Induction of Specific Protein Tyrosine Phosphatase Transcripts during Differentiation of Mouse Erythroleukemia Cells*

We reported previously that most of the phosphotyro- sine-containing cellular proteins were quickly dephosphorylated at the very early stage of erythroid differen- tiation of mouse erythroleukemia (MEL) cells. These and other experimental results implicated a specific protein tyrosine phosphatase(s) (PTPase(s)) involved in the commitment of the erythroid differentiation. We have investigated the pattern of transcripts of PTPases during MEL cell differentiation and found that while the transcripts of most PTPases were unchanged or undetected in the cells, transcripts for two PTPases (PTPp2 and RIP) exhibited distinct patterns of induction at a very early stage of differentiation. Some of the mutant cells defective in differentiation did not show the induc- tion of these PTPase transcripts. We discuss the possible role played by the PTPases in the commitment of MEL cell differentiation.

. This suggests that the molecular cascade is diversified at the initial stage but converges to a common and critical step for cellular commitment to differentiation. Previously, we reported that a series of inhibitors of protein tyrosine kinases are very effective inducers of MEL cell differentiation (6,(16)(17)(18). More recently, we Ministry of Education (to M. 0.) and the Howard Hughes Medical In-* This work was supported by research grants from the Japanese stitute (to M. L. T.). The costs of publication of this article were defrayed hereby marked "uduertisernent" in accordance with 18 U.S.C. Section in part by the payment of page charges. This article must therefore be 1734 solely to indicate this fact.
showed that phosphotyrosine moieties of most of the cellular phosphotyrosine-containing proteins were dephosphorylated at the very early stage of MEL cell differentiation and that Na3V04, a specific inhibitor of protein tyrosine phosphatases (PTPases) (9), prevented the dephosphorylation and erythroid differentiation (8). Mutant MEL cells resistant (defective) to differentiation were also resistant to dephosphorylation (8).
The in vitro interaction between two differentiation inducing factors also indicated the involvement of a dephosphorylation step in MEL cell differentiation (10). Based upon these results, we are inclined to conclude that phosphotyrosine dephosphorylation of specific cellular proteins is a critical reaction that results in the commitment of MEL cell differentiation.
To identify a specific PTPase(s) that is involved in the dephosphorylation step, we examined the level of transcripts of 16 PTPases during MEL cell differentiation. Here we report that while the transcripts of most of the PTPases were unchanged or undetected in the cells, several specific PTPase transcripts exhibited distinct patterns of induction. Some of the mutant MEL cells resistant to differentiation did not show such alterations.
The possible role of the PTPases in MEL cell differentiation is discussed.
EXPERIMENTAL PROCEDURES Muterials-HMBA was a gift from Dr. T. Yamane (Bell Laboratories). Mouse monoclonal anti-phosphotyrosine antibody (PY20, lot 32188) and sheep anti-mouse IgG horseradish peroxidase-conjugated antibody were purchased from ICN and Amersham Corp., respectively. Minimal essential medium was purchased from Nissui Seiyaku. Fetal calf serum was obtained from United Biotechnologies.
Western Blot Analysis-Western blot analysis of phosphotyrosinecontaining proteins was performed as described previously (8).
RNA Isolation and Northern Blot Analysis-Exponentially grown MEL cells were exposed to inducing agents when the cell density reached 2-3 x lo6 celldml. Aliquots (200 ml) were withdrawn, and, after washing twice with cold phosphate-buffered saline, the cells (-5 x lo7 cells) were lysed in 200 p1 of RNA extraction buffer and total RNA was prepared as described (11). Poly(A)+ RNA was obtained by Oligotex-dT30 (Takara Shuzo) from the total RNA preparations according to the manufacturer's instruction. For Northern blot analysis, 2 pg of poly(A)+ RNA was electrophoresed on a 1% formaldehyde-agarose gel, blotted onto a Hybond N+ membrane (Amersham), and hybridized to 32P-labeled PTPase cDNAprobes, which were prepared by random priming kit (Boehringer Mannheim) using [a-32PldCTP. The hybridization was carried out overnight at 42 "C in a hybridization buffer (50% formamide, 5 x SSC, 50 m~ Tris-HC1, pH 7.5, 0.1% SDS, and 0.1 mg/ml sonicated herring sperm DNA). The membranes were washed once in 2 x SSC, 0.1% SDS for 10 min at room temperature, once in 2 x SSC, 0.1% SDS for 15 min at 65 "C, twice in 0.1 x SSC, 0.1% SDS for 15 min at 65 "C, and autoradiographed. The cDNA probes used are given in Table I.

