Biomedical subjects

A R Saltiel

Publications and source records attributed to A R Saltiel.

At least 91 records · Page 5Linked to original sources

PubMed1995

Activation of mitogen-activated protein kinase and phosphatidylinositol 3'-kinase is not sufficient for the hormonal stimulation of glucose uptake, lipogenesis, or glycogen synthesis in 3T3-L1 adipocytes.

The precise mechanism by which insulin regulates glucose metabolism is not fully understood. However, it is known that insulin activates two enzymes, phosphatidylinositol 3'-kinase (PI 3'-K) and mitogen-activated protein kinase (MAPK), which may be involved in stimulating the metabolic effects of insulin. The role of these enzymes in glucose metabolism was examined by comparing the effects of insulin, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) in 3T3-L1 adipocytes. Treatment of the cells with PDGF or EGF for 5 min increased the MAPK activity 3-5-fold, while insulin treatment produced a 2.5-fold increase. The MAPK activity remained elevated for 1 h after either PDGF or insulin treatment. PDGF and insulin, but not EGF, caused a transient increase in the amount PI 3'-K activity coprecipitated with tyrosine phosphorylated proteins. Although PDGF and insulin caused a similar increase in the activities of these two enzymes, only insulin caused substantial increases in glucose utilization. Insulin increased the transport of glucose and the synthesis of lipid 4- and 17-fold, respectively, while PDGF did not affect these processes significantly. Glycogen synthesis was increased 15-fold in response to insulin and only 3-fold in response to PDGF. Thus, the activation of MAPK and PI 3'-K are not sufficient for the complete stimulation of glucose transport, lipid synthesis, or glycogen synthesis by hormones in 3T3-L1 adipocytes, suggesting a requirement for other signaling mechanisms that may be uniquely responsive to insulin.

3T3 Cells↗

PubMed1995

Cell cycle arrest induced by an inhibitor of glucosylceramide synthase. Correlation with cyclin-dependent kinases.

In an attempt to define the basis for sphingolipid regulation of cell proliferation, we studied the effects of glucosylceramide (GlcCer) synthase inhibition by threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on NIH 3T3 cells overexpressing insulin-like growth factor-1 (IGF-1) receptor. PDMP treatment resulted in a time-dependent decrease in GlcCer levels and an increase in cellular ceramide levels. PDMP abolished serum and IGF-1-stimulated cell proliferation, as measured by a reduction in [3H]thymidine incorporation, protein, and DNA levels. However it did not affect IGF-1-mediated early signaling events, including receptor tyrosine kinase, MAP kinase, and phosphatidylinositol 3-kinase activities. Two-color flow cytometry with propidium iodide and 5-bromo-2'-deoxyuridine monophosphate labeling revealed an arrest of the cell cycle at G1/S and G2/M transitions in an asynchronous population of cells. These changes were time dependent, with maximal effects seen by 12-24 h. Removal of PDMP from the cell medium resulted in reversal of the cell cycle changes, with cells re-entering the S phase. The cell cycle arrest at the G1/S and G2/M transitions was confirmed in cells synchronized by pretreatment with nocodazole, aphidicolin, or hydroxyurea, and released from blockade in the presence of PDMP. A decrease in the activities of two cyclin-dependent kinases, p34cdc2 kinase and cdk2 kinase, was observed with PDMP treatment. When cell ceramide levels were increased by N-acetylsphingosine, comparable changes in the cell cycle distribution were seen. However, sphingomyelinase treatment was without effect. Therefore, it appears that ceramide mediates in part the inhibitory effect of GlcCer synthase inhibition on IGF-1-induced cell proliferation in 3T3 cells. The rapid production of decreased cyclin-dependent kinase activities by PDMP suggests that one of the crucial sites of action of the inhibitor lies in this area.

3T3 Cells↗

PubMed1995

Protein-tyrosine-phosphatase SHPTP2 is a required positive effector for insulin downstream signaling.

