©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Neuronal Protein Tyrosine Phosphatase Induced by Nerve Growth Factor (*)

(Received for publication, July 25, 1994; and in revised form, November 2, 1994)

Ela Sharma Paul J. Lombroso (§)

From the Child Study Center, Yale University School of Medicine, New Haven, Connecticut 06520-7900

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A new protein tyrosine phosphatase (PC12-PTP1) was identified in nerve growth factor (NGF)-treated PC12 cells. The mRNA level of PC12-PTP1 is increased 9-fold over the initial 8 h of NGF treatment and then decreases dramatically after 24 h of treatment. In rat brain, three transcripts corresponding to 1.5, 2.6, and 3.0 kilobases (kb) in size are detected by Northern blot analysis. Although the 1.5- and 2.6-kb transcripts are present in brain and other tissues, the 3-kb transcript is exclusively expressed in brain and the expression of this transcript alone increases following NGF treatment. PC12-PTP1 is a non-receptor protein tyrosine phosphatase (PTP) with a 50% sequence homology in the phosphatase domain with several other non-receptor PTPs. PC12-PTP1 fusion protein exhibits tyrosine phosphatase activity, and in vitro translation of the PC12-PTP1 transcript produces a major protein of 39 kDa. The data presented suggest that NGF regulates the expression of PC12-PTP1 during periods of neuronal growth and differentiation.


INTRODUCTION

Several aspects of neuronal differentiation have been studied in the rat pheochromocytoma cell line, PC12, derived from an adrenal tumor of neural crest origin(1) . Treatment of these cells with the neurotrophin nerve growth factor (NGF) (^1)shifts their phenotype from chromaffin-like cells to that of sympathetic neurons. Accompanying this shift is a decrease in cell division, an increase in neurite outgrowth, and an alteration in membrane excitability and neurotransmitter activities(2, 3, 4) . NGF mediates its action by binding to a member of the proto-oncogene family of receptors, trk, resulting in receptor dimerization and autophosphorylation at tyrosine residues ((5) ; reviewed in (6) ). A cascade of phosphorylation events then follows in which multiple target proteins become phosphorylated on tyrosine and serine/threonine residues leading to the stimulation of phospholipase C1(7, 8) , protein kinase C(9, 10) , high molecular weight microtubule-associated protein kinase(11) , and mitogen-activated protein (MAP) kinase(12) , the formation of Ras-GTP complex(13) , and the induction of early response genes(14) .

Protein tyrosine phosphorylation has been demonstrated to play an important role in the regulation of neural growth and differentiation (reviewed in (15) and (16) ). This mechanism is tightly controlled by the opposing activities of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs). The PTPs and PTKs are classified by their structural organization. Receptor-linked PTPs and PTKs contain a large extracellular domain, a single transmembrane domain, and an intracellular region consisting of one or two catalytic domains. Non-receptor PTPs and PTKs lack an extracellular and transmembrane domain and contain a single catalytic domain.

Although the role of PTKs in mediating the cellular responses to NGF has been well characterized (reviewed in (6) and (17) ), relatively less is understood about the involvement of PTPs. In recent studies of PC12 cells treated with NGF, three PTPs have been identified by biochemical fractionation methods that are induced by 2-3-fold (18) , and the mRNA expression of leukocyte common antigen-related receptor-linked PTP shows a 2-fold increase(19) . In both of these studies, PC12 cells were treated with NGF for time points over 24 h. An additional study demonstrated that the tyrosine phosphatase inhibitor orthovanadate can inhibit the NGF-induced differentiation of PC12 cells (20) .

In previous studies, a striatal enriched phosphatase (STEP) was cloned and shown to be highly enriched within the central nervous system (21) where it is mainly localized in dopaminoceptive neurons of the basal ganglia and related structures(22) . In order to identify potential PTPs that might be involved in mediating the early effects of NGF, we screened an NGF-treated (5-10 h) PC12 cDNA library with the STEP cDNA probe. We report here the isolation and characterization of a novel PTP (PC12-PTP1) whose expression is induced by NGF treatment of PC12 cells. The maximal induction is observed after 8 h of NGF treatment, suggesting that PC12-PTP1 may play a role in the early stages of neuronal differentiation.


