Mutation in the glutamate transporter EAAT1 causes episodic ataxia, hemiplegia, and seizures.

BACKGROUND: Transporters, ion pumps, and ion channels are membrane proteins that regulate selective permeability and maintain ionic gradients across cell membranes. Mutations in CACNA1A encoding a neuronal calcium channel and ATP1A2 encoding an ion pump cause episodic ataxia, hemiplegic migraine, and seizures. Mutant gene products of both CACNA1A and ATP1A2 may affect neurotransmission of glutamate, the most abundant excitatory amino acid neurotransmitter. METHODS: We examined our patient population with episodic ataxia and hemiplegic migraine but with no mutation in either CACNA1A or ATP1A2. We looked for mutations in SLC1A3, which encodes the glutamate transporter excitatory amino acid transporter (EAAT) 1 that is important in removing glutamate from the synaptic cleft. RESULTS: A patient with episodic ataxia, seizures, migraine, and alternating hemiplegia has a heterozygous mutation in SLC1A3 that is not present in his asymptomatic parents and controls. Expression studies of the mutant EAAT1 showed decreased expression of the protein with a markedly reduced capacity for glutamate uptake. When coexpressed, the mutant EAAT1 decreased the activity of wild-type EAAT1 but not of two other transporters EAAT2 or EAAT3, suggesting that mutant EAAT1 specifically multimerizes with wild-type EAAT1 to exert its dominant negative effect. CONCLUSION: Our data show that a heterozygous mutation in EAAT1 can lead to decreased glutamate uptake, which can contribute to neuronal hyperexcitability to cause seizures, hemiplegia, and episodic ataxia.

Transduction of human pancreatic tumor cells with vesicular stomatitis virus G-pseudotyped retroviral vectors containing a herpes simplex virus thymidine kinase mutant gene enhances bystander effects and sensitivity to ganciclovir.

We examined the suitability of Moloney murine leukemia virus (MLV) 4070A-, cat endogenous virus (CEV) RD114-, or vesicular stomatitis virus G (VSV-G)-pseudotyped retroviruses containing the humanized enhanced green fluorescent protein (hEGFP) or one of two herpes simplex virus thymidine kinase (HSV-TK) genes to transduce and provide gene expression in human pancreatic tumor cells. Fluorescence-activated cell sorter analysis demonstrated that VSV-G-pseudotyped hEGFP vector infected a greater percentage of cells and generated more robust gene expression than MLV 4070A- or CEV RD114-pseudotyped vectors. Dot blot and Southern blot analysis of genomic DNA revealed up to 10-fold more gene copies in G418-selected VSV-G hEGFP vector-transduced cells compared with genomic DNA from cells transduced with MLV 4070A or CEV RD114 pseudotypes. Cells transduced with VSV-G pseudotypes of HSV-TK(WT) or the HSV-TK30 vectors were 5- to 10-fold more sensitive to ganciclovir (GCV) than other pseudotype-transduced cells. A 40- to 61-fold difference in sensitivity to GCV was observed between cells transduced with VSV-G HSV-TK30 vector and cells transduced with MLV 4070A HSV-TK(WT) vector in vitro. A 13-fold reduction in tumor volume was observed in severe combined immunodeficient mice inoculated with PancTuITK30 cells compared with mice inoculated with PancTuITK(WT) cells during GCV treatment. We conclude that the choice of glycoprotein envelope and the potency of a particular suicide gene were therapeutically additive and increased the number of HSV-TK-positive cells and sensitivity toward GCV in human pancreatic tumors cells for prodrug gene therapy.

Wnt-1 inhibits nerve growth factor-induced differentiation of PC12 cells by preventing the induction of some but not all late-response genes.