SHPTP2 is a ubiquitously expressed tyrosine-specific protein phosphatase that contains two amino-terminal Src homology 2 (SH2) domains responsible for its association with tyrosine-phosphorylated proteins. In this study, expression of dominant interfering mutants of SHPTP2 was found to inhibit insulin stimulation of c-fos reporter gene expression and activation of the 42-kDa (Erk2) and 44-kDa (Erk1) mitogen-activated protein kinases. Cotransfection of dominant interfering SHPTP2 mutants with v-Ras or Grb2 indicated that SHPTP2 regulated insulin signaling either upstream of or in parallel to Ras function. Furthermore, phosphotyrosine blotting and immunoprecipitation identified the 125-kDa focal adhesion kinase (pp125FAK) as a substrate for insulin-dependent tyrosine dephosphorylation. These data demonstrate that SHPTP2 functions as a positive regulator of insulin action and that insulin signaling results in the dephosphorylation of tyrosine-phosphorylated pp125FAK.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Differentiation of peptide molecular recognition by phospholipase C gamma-1 Src homology-2 domain and a mutant Tyr phosphatase PTP1bC215S.

Activated epidermal growth factor receptor (EGFR) undergoes autophosphorylation on several cytoplasmic tyrosine residues, which may then associate with the src homology-2 (SH2) domains of effector proteins such as phospholipase C gamma-1 (PLC gamma-1). Specific phosphotyrosine (pTyr)-modified EGFR fragment peptides can inhibit this intermolecular binding between activated EGFR and a tandem amino- and carboxy-terminal (N/C) SH2 protein construct derived from PLC gamma-1. In this study, we further explored the molecular recognition of phosphorylated EGFR988-998 (Asp-Ala-Asp-Glu-pTyr-Leu-Ile-Pro-Gln-Gln-Gly, I) by PLC gamma-1 N/C SH2 in terms of singular Ala substitutions for amino acid residues N- and C-terminal to the pTyr (P site) of phosphopeptide I. Comparison of the extent to which these phosphopeptides inhibited binding of PLC gamma-1 N/C SH2 to activated EGFR showed the critical importance of amino acid side chains at positions P+2 (Ile994), P+3 (Pro995), and P+4 (Gln996). Relative to phosphopeptide I, multiple Ala substitution throughout the N-terminal sequence, N-terminal sequence, N-terminal truncation, or dephosphorylation of pTyr each resulted in significantly decreased binding to PLC gamma-1 N/C SH2. These structure-activity results were analyzed by molecular modeling studies of the predicted binding of phosphopeptide I to each the N- and C-terminal SH2 domains of PLC gamma-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Insulin stimulates the tyrosine phosphorylation of caveolin.

The specialized plasma membrane structures termed caveolae and the caveolar-coat protein caveolin are highly expressed in insulin-sensitive cells such as adipocytes and muscle. Stimulation of 3T3-L1 adipocytes with insulin significantly increased the tyrosine phosphorylation of caveolin and a 29-kD caveolin-associated protein in caveolin-enriched Triton-insoluble complexes. Maximal phosphorylation occurred within 5 min, and the levels of phosphorylation remained elevated for at least 30 min. The insulin-dose responses for the tyrosine phosphorylation of caveolin and the 29-kD caveolin-associated protein paralleled those for the phosphorylation of the insulin receptor. The stimulation of caveolin tyrosine phosphorylation was specific for insulin and was not observed with PDGF or EGF, although PDGF stimulated the tyrosine phosphorylation of the 29-kD caveolin-associated protein. Increased tyrosine phosphorylation of caveolin, its associated 29-kD protein, and a 60-kD protein was observed in an in vitro kinase assay after incubation of the caveolin-enriched Triton-insoluble complexes with Mg-ATP, suggesting the presence of an intrinsic tyrosine kinase in these complexes. These fractions contain only trace amounts of the activated insulin receptor. In addition, these complexes contain a 60-kD kinase detected in an in situ gel kinase assay and an approximately 60 kD protein that cross-reacts with an antibody against the Src-family kinase p59Fyn. Thus, the insulin-dependent tyrosine phosphorylation of caveolin represents a novel, insulin-specific signal transduction pathway that may involve activation of a tyrosine kinase downstream of the insulin receptor.

3T3 Cells↗

PubMed1994

Identification of amino acids in the N-terminal SH2 domain of phospholipase C gamma 1 important in the interaction with epidermal growth factor receptor.

