEKK biology is that they can potentially serve in more than one pathway. Regulation of MEKK family members often involves LMWG proteins, phosphorylation and subcellular localization. With regard to at least MEKK1, serine/threonine kinases such as NIK, GLK and HPK1 appear also to be important for regulation. Of the MEKK family members, the biological role of MEKK1 is best characterized and studies have shown that MEKK1 is important in mediating survival vs. apoptosis, possibly via its ability to regulate transcription factors, the expression of death receptors and their ligands. The biological roles of MEKK2, 3 and 4 are under investigation and undoubtedly homologous deletion of these MEKK family members will be invaluable at determining the biological functions of these MEKKs. At present, the MEKK family members are characterized as localized sensors that control cell responses at the level of gene expression, metabolism and the cytoskeleton

Anti-apoptotic versus pro-apoptotic signal transduction: checkpoints and stop signs along the road to death.

The activation of caspases is a final commitment step for apoptosis. It is now evident that signal transduction pathways involving specific protein kinases modulate the apoptotic response. Both pro-apoptotic and anti-apoptotic pathways integrate environmental cues that control the decision to undergo apoptosis. Pro- and anti-apoptotic signal pathways regulate the activation of the caspases. In this review we describe our current understanding of apoptotic signal transduction.

14-3-3 proteins interact with specific MEK kinases.

MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) kinases (MEKKs) regulate c-Jun N-terminal kinase and extracellular response kinase pathways. The 14-3-3zeta and 14-3-3epsilon isoforms were isolated in a two-hybrid screen for proteins interacting with the N-terminal regulatory domain of MEKK3. 14-3-3 proteins bound both the N-terminal regulatory and C-terminal kinase domains of MEKK3. The binding affinity of 14-3-3 for the MEKK3 N terminus was 90 nM, demonstrating a high affinity interaction. 14-3-3 proteins also interacted with MEKK1 and MEKK2, but not MEKK4. Endogenous 14-3-3 protein and MEKK1 and MEKK2 were similarly distributed in the cell, consistent with their in vitro interactions. MEKK1 and 14-3-3 proteins colocalized using two-color digital confocal immunofluorescence. Binding of 14-3-3 proteins mapped to the N-terminal 393 residues of 196-kDa MEKK1. Unlike MEKK2 and MEKK3, the C-terminal kinase domain of MEKK1 demonstrated little or no ability to interact with 14-3-3 proteins. MEKK1, but not MEKK2, -3 or -4, is a caspase-3 substrate that when cleaved releases the kinase domain from the N-terminal regulatory domain. Functionally, caspase-3 cleavage of MEKK1 releases the kinase domain from the N-terminal 14-3-3-binding region, demonstrating that caspases can selectively alter protein kinase interactions with regulatory proteins. With regard to MEKK1, -2 and -3, 14-3-3 proteins do not appear to directly influence activity, but rather function as "scaffolds" for protein-protein interactions.

MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42.

MEK kinases (MEKKs) 1, 2, 3 and 4 are members of sequential kinase pathways that regulate MAP kinases including c-Jun NH2-terminal kinases (JNKs) and extracellular regulated kinases (ERKs). Confocal immunofluorescence microscopy of COS cells demonstrated differential MEKK subcellular localization: MEKK1 was nuclear and in post-Golgi vesicular-like structures; MEKK2 and 4 were localized to distinct Golgi-associated vesicles that were dispersed by brefeldin A. MEKK1 and 2 were activated by EGF, and kinase-inactive mutants of each MEKK partially inhibited EGF-stimulated JNK activity. Kinase-inactive MEKK1, but not MEKK2, 3 or 4, strongly inhibited EGF-stimulated ERK activity. In contrast to MEKK2 and 3, MEKK1 and 4 specifically associated with Rac and Cdc42 and kinase-inactive mutants blocked Rac/Cdc42 stimulation of JNK activity. Inhibitory mutants of MEKK1-4 did not affect p21-activated kinase (PAK) activation of JNK, indicating that the PAK-regulated JNK pathway is independent of MEKKs. Thus, in different cellular locations, specific MEKKs are required for the regulation of MAPK family members, and MEKK1 and 4 are involved in the regulation of JNK activation by Rac/Cdc42 independent of PAK. Differential MEKK subcellular distribution and interaction with small GTP-binding proteins provides a mechanism to regulate MAP kinase responses in localized regions of the cell and to different upstream stimuli.

Selective regulation of agrin mRNA induction and alternative splicing in PC12 cells by Ras-dependent actions of nerve growth factor.