The vertebrate Wnt-1 proto-oncogene is expressed transiently in embryonic brain and functions in the development of the central nervous system and neural crest. The role of Wnt-1 in neural crest development appears to be to increase the number of certain progenitor cells by preventing their premature differentiation. To study the mechanism by which this transient Wnt-1 expression inhibits differentiation we have constructed PC12 pheochromocytoma cells in which Wnt-1 expression levels were controlled by use of a tetracycline-responsive transactivator. Induction of Wnt-1 expression by tetracycline withdrawal was followed by activation of the Wnt-1 signalling pathway as shown by activation of the Lef-1/Tcf transcription factor. Wnt-1 expression by these cells resulted in reversible inhibition of NGF-induced neurite outgrowth, but it did not adversely affect the maintenance of previously formed NGF-induced neurites. Wnt-1 expression also partially blocked the ability of NGF to decrease the rate of cell multiplication. Wnt-1 decreased the NGF-induced expression of the late-response gene SCG10 but not of the immediate early genes, fos, Nur77 and UPAR (urokinase-type plasminogen activator receptor) nor of the late-response genes GAP-43 and collagenase. The Wnt-1 expressing PC12 cells multiplied at a greater rate when they expressed Wnt-1 than they did in the absence of Wnt-1 expression, a result that is consistent with the proposal that Wnt-1 may also act as a mitogen.

Wnt signaling induces GLT-1 expression in rat C6 glioma cells.

The regulation of glial and neuronal Na+-dependent glutamate/aspartate transporters is of interest because abnormal glutamate transport may be responsible for certain neurological diseases. Because expression of the Wnt-1 protooncogene results in induction of the glial-type glutamate transporter GLAST in PC12 neuron-like cells, we have evaluated the effect of Wnt-1-induced signaling on glutamate transporter expression in rat C6 glioma cells. C6 cells are known normally to express EAAC1, a neuronal glutamate transporter, but not the GLAST or the GLT-1 glutamate transporter. C6 cells that ectopically expressed Wnt-1 contained a GLT-1 RNA species similar in size (>10 kb) to the GLT-1 transcript present in rat brain, and they also contained a previously unreported 3.3-kb GLT-1 RNA species. Both GLT-1 RNAs contain large parts of the coding region. However, the 3.3-kb GLT-1 species contains at least one small deletion within the coding region. The Wnt-1-expressing C6 cells contained little, if any, GLT-1 protein as determined by immunological techniques. We suggest that one or both of the GLT-1 RNA species induced by Wnt-1 either fail to be translated or yield abnormal translation products that are quickly degraded. Wnt-1-expressing C6 cells may thus represent a novel in vitro system for studying GLT-1 transporter expression at the transcriptional and/or posttranscriptional levels.

Ablation of tumor cells in vivo by direct injection of HSV-thymidine kinase retroviral vector and ganciclovir therapy.

The introduction of therapeutic genes into proliferating tumor cells in vivo by direct intralesional injection of retroviral vectors can provide an effective and valuable approach for the treatment of a variety of solid tumor types. Efficient transduction of tumor cells in situ by direct injection was demonstrated using a retroviral vector containing the beta-galactosidase (beta-gal) gene. Ablation therapy in vivo was demonstrated using a retroviral vector containing the Herpes simplex virus thymidine kinase gene (HSV-TK) to deliver the TK gene into the murine colorectal tumor cell line CT26. Ablation of CT26 tumor cells in situ was achieved by directly injecting high-titer HSV-TK retroviral vector preparations into the site of tumor cell inoculation followed by intraperitoneal (i.p.) delivery of ganciclovir (GCV). This gene therapy strategy demonstrated a markedly lower rate of tumor progression, with several complete regressions, compared to animals in control groups. We also demonstrated that resistance to subsequent challenges with unmodified CT26 cells and an enhanced cellular immune response is associated with tumor regression in immunocompetent animals. Our results demonstrate the feasibility of direct in situ administration of HSV-TK retroviral vectors for the treatment of cancer and suggest that a cellular immune response may be elicited by this therapy.

Impaired differentiation of HPRT-deficient dopaminergic neurons: a possible mechanism underlying neuronal dysfunction in Lesch-Nyhan syndrome.

Lesch-Nyhan syndrome is a hereditary disorder of purine metabolism causing overproduction of uric acid and neurological problems including spasticity, choreoathetosis, mental retardation, and compulsive self-mutilation. The syndrome is caused by a defect in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT), which converts guanine and hypoxanthine to the nucleotides GMP and IMP. There is evidence that the neurological problems are due to an adverse effect of the HPRT deficiency on the survival and/or development of dopaminergic neurons, specifically. Here we report that HPRT-deficient PC12 mutants that have a normal or near normal dopamine content (55-97% of that of wild-type cells) fail to undergo neuronal differentiation induced by nerve growth factor (NGF) when the de novo pathway of purine synthesis is partially inhibited. However, nerve growth factor-induced differentiation is near normal under these conditions in PC12 HPRT-deficient mutants containing much lower dopamine levels (<8% of that of wild type cells), indicating a neurotoxic effect of the endogenous dopamine in the mutants. The degree of inhibition of the de novo pathway of purine synthesis was the same in both classes of HPRT-deficient mutants. Expression of BCl-2 in a PC12 mutant that has a normal dopamine content allowed partial NGF-induced differentiation suggesting that the apoptotic pathway might be involved in the failure of differentiation when the de novo pathway of purine synthesis is partially inhibited.