Photoaffinity labeling and site-directed mutagenesis have been used to identify amino acid residues of the phospholipase C gamma 1 (PLC gamma 1) N-terminal SH2 domain involved in recognition of the activated epidermal growth factor receptor (EGFR). The photoactive amino acid p-benzoylphenylalanine (Bpa) was incorporated into phosphotyrosine-containing peptides derived from EGFR autophosphorylation sites Tyr992 and Tyr1068. Irradiation of these labels in the presence of SH2 domains showed cross-linking which was time-dependent and specific; labeling was inhibited with non-Bpa-containing peptides from EGFR in molar excess. The phosphotyrosine residue on the peptides was important for SH2 recognition, as dephosphorylated peptides did not cross-link. Radiolabeled peptides were used to identify sites of cross-linking to the N-terminal SH2 of PLC gamma 1. Bpa peptide-SH2 complexes were digested with trypsin, and radioactive fragments were purified by HPLC and analyzed by Edman sequencing. These experiments showed Arg562 and an additional site in the alpha A-beta B region of the SH2 domain, most likely Glu587, to be labeled by the Tyr992-derived peptide. Similar analysis of the reaction with the Tyr1068-derived photoaffinity label identified Leu653 as the cross-linked site. Mutation of the neighboring residues of Glu587 decreased photo-cross-linking, emphasizing the importance of this region of the molecule for recognition. These results are consistent with evidence from the v-Src crystal structure and implicate the loop spanning residues Gln640-Ser654 of PLC gamma 1 in specific recognition of phosphopeptides.

Affinity Labels↗

PubMed1994

Activation of the Eck receptor protein tyrosine kinase stimulates phosphatidylinositol 3-kinase activity.

The Eph/Eck subfamily of receptor protein tyrosine kinases is currently the largest subfamily of receptor protein tyrosine kinases with a dozen members (Van der Geer, P., Hunter, T., and Lindberg, R. A. (1994) Annu. Rev. Cell Biol. 10, 251-337). Using the cytoplasmic domain of Eck as bait in a yeast two-hybrid screen of mouse embryonic and T-cell cDNA libraries, it was discovered that the p85 subunit of phosphatidylinositol 3-kinase bound Eck. Further, using glutathione S-transferase fusion proteins, it was found that the C-terminal src homology 2 domain of the p85 subunit specifically interacted with Eck. Additionally, Eck coimmunoprecipitated with p85 in ligand activated cells confirming their interaction in vivo. In keeping with the above observations, activation of Eck by its ligand, B61, increased phosphatidylinositol 3-kinase activity. This is the first description of a signal transduction pathway initiated by any member of the Eph/Eck family.

Animals↗

PubMed1994

Localization of the insulin receptor binding sites for the SH2 domain proteins p85, Syp, and GAP.

The insulin receptor is known to interact with the SH2 domain proteins p85 (the regulatory subunit of phosphatidylinositol 3-kinase), Syp (a tyrosine phosphatase), and GAP (GTPase-activating protein). In this study, we mapped the insulin receptor binding sites for each of these proteins by examining the ability of phosphopeptides, corresponding to insulin receptor phosphorylation sites, and mutant insulin receptors to inhibit an insulin receptor-SH2 domain interaction. Precipitation of partially purified insulin receptors by glutathione S-transferase fusion proteins containing the N-terminal SH2 domains of p85 and GAP and both SH2 domains of Syp was demonstrated. The effect of the addition of each phosphopeptide on insulin receptor precipitation was tested. pY1322, the C-terminal insulin receptor peptide, inhibited insulin receptor precipitation by both p85- and Syp-GST. The NPXY internalization domain peptide inhibited insulin receptor precipitation by GAP-GST. These data were confirmed by mutant insulin receptor experiments. The insulin receptor C-terminal mutants, delta CT and Y/F2, were not precipitated by p85- or Syp-GST and the NPXY mutant insulin receptors, delta Ex16 and HI delta NPEY, were not precipitated by GAP-GST. Therefore, we conclude that p85 and Syp bind to the insulin receptor C terminus at tyrosine 1322 and GAP binds to the insulin receptor NPXY domain at tyrosine 960.

Amino Acid Sequence↗

PubMed1994

Stimulation of glycogen synthesis by insulin in human erythroleukemia cells requires the synthesis of glycosyl-phosphatidylinositol.