MATERIALS AND METHODS

Isolation and Sequencing of PC12-PTP1

PC12 cells were treated with NGF for 5-10 h and an oligo(dT)-primed cDNA library was constructed using ZapII as vector (Stratagene) (library provided courtesy of Dr. Moses Chao). The library was screened with a near full-length STEP cDNA fragment(21) . In order to isolate STEP or STEP-related PTPs, the following conditions were used for this initial screening: 50% formamide, 5 times Denhardt's solution, 5 times SSC, 1% SDS, 100 µg/ml boiled salmon sperm DNA at 42 °C, followed by washes in 1 times SSC, 0.1% SDS at 55 °C. A 3-kb clone that contained the entire open reading frame, as revealed by DNA sequencing, was isolated, purified, and used for subsequent studies.

The nucleotide sequence on both strands of the isolated insert was determined by the chain termination method using Sequenase reagents (version 2, U. S. Biochemical Corp.). Sequence analyses and comparisons with STEP were performed using the software package MacVector (United Biotechnologies), and homologies with other sequences were determined using the GCG software package (University of Wisconsin).

In Vitro Translation

The full-length sense strand of PC12-PTP1 cDNA was transcribed using T(3) RNA polymerase (Stratagene), while the control, antisense message was transcribed by T(7) RNA polymerase. In vitro translation in a rabbit reticulocyte system (Stratagene) was carried as described previously(21) .

Construction and Purification of PC12-PTP1 Fusion Protein

PC12-PTP1 cDNA spanning from 559 bp to 2100 bp was amplified by polymerase chain reaction and cloned in-frame into the polylinker region of the bacterial expression vector pGEX-2T(23) . The fusion protein was purified after induction with isopropyl-beta-galactopyranoside using standard techniques(22) . As a control, glutathione S-transferase protein was similarly purified. The predicted size of the fusion protein was confirmed by SDS-polyacrylamide gel electrophoresis and Coomassie staining.

Phosphatase Activity Assay

Phosphatase activity of PC12-PTP1 fusion protein toward the substrate para-nitrophenyl phosphate (pNPP) was determined as described previously(22) . Phosphorylation of poly(GlubulletTyr) (4:1) with c-Src (Oncogene Sciences) and subsequent dephosphorylation with PC12-PTP1 was carried according to published protocols(24, 25) .

Cell Culture

PC12 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 5% horse serum, and 100 units of penicillin and 100 µg/ml streptomycin (all ingredients from Life Technologies, Inc.) at 37 °C in a humidified atmosphere of 5% CO(2). After plating in 100-cm^2 Petri dishes at 50% confluence, the cells were treated the following day with NGF (Bioproduct for Sciences) (100 ng/ml) and maintained in NGF for varying time intervals before being harvested for RNA isolation.

Northern Analyses

Total RNA was isolated from PC12 cells (RNAzol kit, Tel-Test) following manufacturer's protocol. Twenty-five micrograms of total RNA were electrophoresed on a 1.5% formaldehyde/agarose gel and transferred to nylon membranes (N-Bond, Amersham Corp.). Hybridizations were performed overnight at 42 °C using randomly labeled probes in 50% formamide, 5 times Denhardt's solution, 5 times SSC, 1% SDS, 100 µg/ml boiled salmon sperm DNA. Washes were carried under high stringency conditions as described previously(21) : 1 times SSC, 0.1% SDS, room temperature; 1 times SSC, 0.1% SDS, 65 °C; 0.5 times SSC, 0.1% SDS, 65 °C; 0.1 times SSC, 0.1% SDS, 65 °C. For Northern blot analyses, several probes were employed including a polymerase chain reaction-amplified 1.8-kb PC12 cDNA fragment from 310 bp to 2262 bp (Fig. 1A), a 2.5-kb STEP cDNA fragment, and a 4-kb cDNA corresponding to the 28 S RNA subunit (21) . Poly(A) mRNA from various rat tissues was obtained as a blot (Clontech) for additional Northern analyses.



