BIOCHEMICAL
Vol. 188, No. 3, 1992 No’vember
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
;S, 1992
1305-1311
Differential Regulation of mRNAs Encoding Three Protein-Tyrosine Phosphatases by Insulin and Activation of Protein Kinase C
Naotake Hashimoto and Barry J. Goldstein’ Research Division, Joslin Diabetes Center and Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02215 Received
September
16,
1992
SUMMARY: Protein-tyrosine phosphatases (PTPases) play an essential role in the control of signalling through phosphotyrosine pathways. Since little is known about the regulation of these enzymes, we examined the effect of insulin and phorbol 12-myristate 13-acetate (PMA) treatment of well-differentiated rat hepatoma (Fao) cells on the expression of mRNAs encoding three major PTPase homologs in liver: PTPaselB, an intracellular enzyme with a single conserved PTPase domain, and two tandem-domain, transmembrane PTPases, known as LAR and LRP. Treatment of serum-deprived cells with 100 nM insulin increased the abundance of the 4.3 kb and 1.6 kb mRNAs encoding PTPaselB on Northern analysis by 1.6 and 3.1 -fold, respectively (p (0.02). Similarly, exposure to 100 rig/ml PMA increased the 4.3 and 1.6 kb PTPaselB mRNAs by 4.5 and 5.7-fold, respectively (~50.035). In contrast, treatment with insulin or PMA had no significant effect of the abundance of mRNA encoding either LAR or LRP. PMA appeared to have a transcriptional effect on the PTPaselB gene by a protein kinase C-mediated mechanism. The increase in PTPaselB mRNA expression by insulin and PMA suggests that this PTPase may provide feed-back regulation of signalling through the insulin action pathway as well as a potential link between the action of protein kinase C and the regulation of specific phosphotyrosine residues in cells. 0 199~Academic P~,x;, I-)~
Post-translational modification of proteins by phosphorylation on tyrosine residues is an essential regulatory mechanism for the control of a variety of specialized cellular functions.
Recently, a large family of protein-tyrosine phosphatases (PTPascs; E.C.
3.1.3.48) that reverse the phosphorylation of tyrosyl residues and contribute to the overall control of signal transduction through these pathways has been characterized by enzyme purification and molecular cloning (for recent reviews see refs. 1,2). Our laboratory is particularly interested in PTPases that are involved in the dephosphorylation of phosphotyrosine residues in the signalling pathway for insulin and other related growth tTo whom correspondence should be addressed at Division of Endocrinology and Metabolic Diseases, Jefferson Medical College, Room 349, Alumni Hall, 1020 Locust St. Philadelphia, PA 19107-6799.
Vol.
188, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS . e
factors (3). We have recently identified three PTPase homologs that are highly expressed in liver and muscle and are candidate enzymes for having a physiological role in the regulation of hormone action in these tissues (46).
These enzymes include PTPaselB, which has a
single conserved PTPase domain and is associated with the endoplasmic reticulum (7-lo), and two PTPases that have a receptor-like transmembrane structure, including the tandem PTPase domain enzymes LAR (for Leukocyte Antigen Related) and LRP (for LCA-related phosphatase; also called RPTP-a) (1 l-14). To gain some initial insight into the potential regulation of these PTPases at the level of mRNA expression, we examined the effect of insulin and phorbol 12-my&ate (PMA) on PTPase mRNA expression in well-differentiated
13-acetate
rat hepatoma (Fao) cells. The
mRNA for these PTPases was found to be differentially regulated. Messenger RNA for LAR and LRP was not affected by these agents. In contrast, the mRNAs encoding PTPaselB mRNA were increased by treatment with insulin or PMA, suggesting a potential mechanism for feed-back desensitization of signalling through the insulin action pathway as well as a potential link between the action of protein kinase C and the cellular regulation of specific phosphotyrosine signals. METHODS
AND MATERIALS
Cell Culture: The well-differentiated rat hepatoma Fao cell line (15) was kindly provided by Dr. C. Ronald Kahn (Joslin Diabetes Center). Fao cells were grown in monolayer using RPMI-1640 medium (GIBCO-BRL, Gaithersburg, MD) containing 10% fetal bovine serum to 80% confluence, washed with phosphate-buffered saline (PBS) and maintained in serum-free medium for 30-40 hr prior to the addition of the indicated experimental agent. Northern Blot Analysis: After removing the medium, the cell monolayer was washed with PBS and total RNA was extracted by homogenization in buffered 4M guanidinium thiocyanate, extraction with phenol/chloroform and precipitation from isopropanol (16). Electrophoresis of samples containing 20 pg of total RNA was performed in 1% agarose/O.66M formaldehyde gels as described (17). The gels were rinsed twice for 5 min in water, soaked for 30 min in 0.15M NaCl-O.OSM NaOH, rinsed and soaked again for 30 min in 0.15M NaCl-O.lM Tris-HCl, pH 8.0. Capillary transfer to Duralon-UV nylon membranes (Stratagene, La Jolla, CA) was performed with 10X SSC (1X SSC = 0.15M NaCl in 0.015M Na&I!itrate, pH 7.0). After transfer, the RNA was bound to the membrane by UV crosslinking. Prehybridization was performed for 2 hr at 42” C in a solution containing 50% formamide, 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS), 1M NaCl, and 100 pg/ml denatured salmon sperm DNA. Rat cDNA inserts encoding each of the PTPases were obtained by cDNA amplification or cDNA library screening as described previously (4) and labeled by random hexamer priming with a-[?I-dCTP (18). Hybridization was performed by adding 0.5 to 1 x 106 cpm of probe per ml of prehybridization solution and incubating at 42°C for 20 hr. Filters were washed three times in 2X SSC/O. 1% SDS at room temperature for 15 min each and then in 0.1X SSC/O. 1% SDS 1306
Vol.