Although the insulin-dependent hydrolysis of glycosyl-phosphatidylinositol (GPI) may play an important role in insulin action, an absolute requirement for this glycolipid has not been demonstrated. Human K562 cells were mutated to produce a cell line (IA) incapable of the earliest step in PI glycosylation, the formation of PI-GlcNAc. Another cell line (IVD) was deficient in the deacetylation of PI-GlcNAc to form PI-GlcN and subsequent mannosylated species. Each line was transfected with wild-type human insulin receptors. Similar insulin-stimulated receptor autophosphorylation was observed in all three lines, along with a nearly identical increase in the association of phosphorylated insulin receptor substrate 1 with endogenous PI 3-kinase. Both normal and GPI-defective lines also displayed a similar 2- to 3-fold increase in phosphorylation of the Shc protein and its association with growth factor receptor-bound protein 2 in response to insulin. In contrast to these results, striking differences were noted in insulin-stimulated glycogen synthesis. In normal cells, glycogen synthesis was significantly increased by insulin, whereas no insulin stimulation was observed in GPI-deficient IA cells, and only a trace of stimulation was detected in IVD cells. These results indicate that tyrosine phosphorylation produced by insulin is not dependent on GPI synthesis, and this effect is not sufficient to elicit at least some of the metabolic effects of the hormone. In contrast, GPI synthesis is required for the stimulation of glycogen synthesis by insulin in these cells. These findings support the existence of divergent pathways in the action of insulin.

Cell Line↗

PubMed1994

Expression of catalytically inactive Syp phosphatase in 3T3 cells blocks stimulation of mitogen-activated protein kinase by insulin.

To explore the role of the protein tyrosine phosphatase Syp in insulin signaling, a catalytically inert mutant Syp protein was expressed under an inducible promoter in cells transfected with the human insulin receptor. Expression of the mutant phosphatase significantly reduced the stimulation of mitogenesis by insulin, indicating that the mutation produced a dominant negative phenotype. Tyrosine phosphorylation of both the insulin receptor and its major substrates, Shc and insulin receptor substrate-1, were unaffected by the mutant phosphatase. However, both the insulin-dependent tyrosine phosphorylation and activation of mitogen-activated protein kinase were markedly attenuated. Expression of the mutant phosphatase allowed the detection of a 120-kDa protein phosphorylated in response to insulin that associated with the src homology (SH) 2 domains of the phosphatase, suggesting a possible regulatory role for this protein. These results indicate that the activity of Syp plays a critical part in the mitogenic actions of insulin.

3T3 Cells↗

PubMed1994

Growth factor induced MEK activation is primarily mediated by an activator different from c-raf.

Activation of the mitogen-activated protein kinases (MAPKs) is a common event of many signal transduction pathways. MAPKs are phosphorylated and activated by an immediate upstream activating kinase, MEK. The proto-oncogene c-raf, encoding a serine/threonine kinase, has been reported to be a direct activator of MEK. In this paper, it is shown that growth factors activate MEK by stimulating c-raf and a raf-independent MEK activator. Treatment of Swiss3T3 cells with epidermal growth factor (EGF) rapidly increased the activity of MEK activator. Maximal activation was detected by 2.5 min and declined to the prestimulated level within 10 min. This stimulation of the MEK activator was temporally followed by increased activities of MEK and MAPK. The activation of MEK was accompanied by phosphorylation of this protein. To determine the relationship of this MEK activator and the c-raf kinase, cell lysates were immunoprecipitated with anti-raf antibody and assayed for MEK activation. Only a fraction (< 20%) of the MEK activating activity was detected in anti-raf immunoprecipitates from EGF-stimulated Swiss3T3 cells. Similar experiments with nerve growth factor stimulated pheochromocytoma 12 (PC-12) cells revealed that the raf kinase contributed less than 5% of the total MEK activating activity while the overwhelming majority of MEK activating activity remained in the postimmunoprecipitation supernatant in which the raf protein had been quantitatively depleted. These data demonstrate that Swiss3T3 and PC-12 cells contain at least two different growth factor sensitive MEK activators, one residing in anti-raf immunoprecipitates and a second activity that is separate from raf.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1994

Evidence for a functional role of Shc proteins in mitogenic signaling induced by insulin, insulin-like growth factor-1, and epidermal growth factor.