Figure 1: Sequence of PC12-PTP1. A, complete nucleotide and amino acid sequence of rat PC12-PTP1. The 12-amino acid consensus sequence of the catalytic site in the phosphatase domain is boxed. Five potential initiating methionines are underlined. Polyadenylation signal is in bold. *, stop codon. B, alignment of PC12-PTP1 amino acid sequence with rat striatal enriched PTP (rSTEP; (21) ), human hematopoietic PTP (hHePTP; (29) ), and human leukocyte PTP (hLCPTP; (30) ). The letters in the consensus sequence represent amino acids that are identical in all four PTPs. Dots denote amino acids conserved in at least three of the PTPs. The amino acids between the arrows demarcate the approximately 240-amino acid phosphatase domain. The 12-amino acid catalytic domain is underlined.




RESULTS AND DISCUSSION

We screened an NGF-treated PC12 library, using the brain-specific PTP STEP as a probe, and isolated a new PTP termed PC12-PTP1. The DNA of a 3-kb cDNA clone was purified and sequenced in both directions. The presence of an in-frame stop codon located immediately upstream of the putative initiating methionine as well as the presence of a polyadenylation signal followed by a poly(A) tail suggested that the full-length cDNA had been obtained (Fig. 1A). In addition, the size of the cDNA clone is in close agreement with the size of the message seen on Northern blot analysis (see below).

PC12-PTP1 has a predicted open reading frame of 412 amino acids, beginning at the first available ATG at base pair 563 (Fig. 1A). The highly conserved 12-amino acid catalytic sequence present in all PTPs reported to date (26) is highlighted. The absence of a membrane-spanning, hydrophobic region at its N-terminal end, and the presence of a single phosphatase domain suggests that PC12-PTP1 is a non-receptor intracellular PTP. All previously cloned PTPs from PC12 cells are members of the receptor-linked family(19, 27, 28) .

PC12-PTP1 is a new and distinct member of the PTP family of enzymes. All members of this family share a conserved phosphatase domain of approximately 240 amino acids. Amino acid similarities of up to 65% are present in this region among different PTPs(26) . The 12-amino acid catalytic sequence within this domain shows an even higher degree of homology of greater than 75%. Comparison of PC12-PTP1 and STEP amino acid sequences revealed a 40% sequence identity and a 46% similarity over the entire amino acid sequence (Fig. 1B). The region of highest identity was found within the 240-amino acid conserved phosphatase domain (57%). Further alignment of PC12-PTP1 amino acid sequence with GenBank gave the highest similarity scores of 49% with two human non-receptor PTPs, hematopoietic PTP (HePTP; (29) ), and leukocyte PTP (LCPTP; (30) ), which are very similar to each other. Regions of highest identity between PC12-PTP1 and these two PTPs are again localized within the phosphatase domain (56%). In addition, these PTPs (STEP, HePTP, and LCPTP) have different transcript sizes and tissue distributions from those observed with PC12-PTP1 transcripts on high stringency Northern blot analyses(21, 29, 30) . Taken together, the results suggest that different genes encode for PC12-PTP1 and these other PTPs.

The predicted size of PC12-PTP1 protein is 47 kDa from the first potential initiator methionine. To determine the size of the PC12-PTP1 protein, in vitro translation was carried out using a rabbit reticulocyte translation system. The full-length sense strand of PC12-PTP1 cDNA transcribed under the control of a T(3) polymerase promoter synthesized a major protein corresponding to 39 kDa and four less prominent proteins of larger sizes (Fig. 2). The existence of these minor proteins can be best explained by the presence of five potential initiator methionines within the first 400 bases of the open reading frame (Fig. 1, underlined). Although only one of these is preferentially utilized to produce the major protein product of 39 kDa, the four other methionines appear to also initiate minimal transcription in the in vitro translation system. The generation of PC12-PTP1-specific antibodies will determine the actual protein size in vivo.


