Ribosomal protein S6 phosphorylation and function during late gestation liver development in the rat.

The phosphorylation of ribosomal protein S6 is thought to be required for biosynthesis of the cell's translational apparatus, a critical component of cell growth and proliferation. We have studied the signal transduction pathways involved in hepatic S6 phosphorylation during late gestation in the rat. This is a period during which hepatocytes show a high rate of proliferation that is, at least in part, independent of mitogenic signaling pathways that are operative in mature hepatocytes. Our initial studies demonstrated that there was low basal activity of two S6 kinases in liver, S6K1 and S6K2, on embryonic day 19 (2 days preterm). In addition, insulin- and growth factor-mediated S6K1 and S6K2 activation was markedly attenuated compared with that in adult liver. Nonetheless, two-dimensional gel electrophoresis demonstrated that fetal liver S6 itself was highly phosphorylated. To characterize the fetal hepatocyte pathway for S6 phosphorylation, we went on to study the sensitivity of hepatocyte proliferation to the S6 kinase inhibitor rapamycin. Unexpectedly, administration of rapamycin to embryonic day 19 fetuses in situ did not affect hepatocyte DNA synthesis. This resistance to the growth inhibitory effect of rapamycin occurred even though S6K1 and S6K2 were inhibited. Furthermore, fetal hepatocyte proliferation was sustained even though rapamycin administration resulted in the dephosphorylation of ribosomal protein S6. In contrast, rapamycin blocked hepatic DNA synthesis in adult rats following partial hepatectomy coincident with S6 dephosphorylation. We conclude that hepatocyte proliferation in the late gestation fetus is supported by a rapamycin-resistant mechanism that can function independently of ribosomal protein S6 phosphorylation.

Identification of candidate growth-regulating genes that are overexpressed in late gestation fetal liver in the rat.

We have shown previously that hepatocyte proliferation in the late gestation fetal rat is mediated by growth factor-independent mechanisms that are distinct from the signaling pathways that promote proliferation of adult rat hepatocytes. In the present studies, we identified six candidate growth-regulating genes that are overexpressed in fetal rat liver (embryonic day 19, 2 days pre-term) relative to adult rat liver using suppressive subtractive hybridization. These included the following: Grb10, a growth factor receptor binding protein; eps15, a growth factor receptor substrate; nuc2+, a retinoblastoma protein binding protein; cdc25B, a cell cycle tyrosine phosphatase; the peroxisome proliferator-activated receptor PPAR alpha; and a deoxyuridine triphosphatase that functions as a PPAR alpha binding partner. In every case, the ontogeny of the expression of these genes declined postnatally in a manner consistent with the transition from a fetal to an adult hepatocyte phenotype. None were found to be cell cycle-dependent, in that they did not show expression that followed perinatal changes in hepatocyte cell cycle activity. Based on our identification of these genes and previous work characterizing their role in growth regulation, we conclude that they may contribute to the mitogenic signaling phenotype of fetal rat hepatocytes.

Growth regulation via p38 mitogen-activated protein kinase in developing liver.

During normal development in the rat, hepatocytes undergo marked changes in the rate of proliferation. We have previously observed transient G(1) growth arrest at term, re-activation of proliferation immediately after birth, and a gradual transition to the quiescent adult hepatocyte phenotype after postnatal day 4. We hypothesized that these changes in proliferation are due in part to growth inhibitory effects mediated by the p38 mitogen-activated protein kinase pathway. p38 kinase activity measurements showed an inverse relationship with hepatocyte proliferation during the perinatal and postnatal transitions, whereas p38 content remained constant. Anisomycin activated the p38 pathway in fetal hepatocyte cultures while inducing growth inhibition that was sensitive to the p38 inhibitor, SB203580. Activation of p38 in these cultures, via transient transfection with a constitutively active form of its upstream kinase MKK6, also inhibited DNA synthesis as well as reducing cyclin D1 content. Transfection with inactive MKK6 did neither. Furthermore, MKK6-induced growth arrest was sensitive to SB203580. Finally, administration of SB203580 to near-term fetal rats in utero abrogated the transient hepatocyte growth arrest that occurs at term. These findings indicate a role for the p38 mitogen-activated protein kinase pathway in the physiological regulation of hepatocyte proliferation during normal development in the rat.