Shc proteins contain a single SH2 domain, lack catalytic activity, and are substrates for activated receptors for insulin, insulin-like growth factor-1 (IGF-1), and epidermal growth factor (EGF). Treatment with these growth factors induced rapid tyrosine phosphorylation of Shc. We investigated the potential role of Shc in mitogenic signaling. Affinity-purified antibodies were microinjected into living Rat1 fibroblasts overexpressing human insulin receptors. Bromodeoxyuridine incorporation into newly synthesized DNA was subsequently studied to assess the importance of Shc. Cellular microinjection of anti-Shc antibody inhibited BrdU incorporation induced by insulin, IGF-1, and EGF, but did not affect cells stimulated by fetal calf serum. Microinjection of an oncogenic p21ras protein (T24) into quiescent cells produced constitutively active mitogenic signaling, and comicroinjection of T24 with the anti-Shc antibody restored insulin and EGF stimulation of DNA synthesis. Immunoprecipitates of Shc from lysates of insulin-stimulated cells removed 70-80% of guanine nucleotide-releasing factor activity. These results indicate that Shc is an important component in a mitogenic signal transduction pathway that is shared by insulin, IGF-1, and EGF. The functional locus of Shc is either upstream of p21ras or lies on a distinct branch of the pathway leading to cell cycle progression.

Animals↗

PubMed1994

Mutation of the two carboxyl-terminal tyrosines in the insulin receptor results in enhanced activation of mitogen-activated protein kinase.

Activation of mitogen-activated protein (MAP) kinase represents an important mechanism in hormonal regulation. To clarify the role of MAP kinase activation in insulin action, we compared the activation of the enzyme in Rat-1 cells transfected with wild-type (Hirc) and mutant insulin receptors in which the 2 carboxyl-terminal tyrosines were substituted with phenylalanine (Y/F2). Expression of the Y/F2 mutant receptor enhanced the responsiveness of MAP kinase to insulin. Moreover, the insulin responsiveness of the activator of this enzyme, MAP kinase kinase, was also increased in these cells. To explore the early signaling events that might account for this increase in responsiveness, we evaluated the tyrosine phosphorylation of the insulin receptor substrate, IRS-1, and its subsequent association with phosphatidylinositol (PI)-3 kinase. In both cell types, insulin led to a dose-dependent increase in the association of tyrosine phosphorylated IRS-1 with the SH2 domain of the p85 regulatory subunit of PI-3 kinase, and also increased the amount of PI kinase activity detected in anti-IRS-1 immunoprecipitates. The effect of insulin was significantly greater in Y/F2 cells, as determined in both assays. In previous studies, cells bearing this receptor mutant exhibited an identical metabolic response but enhanced mitogenic response to insulin when compared with wild-type receptor. These data provide further evidence for divergence of the mitogenic and metabolic signaling pathways at or near the insulin receptor.

Animals↗

PubMed1994

Thyrotropin-releasing hormone stimulates MAP kinase activity in GH3 cells by divergent pathways. Evidence of a role for early tyrosine phosphorylation.

Regulation of the mitogen-activated protein (MAP) kinase by thyrotropin-releasing hormone (TRH) in GH3 rat pituitary tumor cells was investigated. Both TRH and epidermal growth factor (EGF) acutely activated this enzyme, via tyrosine and serine/threonine phosphorylation. Down-regulation of cellular protein kinase C (PKC) only partly inhibited the phosphorylation of MAP kinase by TRH, suggesting both PKC-dependent and -independent pathways. Both TRH and EGF similarly increased the phosphorylation of raf-1, by a PKC-independent mechanism. Both TRH and EGF stimulated the formation of a ras-GTP complex. This activation of ras by growth factors is thought to involve the tyrosine phosphorylation of Shc. EGF stimulated the tyrosine phosphorylation of three Shc proteins and their subsequent association with its receptor. TRH stimulated the tyrosine phosphorylation of the 52-kDa Shc protein, although neither phorbol esters nor the calcium ionophore A23187 had any effect, indicating that this effect of TRH was not dependent on PKC. Both TRH and EGF induced the association of tyrosine phosphorylated Shc proteins with a fusion protein containing SH2 and SH3 domains of Grb2, another important component in ras activation. These results provide evidence that MAP kinase is acutely activated by TRH through a PKC-dependent pathway as well as a second pathway possibly involving tyrosine phosphorylation.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Insulin receptor substrate 1 is required for insulin-mediated mitogenic signal transduction.