Figure 2: In vitro translation of PC12-PTP1 RNA transcripts. In vitro transcribed RNA was translated in a rabbit reticulocyte system in the presence of [S]methionine and analyzed on a 15% SDS-polyacrylamide gel electrophoresis. Lane 1, no added RNA; lane2, 2 µg of antisense RNA; lane3, 2 µg of sense RNA. Autoradiograph was exposed overnight.



The phosphatase activity of PC12-PTP1 fusion protein toward the substrate pNPP is demonstrated in Fig. 3. PC12-PTP1 activity was strongly inhibited by nanomolar concentrations of sodium vanadate (IC = 50 nM) and ammonium molybdate (IC = 1 µM), potent inhibitors of all biochemically characterized PTPs. The enzymatic activity of PC12-PTP1 was not inhibited by heparin, poly(GlubulletTyr) (4:1), or ZnCl(2), which are known to inhibit PTP1B(31) , PTP5(25) , and a PTP associated with the acetylcholine receptor(24) . Similar results were observed previously for STEP(22) . Modulators of serine/threonine phosphatases such as CaCl(2), MgCl(2), and NaF had minimal affect on PC12-PTP1 activity. Tyrosine phosphatase activity of PC12-PTP1 was also confirmed by the dephosphorylation of c-Src-phosphorylated poly(GlubulletTyr) (4:1) (data not shown).


Figure 3: Phosphatase activity of PC12-PTP1. PC12-PTP1 fusion protein was affinity-purified and assayed for phosphatase activity. Glutathione S-transferase alone was used as a control and had no detectable phosphatase activity.



To elucidate the role of PC12-PTP1, PC12 cells were treated with NGF for varying time intervals and analyzed for mRNA expression (Fig. 4). A 2-fold increase in PC12-PTP1 mRNA was evident after 2 h of NGF treatment as compared to untreated cells. A maximal increase of 9-fold was observed after 8 h of treatment followed by a decrease in message by 24 h. Continued NGF treatment for 72 h did not have a further affect on PC12-PTP1 mRNA expression. It should be pointed out that in this study we have only shown an increase in mRNA levels in response to NGF. Specific antibodies to PC12-PTP1 will demonstrate whether the increase in transcription shown here is followed by a corresponding increase in protein product and enzymatic activity.


Figure 4: Northern blot analysis of PC12-PTP1 mRNA expression in PC12 cells. PC12 cells were treated with NGF (100 µg/ml) for the indicated time periods. 0 h time point corresponds to untreated cells. 25 µg of total RNA was loaded/lane. The blot was probed with a 2.5-kb randomly labeled PC12-PTP1 cDNA and washed under high stringency conditions. The experiment was repeated four times with similar results observed. The location of RNA size markers (in kb) is shown on the left. As a control for RNA quantity, the identical blot was hybridized with a cDNA corresponding to the 28 S RNA subunit (lower panel). Autoradiographs were exposed overnight and then quantified using a Ultrascan Densitometer and normalized for the amount of total RNA loaded.



The distribution of PC12-PTP1 mRNA in rat brain and several peripheral tissues was determined (Fig. 5). In brain, three messages of approximately 1.5, 2.6, and 3 kb were detected. The 3-kb transcript was brain-specific and the only one induced by NGF. The 2.6-kb transcript was found enriched in brain and was detectable in kidney, while the 1.5-kb transcript was enriched in heart, kidney, and skeletal muscle with a weak expression in brain and lung. One possible explanation for the existence of three transcripts is that they represent alternatively spliced transcripts of a single gene. That the observed transcripts are not the result of cross-hybridization with previously characterized PTPs is suggested by the high stringency conditions used on all Northern analyses. In addition, the most closely homologous PTPs (STEP, HePTP, and LCPTP) have different transcript sizes and tissue distributions from those observed with PC12-PTP1 transcripts(21, 29, 30) .