Establishment of parameters for optimal transduction efficiency and antitumor effects with purified high-titer HSV-TK retroviral vector in established solid tumors.

Suicide gene therapy using the herpes simplex thymidine kinase gene and ganciclovir is an attractive strategy for solid tumors. Early animal studies involved intratumoral injection of retroviral producer cells or unprocessed supernatant to generate an antitumor effect. Xenotransplantation of producer cells proved effective in several models, but the crude supernatants from the same cells were of insufficient titer to produce antitumor effects. We have developed new non-murine producer lines that yield replication-defective retroviral vectors encoding thymidine kinase at high titer which are then further purified and processed, resulting in pharmaceutical grade retroviral vectors with titers of up to 10(8) cfu/ml. Purified, high-titer retroviral preparations were injected directly into solid tumors in two syngeneic mouse tumor models. Significant antitumor responses and some cures were observed following systemic ganciclovir therapy. Assays using monoclonal antibodies to measure thymidine kinase protein expression at the single cell level in vitro and in vivo were developed so that therapeutic transgene expression could be quantified. Intralesional delivery resulted in transduction of over 20% of tumor cells in a protocol designed to maximize transduction on the basis of separate analyses of route, dosage, and schedule of vector administration. A consensus strategy evolved in which the combined effects of increased titer and a longer duration of retroviral vector administration interact to maximize transduction efficiency. These results indicate that purified high-titer retroviral vectors have the potential to transfer effective quantities of therapeutic genes into solid tumors in human subjects and highlight some pharmacologic factors that could be valuable in the design of clinical gene therapy protocols.

Rat C6 and human astrocytic tumor cells express a neuronal type of glutamate transporter.

C6 glioma cells take up aspartate and glutamate by a Na(+)-dependent transporter. Using the polymerase chain reaction and degenerate oligonucleotide primers corresponding to conserved regions of previously cloned glutamate transporters, we isolated from these cells a partial cDNA clone with a sequence of the neuronal type EAAC1 glutamate transporter. The cells express a 4.4 kb message that hybridizes to this cDNA, and they do not express either of the previously described glial type glutamate transporters, GLT-1 or GLAST. The cells were sensitive to the toxic aspartate analog alanosine, which enters the cells by a glutamate transporter. Several human brain tumors examined, including some astrocytic tumors, expressed the EAAT3 glutamate transporter, which is the human homolog of the rodent EAAC1 transporter. Some of the tumors also expressed the other types of glutamate transporter.

The induction of ret by Wnt-1 in PC12 cells is atypically dependent on continual Wnt-1 expression.

Wild type PC12 pheochromocytoma cells that had been infected with a Wnt-1-carrying virus and thus express Wnt-1 (PC12/Wnt-1) are known to acquire the same flat cell phenotype as that of spontaneously occurring PC12 flat cell variants except that the latter do not presently express Wnt-1. Flat cell variants of PC12 cells exhibit markedly altered morphology and gene expression. In order to assess the possibility that the spontaneously occurring flat cell variants could have been induced in wild type PC12 cells by previous transient expression of the cell's endogenous Wnt-1, we have isolated PC12/Wnt-1 cells expressing little or no Wnt-1. In spite of absent Wnt-1 expression, they retained their flat cell morphology, glutamate/aspartate transporter activity, increased neu mRNA levels and lack of both norepinephrine transporter activity and nerve growth factor-induced differentiation. Thus, Wnt-1 expression is not required to maintain the flat cell phenotype. However, we identified one gene, ret, whose mRNA level in PC12 was not only increased by Wnt-1 expression, but whose increased mRNA level was also dependent on continual Wnt-1 expression. This finding suggests that the induction of ret by Wnt-1 can be used to elucidate the Wnt-induced signalling pathway in mammalian cells.