The extracellular matrix protein agrin plays an important role in the formation and maintenance of the neuromuscular junction. However, regulation of agrin gene expression and pre-mRNA splicing, important in determining the biological actions of agrin, is not well understood. To begin to identify mechanisms controlling agrin expression, quantitative polymerase chain reaction techniques were used to analyze the effect of growth factors on the expression of agrin mRNA isoforms in rat pheochromocytoma (PC12) cells. Agrin transcripts in untreated cells lacked inserts in the Y and Z sites (agriny0z0), encoding agrin isoforms with low acetylcholine receptor aggregating activity and a primarily non-neuronal tissue distribution. Transcripts encoding isoforms with high aggregating activity and neuronal tissue distribution (agriny4z8, agriny4z11, and agriny4z19) were not detected. Treatment of PC12 cells with nerve growth factor (NGF) caused a significant increase in total agrin mRNA. In contrast, exposure to epidermal growth factor had no effect. Analysis of alternative splicing of agrin mRNA revealed that NGF elicited a specific increase in agriny4 and agrinz8 mRNAs that did not occur in the presence of epidermal growth factor, insulin, dexamethasone, or retinoic acid. Analysis of PC12 sublines stably overexpressing a dominant inhibitory form of p21 Ras indicated that NGF induced changes in levels of agrin mRNA and alternative splicing required Ras activity. The results show that NGF can influence important aspects of neuronal differentiation by regulating alternative splicing. Furthermore, these data provide insight into the mechanisms governing agrin gene expression and suggest that neurotrophic factors may play a role in regulating agrin expression in vivo.

Mast cell tumor necrosis factor alpha production is regulated by MEK kinases.

Mast cells synthesize and secrete specific cytokines and chemokines which play an important role in allergic inflammation. Aggregation of the high-affinity Fc receptor (FcepsilonRI) for immunoglobulin E (IgE) in MC/9 mouse mast cells stimulates the synthesis and secretion of tumor necrosis factor alpha (TNF-alpha). FcepsilonRI aggregation activates several sequential protein kinase pathways, leading to increased activity of extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), and the p38 mitogen-activated protein (MAP) kinase. Inhibition of ERKs with the compound PD 098059 had little effect on FcepsilonRI-stimulated TNF-alpha production. Aggregation of FcepsilonRI stimulated MEK kinase 1 (MEKK1) activity, which activates JNK kinase (JNKK), the kinase that phosphorylates and activates JNKs. Expression of activated MEKK1 (DeltaMEKK1) in MC/9 cells strongly stimulated JNK activity but only weakly stimulated p38 activity, and it induced a large activation of TNF-alpha promoter-regulated luciferase gene expression. Inhibitory mutant JNK2 expressed in MC/9 cells significantly blunted FcepsilonRI stimulation of TNF-alpha promoter-driven luciferase expression. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, diminished FcepsilonRI-mediated TNF-alpha synthesis, significantly blunted JNK activation and TNF-alpha promoter-driven luciferase expression, and only weakly inhibited p38 kinase activation. Inhibition of NFkappaB activation resulting from DeltaMEKK1 expression or FcepsilonRI stimulation did not affect TNF-alpha promoter-driven luciferase expression. Our findings define a MEKK-regulated JNK pathway activated by FcepsilonRI that regulates TNF-alpha production in mast cells.

MEKKs, GCKs, MLKs, PAKs, TAKs, and tpls: upstream regulators of the c-Jun amino-terminal kinases?

Regulation of the mitogen-activated protein kinase (MAPK) family members - which include the extracellular response kinases (ERKs), p38/HOG1, and the c-Jun amino-terminal kinases (JNKs) - plays a central role in mediating the effects of diverse stimuli encompassing cytokines, hormones, growth factors and stresses such as osmotic imbalance, heat shock, inhibition of protein synthesis and irradiation. A rapidly increasing number of kinases that activate the JNK pathways has been described recently, including the MAPK/ERK kinase kinases, p21-activated kinases, germinal center kinase, mixed lineage kinases, tumor progression locus 2, and TGF-beta-activated kinase. Thus, regulation of the JNK pathway provides an interesting example of how many different stimuli can converge into regulating pathways critical for the determination of cell fate.

Analysis of mutant platelet-derived growth factor receptors expressed in PC12 cells identifies signals governing sodium channel induction during neuronal differentiation.

The mechanisms governing neuronal differentiation, including the signals underlying the induction of voltage-dependent sodium (Na+) channel expression by neurotrophic factors, which occurs independent of Ras activity, are not well understood. Therefore, Na+ channel induction was analyzed in sublines of PC12 cells stably expressing platelet-derived growth factor (PDGF) beta receptors with mutations that eliminate activation of specific signalling molecules. Mutations eliminating activation of phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein (GAP), and Syp phosphatase failed to diminish the induction of type II Na+ channel alpha-subunit mRNA and functional Na+ channel expression by PDGF, as determined by RNase protection assays and whole-cell patch clamp recording. However, mutation of juxtamembrane tyrosines that bind members of the Src family of kinases upon receptor activation inhibited the induction of functional Na+ channels while leaving the induction of type II alpha-subunit mRNA intact. Mutation of juxtamembrane tyrosines in combination with mutations eliminating activation of PI3K, PLC gamma, GAP, and Syp abolished the induction of type II alpha-subunit mRNA, suggesting that at least partially redundant signaling mechanisms mediate this induction. The differential effects of the receptor mutations on Na+ channel expression did not reflect global changes in receptor signaling capabilities, as in all of the mutant receptors analyzed, the induction of c-fos and transin mRNAs still occurred. The results reveal an important role for the Src family in the induction of Na+ channel expression and highlight the multiplicity and combinatorial nature of the signaling mechanisms governing neuronal differentiation.