Insulin treatment of mammalian cells immediately stimulates the tyrosine phosphorylation of a cellular protein of 185 kDa referred to as pp185 or IRS-1 (insulin receptor substrate 1). The potential role of the IRS-1 protein in insulin signaling has been examined by microinjecting affinity-purified antibodies into living cells. Stably transfected Rat-1 fibroblasts, which overexpress the human insulin receptor, were microinjected and subsequently stimulated with insulin or other growth factors. Progression through the cell cycle was monitored by using a single-cell assay, which employs bromodeoxyuridine labeling of DNA and analysis with immunofluorescence microscopy. Microinjection of anti-IRS-1 antibody completely inhibited incorporation of bromodeoxyuridine into the nuclei of cells stimulated with insulin or insulin-like growth factor I but did not affect cells stimulated with serum or a variety of purified growth factors. These studies indicate that IRS-1 is a critical component of the insulin and insulin-like growth factor I signaling pathways, which lead to DNA synthesis and cell growth.

Amino Acid Sequence↗

PubMed1994

Growth factors differentially stimulate the phosphorylation of Shc proteins and their association with Grb2 in PC-12 pheochromocytoma cells.

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including two proteins, Shc and Grb2, that are thought to lie upstream from the ras protooncogene in a signaling cascade. The phosphorylation and molecular associations of these proteins were evaluated in PC-12 pheochromocytoma cells treated with nerve growth factor (NGF), epidermal growth factor (EGF), and insulin. Both NGF and EGF stimulated the tyrosine phosphorylation of Shc proteins and their subsequent association with the receptors. In contrast, insulin had no effect on Shc phosphorylation, despite the expression of functional insulin receptors in these cells at levels comparable to those observed for NGF and EGF. NGF and EGF also induced the association of Shc proteins with a Grb2 fusion protein or endogenous Grb2, whereas insulin had no effect. All of the tyrosine-phosphorylated Shc proteins associated with the Grb2 fusion protein, although only about half of the endogenous Shc was phosphorylated in response to NGF or EGF. However, all three hormones induced the association of several additional tyrosine phosphorylated proteins with Grb2, some of which also coprecipitated with antiserum against the 85-kDa subunit of phosphatidylinositol-3 kinase. Moreover, these growth factors stimulated the association of phosphatidylinositol-3 kinase activity with the Grb2 fusion protein, although this activity was not detected in anti-Shc immunoprecipitates. These results provide further evidence for the divergence of signaling pathways in insulin action, and suggest that Grb2 forms separate complexes with tyrosine-phosphorylated proteins in PC-12 cells.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Cellular mechanisms of signal transduction for neurotrophins.

The molecular cloning of new neuroactive growth factors and their receptors has greatly enhanced our understanding of important interactions among receptors and signaling molecules. These studies have begun to illuminate some of the mechanisms that allow for specificity in neuronal signaling. Model cell systems, such as the PC-12 pheochromocytoma cell line, express receptors for these different neurotrophic factors, leading to comparisons of signaling pathways for these factors. Upon binding their ligands, these receptors undergo phosphorylation on tyrosine residues, which directs their interaction with signaling proteins containing src homology (SH2) domains, sequences that mediate associations with tyrosine-phosphorylated proteins. These SH2 proteins translate the tyrosine kinase activity of receptors into downstream events that result in the specific cellular response. Investigations such as these have revealed that molecular specificity in signaling pathways may arise from combinatorial diversity in interactions between receptors and key regulatory proteins.

Amino Acid Sequence↗

PubMed1994

c-Myc does not require max for transcriptional activity in PC-12 cells.

The c-Myc proto-oncogene is a basic helix-loop-helix leucine zipper (b/HLH/LZ) protein that participates in cellular growth and differentiation. The expression of c-Myc mRNA is rapidly induced by nerve growth factor (NGF) and epidermal growth factor (EGF) in PC-12 pheochromocytoma cells. In most cell types, c-Myc forms a sequence-specific DNA binding complex with the stable, constitutively expressed Max. This complex can function as a transcriptional regulator. We show here that the expression of Max mRNA or protein was not detected in PC-12 cells. Nevertheless, treatment of PC-12 cells with NGF and serum caused an increase in the expression of the c-Myc protein and the transcription of a reporter gene linked to the Myc/Max DNA binding site. Transcription from the same reporter gene is stimulated by over-expression of c-Myc. These results suggest that c-Myc protein functions as a transcriptional regulator in PC-12 cells despite the lack of Max protein. Therefore, Myc/Max complexes may not be an absolute requirement for Myc-dependent gene expression.

Animals↗