K252a-potentiation of EGF-induced neurite outgrowth from PC12 cells is not mimicked or blocked by other protein kinase activators or inhibitors.

Epidermal growth factor (EGF) has recently been shown to cause certain strains of PC12 cells to extend short neurites. This EGF-induced differentiation of PC12 was found to be potentiated by the protein kinase inhibitor, K252a, in that PC12 cells treated with both EGF and K252a extended long branched neurites similar to those induced by nerve growth factor (NGF). As reported here no other protein kinase inhibitor or activator mimicked or blocked the effect of K252a on EGF-induced PC12 differentiation. Cyclic adenosine 3',5'-monophosphate (cAMP) also potentiated EGF-induced neurite outgrowth from PC12 cells, but the mechanism of this potentiation was different from that of K252a. Cells that had been exposed to EGF and then stripped of their neurons extended neurites again when retreated with EGF in the absence of RNA synthesis or when treated with NGF in the absence of RNA synthesis. Thus EGF can prime PC12 cells for either EGF or for NGF, a finding that further suggests that EGF and NGF use similar signaling pathways to induced neuronal differentiation of PC12.

Cytotoxic T lymphocyte and antibody responses generated in rhesus monkeys immunized with retroviral vector-transduced fibroblasts expressing human immunodeficiency virus type-1 IIIB ENV/REV proteins.

The immune response against human immunodeficiency virus type-1 (HIV-1) is believed to play a role in controlling the early stages of disease progression. The cellular immune response, in particular cytotoxic T lymphocyte (CTL) activity, may be important for eliminating virally infected cells in HIV-1-infected individuals. Genetic immunization using retroviral vectors provides an effective means of introducing antigens into the antigen presentation pathways for T cell stimulation. A nonreplicating, amphotropic murine retroviral vector containing the HIV-1 IIIB env gene has been used to transduce primary rhesus monkey fibroblasts for the expression of HIV-1 antigenic determinants. Rhesus monkeys were immunized with four doses of either vector-transduced autologous fibroblasts (VTAF) expressing the HIV-1 IIIB ENV/REV proteins or nontransduced autologous fibroblasts (NTAF) administered at 2-week intervals. The animals were evaluated for both the induction of HIV-1-specific immune responses and potential toxicity associated with this ex vivo treatment. The VTAF-immunized monkeys generated CTL responses specific for HIV-1 ENV/REV expressing autologous target cells, whereas, NTAF-immunized monkeys showed negligible CTL activity. The cytotoxic activity was mediated by CD8+, major histocompatibility complex (MHC)-restricted CTL. In addition, antibody responses directed against the HIV-1 gp120 protein were also detected in the sera of VTAF-immunized monkeys. Clinical and histopathological evaluation of immunized monkeys showed no evidence of significant adverse events. Several animals that received either VTAF or NTAF had detectable anti-cytoplasmic antibodies, but were not positive for anti-nuclear antibodies or rheumatoid factor. Subsequent evaluation of renal, synovial, and hepatic tissue samples from these monkeys revealed no autoimmune disease-associated lesions. This study demonstrates the safety and ability of autologous retroviral vector-transduced cells expressing HIV-1 IIIB ENV/REV proteins to stimulate immune responses in a non-human primate model, and provides a basis for this form of genetic immunization in HIV-infected humans.

PC12 variants deficient in norepinephrine transporter mRNA have wild type activities of several other related transporters.

Wild type PC12 pheochromocytoma cells express a Na(+)-dependent norepinephrine transporter that operates in the uptake of catecholamines. In addition to the previously described Na(+)-dependent system A for the uptake of alpha-amino-isobutyric acid and system Gly for glycine, we have identified two other Na(+)-dependent transporter systems for amino acid uptake in these cells: 1) system beta for beta-alanine and taurine; and 2) a system for creatine. Uptake of alpha-amino-isobutyric acid, glycine, beta-alanine, and creatine is not affected in some PC12 variants that were previously shown to be deficient in catecholamine uptake and to have decreased levels of norepinephrine transporter mRNA. We have isolated two PC12 cDNA clones that are essentially identical in sequence to recently reported cDNAs for rat brain taurine and creatine transporters, respectively, and a third cDNA that appears to code for a novel transporter. mRNAs for these three transporters are present at wild type levels in those variants that express no or little norepinephrine transporter mRNA. These results support the notion that the expression of catecholamine reuptake transporters may be particularly susceptible to down-regulation.

