(Received for publication, July 25, 1994; and in revised form, November 2, 1994)
From the
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.
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) ()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
C
1(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.
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).
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.
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 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(Glu
Tyr) (4:1), or
ZnCl
, 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
, MgCl
, 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(Glu
Tyr) (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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U14914[GenBank].