Figure 5: Northern blot analyses of PC12-PTP1 mRNA expression in various rat tissues. 2 µg of poly(A) RNA from various tissues was loaded/lane. Blots were hybridized with a 2.5-kb randomly labeled PC12-PTP1 cDNA. Lower panel is 28 S RNA subunit control. Autoradiograph was exposed for 24 h. Prolonged exposure for 3 days did not reveal any additional messages.



A number of others PTPs, including CD45 (32) and two receptor-linked PTPs recently reported in PC12 cells, have alternatively spliced transcripts(27, 30) . Alternative splicing has also been suggested to occur with leukocyte common antigen-related receptor-linked PTP (19) and STEP(21, 22) . To further study this possible explanation, Southern blot analysis of rat genomic DNA was performed. Rat genomic DNA was digested with either BamHI or EcoRI and hybridized under high stringency conditions with the full-length PC12-PTP1 cDNA. Two DNA fragments were detected with each of the digest (data not shown). As both of these restriction enzymes have single sites within the PC12-PTP1 cDNA, these preliminary results are consistent with the hypothesis that the three transcripts are the product of a single gene. Isolation and sequencing of the cDNAs corresponding to the two additional observed transcripts (1.5 and 2.6 kb) will be necessary to establish their relationship to the 3-kb transcript induced by NGF.

NGF-induced morphological differentiation of PC12 cells, indicated by neurite outgrowth, is observed after 48-72 h of treatment, whereas maximal induction of PC12-PTP1 is observed after 8 h of NGF treatment. Although the precise signal transduction mechanism following the binding of NGF to its neuronal cell surface receptor is not fully understood, some of the known early responses are phosphorylation of specific proteins(33) , induction of early response genes (14) and MAP kinase activator(12) , and formation of Ras-GTP complex(15) . Activation of MAP kinase activator occurs within 30 s returning to base-line level by 20 min, while the Ras-GTP complex is formed within 2 min and disappears after 10 min. The early response genes are stimulated within 5 min with an induction peak by 2-3 h. These latter results are consistent with the observed initial induction of PC12-PTP1 after 2 h of NGF treatment. Many of these early responses to NGF treatment are also seen after stimulation of PC12 cells with epidermal growth factor and basic fibroblast growth factor(20, 34) . At present, it is not known whether the induction of PC12-PTP1 is specific to NGF or will also be seen in response to these other growth factors.

The longer term responses to NGF, such as induction of a high molecular weight microtubule-associated protein (35) and the incorporation of fucose into Thy-1 glycoprotein(36) , occur over a time course of hours to days. This time course is more consistent with the observed induction of PC12-PTP1 after 2 h and its decline by 24 h. Although we do not yet have a mechanism to explain the role(s), if any, played by PC12-PTP1 in these events, it is clear that PC12-PTP1 mRNA is regulated by NGF.

In conclusion, we report here a PTP transcript (3 kb) that is exclusively expressed in the brain and induced by NGF. We also describe the time course induction of mRNA expression in response to varying periods of NGF treatment in PC12 cells. Further insight into the mechanisms that determine neuronal responses to NGF will be gained by clarifying the role of PC12-PTP1 in NGF signaling pathways and identifying its substrates.


FOOTNOTES

*
This work was supported by NIMH Grants MH00856 and MH49351, and the NARSAD Foundation (to P. J. L.), and NIMH Postdoctoral Training Fellowship Grant MH18268 (to E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U14914[GenBank].

§
To whom reprint requests should be addressed: Child Study Center, Yale University School of Medicine, 230 S. Frontage Rd., New Haven, CT 06520-7900. Tel.: 203-737-2224; Fax: 203-785-7611.

(^1)
The abbreviations used are: NGF, nerve growth factor; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; STEP, striatum-enriched phosphatase; MAP, mitogen-activated protein; pNPP, para-nitrophenyl phosphate; kb, kilobase(s); bp, base pair(s).


ACKNOWLEDGEMENTS

We are very grateful to Moses Chao for providing the PC12 cDNA library. We thank Drs. Janice Naegele, Flora Vaccarino, and Lawrence Robel for their helpful comments on the manuscript.


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