188,
No.
3,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
at 55” C for 30 min. The damp filters were then exposed to Kodak X-Omat AR film at with a DuPont Cronex Lightning Plus intensifying screen at -80” C. Data Analysis: Autoradiograms were quantitated by scanning on a computing densitometer (Molecular Dynamics). Sets of data were compared with two-tailed Student’s ttests using Instat software (GraphPad, San Diego, CA).
RESULTS Expression of PTPase mRNAs in rat Fao hepatoma cells: PTPaselB mRNA was expressed as two transcripts of 4.3 and 1.6 kb, as we have found in normal rat tissues (4) and as also reported by Guan et al. (7) (Figure 1). The mRNA transcripts for rat LRP and LAR were expressed in Fao cells as 3.0 kb and 8.0 kb transcripts, respectively, identical to the major mRNA species for these PTPases in normal rat liver tissue (5)‘. The strength of the signal from each of the Northern blots using 20 pg of total RNA is a indication of the relative abundance of mRNA for these PTPases in the hepatoma cells. Effect of insulin on PTPase mRNA expression:
Cells were grown to 80%
confluence and made quiescent by serum starvation for 40h. At that time, insulin was added at 100 nM, and mRNA was prepared from the cells after an additional 3h of incubation. Northern blot analysis showed that the expression of both PTPaselB transcripts was increased significantly by insulin treatment (Figure 1). Quantitation of the Northern blots revealed that the 4.3 kb mRNA was increased by 1.6-fold (n=6; p=O.O20), and the 1.6 kb mRNA was increased by a mean of 3. l-fold (n=6; p
Vol. 188, No. 3, 1992 No’vember
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
;S, 1992
1305-1311
Differential Regulation of mRNAs Encoding Three Protein-Tyrosine Phosphatases by Insulin and Activation of Protein Kinase C
Naotake Hashimoto and Barry J. Goldstein’ Research Division, Joslin Diabetes Center and Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02215 Received
September
16,
1992
SUMMARY: Protein-tyrosine phosphatases (PTPases) play an essential role in the control of signalling through phosphotyrosine pathways. Since little is known about the regulation of these enzymes, we examined the effect of insulin and phorbol 12-myristate 13-acetate (PMA) treatment of well-differentiated rat hepatoma (Fao) cells on the expression of mRNAs encoding three major PTPase homologs in liver: PTPaselB, an intracellular enzyme with a single conserved PTPase domain, and two tandem-domain, transmembrane PTPases, known as LAR and LRP. Treatment of serum-deprived cells with 100 nM insulin increased the abundance of the 4.3 kb and 1.6 kb mRNAs encoding PTPaselB on Northern analysis by 1.6 and 3.1 -fold, respectively (p (0.02). Similarly, exposure to 100 rig/ml PMA increased the 4.3 and 1.6 kb PTPaselB mRNAs by 4.5 and 5.7-fold, respectively (~50.035). In contrast, treatment with insulin or PMA had no significant effect of the abundance of mRNA encoding either LAR or LRP. PMA appeared to have a transcriptional effect on the PTPaselB gene by a protein kinase C-mediated mechanism. The increase in PTPaselB mRNA expression by insulin and PMA suggests that this PTPase may provide feed-back regulation of signalling through the insulin action pathway as well as a potential link between the action of protein kinase C and the regulation of specific phosphotyrosine residues in cells. 0 199~Academic P~,x;, I-)~
Post-translational modification of proteins by phosphorylation on tyrosine residues is an essential regulatory mechanism for the control of a variety of specialized cellular functions.
Recently, a large family of protein-tyrosine phosphatases (PTPascs; E.C.
3.1.3.48) that reverse the phosphorylation of tyrosyl residues and contribute to the overall control of signal transduction through these pathways has been characterized by enzyme purification and molecular cloning (for recent reviews see refs. 1,2). Our laboratory is particularly interested in PTPases that are involved in the dephosphorylation of phosphotyrosine residues in the signalling pathway for insulin and other related growth tTo whom correspondence should be addressed at Division of Endocrinology and Metabolic Diseases, Jefferson Medical College, Room 349, Alumni Hall, 1020 Locust St. Philadelphia, PA 19107-6799.