RESULTS AND DISCUSSION
As we reported recently, dephosphorylation of phosphotyrosine-containing proteins was observed as early as 12 h after addition of inducers, and most of the phosphotyrosine-containing proteins were dephosphorylated by 24 h of incubation (8). Mutant MEL cells defective in differentiation did not exhibit such extensive dephosphorylation (Fig. 1 At the times (h) indicated, samples were withdrawn and cell-free extracts were prepared. Proteins (50 pg) were separated on SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membrane, and immunoblotted using anti-phosphotyrosine antibody as described under "Experimental Procedures." Na3V04, a specific inhibitor of PTPases, not only inhibited the dephosphorylation but also the differentiation itself (8). As a natural extension of these results, we sought to identify a PTPase(s) that is responsible for the dephophorylation and possibly for triggering the differentiation. Initially, we examined whether the transcripts of any specific PTPases are induced during MEL cell differentiation, particularly at the early stage (0-12 h) of differentiation when the pattern of cellular phosphotyrosine-containing proteins started to be altered. MEL cells were incubated in the presence of Me,SO, a typical erythroid-inducing agent, and poly(A)+ RNA were prepared at different time intervals up to 48 h. Under the condition employed, the differentiation was completed in 100-120 h but the cellular commitment to differentiation should occur by 20-30 h (12). The RNA were then subjected to Northern blot analysis. For the PTPase probes, we used 16 different mammalian PTPase cDNA clones, which included many recently cloned PTPase cDNAs. The cDNA clones used as probes are listed in Table I.
We found that PTPase transcripts were roughly classified into five groups according to their patterns of alteration during the early stage (0-48 h) of differentiation. The patterns of typical PTPase transcripts detected up to 48 h after addition of the inducer is shown in Fig. 2. Transcripts for several PTPases exemplified by LRP and SHP in Fig. 2 were present in the control (uninduced) cells, but their level was virtually unchanged at least for the early stage of differentiation (group 1). PTPases for which transcripts exhibited similar patterns to LRP and SHP include STEP and PTP-S (MPTP) (data not shown). Transcripts for Cdc25M2 (shown in Fig. 2 Fig. 2, transcripts for these PTPases were at very low level before addition of Me2S0, but sharply increased between 6 and 12 h (PTPp2 transcripts exhibited a slight decline at 6 h of incubation). Transcripts for PTPE, which were apparent as two molecular weight forms, decreased im-  2) gradually increased up to 72 h and decreased thereafter. For the group 3, the transcripts for PTPp2 and RIP were maintained at the induced level but the level of CD45 transcripts started to decrease at 24 h and was barely detectable at 120 h.
The P T P E transcripts, which exhibited immediate decline after addition of Me2S0 and sharp recovery to the original level between 18 and 24 h, decreased again thereafter and were not detectable at 120 h of incubation. This pattern of PTPe transcripts was essentially the same as that of c-myc transcripts during MEL cell differentiation as we reported before (13).