Contrasting morphological changes in PC12 flat cells expressing two different forms of exogenous oncogenic ras.

Oncogenic ras is known to transform certain cells, whereas it induces terminal differentiation of others, e.g., neuronal differentiation of PC12 cells. MPT1 is a PC12 flat cell variant that extends glial-like processes and exhibits some properties of noncancer cells in culture, e.g., absence of anchorage-independent growth. Expression of oncogenic ras by MPT1 cells failed to result in neuronal differentiation, but such cells exhibited two contrasting morphological changes under certain conditions. First, they retained their extended processes in the presence of dexamethasone, unlike MPT1 cells not expressing oncogenic ras. Second, confluent cultures of ras-expressing MPT1 cells contained foci of transformed-looking cells that were refractile and grew in multiple layers. Thus, ras seemed to induce both a kind of differentiation and transformation of MPT1 cells. MPT1 cells were transfected with a plasmid carrying an oncogenic Harvey ras gene under transcriptional control of the metallothionein promoter. Two subclones of the transfected cells exhibited different responses to the induction of ras and expressed two different forms of the ras gene product. One clone extended dexamethasone-resistant processes and the second clone exhibited a more transformed phenotype. The ras gene product expressed in these two clones differed in migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in the extent of phosphorylation. These results suggest that ras protein phosphorylation may be important in determining whether a ras-mediated response is differentiation or transformation.

K252a potentiates epidermal growth factor-induced differentiation of PC12 cells.

Epidermal growth factor (EGF) induced short neurites in two different strains of PC12 cells. The length of the EGF-induced neurites was markedly increased in the presence of the protein kinase inhibitor K252a, which is known to inhibit differentiation induced by nerve growth factor (NGF). EGF-induced differentiation of PC12 required RNA synthesis and activity of the ras proto-oncogene product. EGF increased the levels of three neurofilament proteins and the mRNA level of two late response genes (SCG10 and 63) known to be induced by NGF. Together, EGF and K252a caused a greater increase in these mRNAs than did either agent alone. K252a did not alter the extent of EGF-induced autophosphorylation of the EGF receptor, but it did decrease the extent of receptor phosphorylation in the absence of added EGF. Thus, the ability of the EGF receptor to trigger neuronal differentiation may depend on the state of its phosphorylation at serine and/or threonine residues. Two other strains of PC12 did not extend neurites when exposed to EGF, even when K252a was also present. Thus, the differentiating effect of EGF on PC12 is PC12 strain-specific.

Differential expression of transporters for norepinephrine and glutamate in wild type, variant, and WNT1-expressing PC12 cells.

Wild type PC12 pheochromocytoma cells express a Na(+)-dependent norepinephrine transporter that operates in the uptake of catecholamines, including dopamine. This transporter is not expressed in two spontaneously occurring flat cell variants of PC12 or in two other flat cell variants whose phenotype was induced by expression of the Wnt-1 oncogene. However, each of the flat cell variants, including those that express Wnt-1, exhibit a Na(+)-dependent, Cl(-)-independent glutamate/aspartate transporter activity that is not present in wild type PC12 cells. The flat cell variants took up glycine by a Na(+)-dependent process as well as did wild type cells. All of the flat cell variants have decreased levels of norepinephrine transporter mRNA but normal levels of glycine transporter mRNA. Glutamate/aspartate transporter mRNA was detected only in the variants that exhibited glutamate/aspartate transporter activity, and the nucleotide sequence of a partial glutamate/aspartate transporter cDNA from these cells demonstrated that it was the glial form of the transporter that was expressed. These variants were more sensitive than was wild type PC12 to alanosine, a toxic aspartate analog that enters cells by a transporter-mediated system such as the glutamate/aspartate transporter; however, these variants were as sensitive as wild type cells to another toxic aspartate analog, N-(phosphonacetyl)-L-aspartic acid, which is believed to enter cells by endocytosis. We suggest that the Wnt-1 gene product, or a homolog, may be involved in glial differentiation and that the mechanisms that alter the expression of the norepinephrine and glutamate/aspartate transporters in wild type and variant PC12 cells may also operate to regulate neurotransmitter transporter expression in vivo.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-resistant, flat-cell PC12 variants having a partial loss of transformed phenotype.