Vol.
188, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS . e
factors (3). We have recently identified three PTPase homologs that are highly expressed in liver and muscle and are candidate enzymes for having a physiological role in the regulation of hormone action in these tissues (46).
These enzymes include PTPaselB, which has a
single conserved PTPase domain and is associated with the endoplasmic reticulum (7-lo), and two PTPases that have a receptor-like transmembrane structure, including the tandem PTPase domain enzymes LAR (for Leukocyte Antigen Related) and LRP (for LCA-related phosphatase; also called RPTP-a) (1 l-14). To gain some initial insight into the potential regulation of these PTPases at the level of mRNA expression, we examined the effect of insulin and phorbol 12-my&ate (PMA) on PTPase mRNA expression in well-differentiated
13-acetate
rat hepatoma (Fao) cells. The
mRNA for these PTPases was found to be differentially regulated. Messenger RNA for LAR and LRP was not affected by these agents. In contrast, the mRNAs encoding PTPaselB mRNA were increased by treatment with insulin or PMA, suggesting a potential mechanism for feed-back desensitization of signalling through the insulin action pathway as well as a potential link between the action of protein kinase C and the cellular regulation of specific phosphotyrosine signals. METHODS
AND MATERIALS
Cell Culture: The well-differentiated rat hepatoma Fao cell line (15) was kindly provided by Dr. C. Ronald Kahn (Joslin Diabetes Center). Fao cells were grown in monolayer using RPMI-1640 medium (GIBCO-BRL, Gaithersburg, MD) containing 10% fetal bovine serum to 80% confluence, washed with phosphate-buffered saline (PBS) and maintained in serum-free medium for 30-40 hr prior to the addition of the indicated experimental agent. Northern Blot Analysis: After removing the medium, the cell monolayer was washed with PBS and total RNA was extracted by homogenization in buffered 4M guanidinium thiocyanate, extraction with phenol/chloroform and precipitation from isopropanol (16). Electrophoresis of samples containing 20 pg of total RNA was performed in 1% agarose/O.66M formaldehyde gels as described (17). The gels were rinsed twice for 5 min in water, soaked for 30 min in 0.15M NaCl-O.OSM NaOH, rinsed and soaked again for 30 min in 0.15M NaCl-O.lM Tris-HCl, pH 8.0. Capillary transfer to Duralon-UV nylon membranes (Stratagene, La Jolla, CA) was performed with 10X SSC (1X SSC = 0.15M NaCl in 0.015M Na&I!itrate, pH 7.0). After transfer, the RNA was bound to the membrane by UV crosslinking. Prehybridization was performed for 2 hr at 42” C in a solution containing 50% formamide, 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS), 1M NaCl, and 100 pg/ml denatured salmon sperm DNA. Rat cDNA inserts encoding each of the PTPases were obtained by cDNA amplification or cDNA library screening as described previously (4) and labeled by random hexamer priming with a-[?I-dCTP (18). Hybridization was performed by adding 0.5 to 1 x 106 cpm of probe per ml of prehybridization solution and incubating at 42°C for 20 hr. Filters were washed three times in 2X SSC/O. 1% SDS at room temperature for 15 min each and then in 0.1X SSC/O. 1% SDS 1306
Vol.
188,
No.
3,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
at 55” C for 30 min. The damp filters were then exposed to Kodak X-Omat AR film at with a DuPont Cronex Lightning Plus intensifying screen at -80” C. Data Analysis: Autoradiograms were quantitated by scanning on a computing densitometer (Molecular Dynamics). Sets of data were compared with two-tailed Student’s ttests using Instat software (GraphPad, San Diego, CA).
RESULTS Expression of PTPase mRNAs in rat Fao hepatoma cells: PTPaselB mRNA was expressed as two transcripts of 4.3 and 1.6 kb, as we have found in normal rat tissues (4) and as also reported by Guan et al. (7) (Figure 1). The mRNA transcripts for rat LRP and LAR were expressed in Fao cells as 3.0 kb and 8.0 kb transcripts, respectively, identical to the major mRNA species for these PTPases in normal rat liver tissue (5)‘. The strength of the signal from each of the Northern blots using 20 pg of total RNA is a indication of the relative abundance of mRNA for these PTPases in the hepatoma cells. Effect of insulin on PTPase mRNA expression:
Cells were grown to 80%
confluence and made quiescent by serum starvation for 40h. At that time, insulin was added at 100 nM, and mRNA was prepared from the cells after an additional 3h of incubation. Northern blot analysis showed that the expression of both PTPaselB transcripts was increased significantly by insulin treatment (Figure 1). Quantitation of the Northern blots revealed that the 4.3 kb mRNA was increased by 1.6-fold (n=6; p=O.O20), and the 1.6 kb mRNA was increased by a mean of 3. l-fold (n=6; p