Among the PTPases described above, we extended our studies primarily to the ones that exhibited drastic induction at the early stage of differentiation (PTPp2, CD45, RIP, and PTPe).To confirm that the induction of the transcripts was specific to MEL cell differentiation rather than due to one of the physiological effects of Me2S0, poly(A)+ RNA from the cells induced by HMBA, another potent inducer of MEL cell differentiation, was subjected to Northern blot analysis as done before. Quite surprisingly, the level of CD45 transcripts did not increase at all up to 24 h of incubation, whereas those for PTPp2 and RIP increased as observed with the cells treated with Me2S0 (Fig.  4). We, therefore, eliminated CD45 from the candidates of PTPases responsible for MEL cell differentiation. The pattern of PTPe transcripts in the HMBA-treated cells was also similar to that of Me2SO-treated cells (Fig. 4). Essentially the same pattern of alteration of transcripts was observed with PTPp2, RIP and PTPe after treating the cells with another inducer, sodium butyrate, although degradation of poly(A)+ RNA from sodium butyrate-treated cells made a definite conclusion somewhat difficult (data not shown). Thus, among the 16 PTPases so far examined, transcripts for two PTPases, PTPp2 and RIP, exhibited very early and drastic increase in response to inducers of MEL cell differentiation. Importantly, the timing of the induction apparently corresponded to, or preceded, the start of the dephosphorylation of phosphotyrosine-containing proteins.
In order to obtain further evidence for the possible involvement of PTPp2 and/or RIP in MEL cell differentiation, we examined the level of transcripts of PTPp2 and RIP in differentiation-resistant (defective) mutant MEL cells. Poly(A)+ RNA were prepared from four independently isolated mutant MEL cells (Dif-1, -2, -3, and -4) after addition of Me2S04 and subjected to Northern blot analysis. These mutant MEL cells are resistant (defective) to erythroid differentiation, as well as dephosphorylation of phosphotyrosine-containing proteins induced by Me2S0, HMBA, and other typical inducing agents (8). After addition of Me2S0, the transcripts of these PTPases increased in two of the mutant cells, Dif-3 and Dif-4, the pattern being not significantly different from those of the control (745A) cells (data not shown). In Dif-1 and Dif-2, the level of PTPp2 transcripts exhibited a similar pattern to that of the wild type (745A) cells (data not shown); however, no RIP transcripts were detected in Dif-1 even after 48 h of incubation with Me2S0, and significantly lower levels of transcripts were detected in Dif-2 at least up to 12 h of incubation, although the level may be increased at the later stage of differentiation (Fig. 5). Patterns similar to those shown in Fig. 5 were also observed with poly(A)+ RNA from HMBA-treated Dif-1 and -2 cells (data not shown). It seems that the induction of transcription of RIP was affected by the introduction of the mutation(s), which results in the deficiency of erythroid differentiation.
We examined whether the level of any of the PTPase transcripts increased or was altered during MEL cell differentiation. Among 16 PTPases examined, PTPp2 and RIP PTPases exhibited drastic increase of their transcripts at the very early stage of differentiation. Among a number of biochemical, morphological, and physiological changes specific to MEL cell differentiation, dephosphorylation of phosphotyrosine-containing proteins and the increase of specific PTPase transcripts reported here are the earliest events so far known, preceding by at least 10 h the cellular commitment to differentiation that occurs between 20 and 30 h after incubation with inducing agents. One of the cytoplasmic differentiation inducing factor (Dif-11) implicated by our previous cell and cytoplast fusion also reached the maximum approximately 6-8 h after addition of inducers (14,15). The very early increase of the transcripts of these PTPases and coincidence of the timing with the start of the dephosphorylation of phosphotyrosine containing proteins, as well as the induction of the putative differentiation inducing factor, may support the view that the induction of specific PTPases is the earliest molecular event to trigger a cascade of reactions leading to MEL cell differentiation by dephosphorylating specific cellular proteins. In this connection, it is interesting that mutant MEL cells (Dif-1) defective in differentiation apparently lacked the transcripts for RIP and had no signs of their induction after MezSO or HMBA treatment. This suggests that the mutation responsible for the defectiveness in Dif-1 is located either at or before the dephosphorylation step by RIP in the differentiation cascade, although the possibility that the inhibition of the differentiation cascade somehow suppresses the transcription of RIP gene cannot be excluded. From this rather circumstantial evidence alone, it is too early to conclude that RIP andor FTPp2 are responsible for dephosphorylation of phosphotyrosine-containing cellular proteins, which leads to cellular commitment (6,8,(16)(17)(18). We are currently in the process of determining whether expression of PTPp2 and RIP can induce MEL cell differentiation.