We have cloned and characterized two variants of PC12 cells. MPT1 cells were selected by their resistance to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and variant 2068 was isolated nonselectively as a large, flat-cell variant commonly occurring in PC12 cultures. Variant 2068 cells also exhibit resistance to MPTP. Karyotype analysis revealed that these variants are true derivatives of wild-type PC12 cells; however, each variant is tetraploid, whereas the wild-type parent is diploid. The two variants contain an altered level and composition of lactate dehydrogenase isoenzymes, which could account for a previously described difference in lactate metabolism. Both variants exhibit a partial loss of transformed phenotype in culture in that they are nonrefractile, grow in monolayers, and fail to multiply in soft agar. We suggest that this alteration in transformed phenotype may result in altered mitochondria and lactate dehydrogenase and thus account for their resistance to MPTP. Compared with wild-type PC12 cells, MPT1 cells have a decreased level of fos mRNA and an increased level of myc mRNA; the latter results from an increased level of transcription of exon 1 of the myc gene. Studies with hybrid cells obtained by fusing MPT1 cells with wild-type-like cells show that most, but not all, of the parameters of the MPT1 phenotype predominate.

Covalent labeling of the cocaine-sensitive catecholamine transporter.

Xylamine is an alkylating agent that is a substrate for and specific irreversible inhibitor of the cocaine-sensitive catecholamine transporter that functions in catecholamine reuptake into neurons and PC12 cells. [3H]xylamine prominently labels nine PC12 proteins; the relative xylamine-alkylation of a Mr 54,000 protein was decreased by cocaine and increased in the case of a PC12 variant, B9, which is deficient in catecholamine transport. [3H]xylamine labels no such protein in another transport variant, MPT1. We propose that this Mr 54,000 protein 1) is a component of the catecholamine transporter, 2) is present in B9 cells but in a conformation that reduces transporter activity and makes alkylation by xylamine more likely, and 3) is absent in MPT1 cells. Nerve growth factor treatment restores transporter activity in B9 cells but not in other transporter-deficient variants.

Transforming growth factor alpha and a PC12-derived growth factor induce neurites in PC12 cells and enhance the survival of embryonic brain neurons.

We have identified and characterized a 5000-Da protein that induces neurite outgrowth from PC12 pheochromocytoma cells, enhances the survival of embryonic rat brain neurons in primary culture, and induces the multiplication of embryonic rat brain astrocytes in primary culture. The factor is produced by a flat cell PC12 variant that expresses the activated ras oncogene after transfection of the gene. The factor resembles transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) in that it induces anchorage-independent colony formation of normal rat kidney cells in soft agar and competes with EGF for binding to the EGF receptor. Rat TGF alpha and human TGF alpha also induce neurite outgrowth from PC12 and enhance the survival of embryonic brain neurons. The PC12 variant-derived factor can be distinguished from TGF alpha and EGF immunologically and by migration rates on reversed-phase high-performance liquid chromatography.

Phosphotyrosine-containing lactate dehydrogenase is restricted to the nuclei of PC12 pheochromocytoma cells.

There are five lactate dehydrogenase (LDH) isoenzymes, composed of various combinations of two types of subunits. LDH-5, which contains only the LDH A subunit, is known to be present in both the cytoplasm and the nucleus, to act as a single-stranded DNA-binding protein possibly functioning in transcription and/or replication, and to undergo phosphorylation of tyrosine 238 in approximately 1% of the enzyme after cell transformation by certain tumor viruses. We have characterized LDH from wild-type PC12 pheochromocytoma cells and from a PC12 variant (MPT1) that exhibits altered lactate metabolism and altered expression of multiple genes. Wild-type and MPT1 cells contain different proportions of LDH isoenzymes, with LDH-5 being more predominant in wild-type cells than in the variant. A small fraction of LDH from PC12 cells contains phosphotyrosine. Approximately 99% of the total LDH activity is located in the cytoplasm, but all of the phosphotyrosine-containing LDH is located in the nucleus. Furthermore, essentially all of the nuclear LDH contains phosphotyrosine. These results suggest that tyrosine phosphorylation can affect its role in the nucleus.