Hepatic epidermal growth factor-regulated mitogen-activated protein kinase kinase kinase activity in the rat: lack of identity with known forms of raf and MEKK.

Mitogenic signaling involves protein kinases that phosphorylate the mitogen-activated protein kinase (MAPK) activator, MEK. In rats, basal hepatic MEK kinase activity is low in vivo in both adult rats and late gestation fetal rats, and is markedly stimulated by intraperitoneal administration of epidermal growth factor (EGF). The level of stimulated MEK phosphorylating activity is approximately 15 times higher in fetal liver than in adult liver. To identify regulated forms of the two categories of MEK kinase, Raf and MEKK, Western immunoblotting, immunoprecipitation kinase assays and immunodepletion studies were performed. Western immunoblotting confirmed that Raf-1, A-Raf, B-Raf, MEKK1 and MEKK2 were present at similar levels in E19 and adult liver. However, specific immunoprecipitation kinase assays did not detect any kinases that could account for marked EGF sensitivity or the higher level of activity in E19 fetuses. Immunodepletion studies produced a marked reduction in immunoreactive Raf/MEKK content and activity, but a minimal decrease in the ability of chromatography fractions to phosphorylate and activate recombinant MEK-1. Our results indicate that hepatic, EGF-sensitive MEK kinase activity may reside with a previously unidentified and physiologically relevant form of Raf and/or MEKK.

Uncoupling of hepatic, epidermal growth factor-mediated mitogen-activated protein kinase activation in the fetal rat.

Stimulation of cell proliferation by mitogens involves tyrosine phosphorylation of proteins at the cell membrane by receptor tyrosine kinases. This promotes formation of multi-protein complexes that can activate the small G-protein, Ras. Activation of Ras, in turn, leads to sequential activation of the following three serine-threonine kinases: Raf, extracellular signal-regulated kinase kinase (MEK), and members of the family of mitogen-activated protein (MAP) kinases. Prior studies have shown that intraperitoneal injection of epidermal growth factor (EGF) leads to rapid activation of hepatic MAP kinases in adult rats but not in late gestation (E19) fetal rats (Boylan, J. M., and Gruppuso, P. A. (1996) Cell Growth & Differ. 7, 1261-1269). The present studies were undertaken to determine the mechanism for this "uncoupling" of the MAP kinase pathway. E19 fetal rats and adult male rats were injected with EGF (0.5 microg/g body weight, intraperitoneally) or with saline. After 15 min, livers were removed and prepared for kinase analyses. EGF injection led to a rapid and marked activation of hepatic Raf and MEK in both fetal and adult rats, whereas MAP kinase activation was minimal in fetal as opposed to adult rats. Examination of the ontogeny of this dissociation of MAP kinase activation from MEK activation showed gradual acquisition of intact signaling as an adult hepatocyte phenotype was attained during the first 4 postnatal weeks. Over this period, MAP kinase content as determined by Western immunoblotting was constant. Recombination experiments using partially purified fetal and adult rat liver MEK and MAP kinase showed intact MAP kinase activation in vitro, indicating that neither enzyme was irreversibly altered in the fetus. In studies using primary cultures of E19 fetal rat hepatocytes, uncoupling of MAP kinase activation from MEK activation could be induced by incubation of fetal hepatocytes for 24 h with a potent fetal hepatocyte mitogen, transforming growth factor-alpha. These findings indicate that a novel negative feedback mechanism for MAP kinase regulation may be active in developing rat hepatocytes.

Modulation of mitogen-independent hepatocyte proliferation during the perinatal period in the rat.

Late gestation fetal rat hepatocytes can proliferate under defined in vitro conditions in the absence of added mitogens. However, this capacity declines with advancing gestational age of the fetus from which the hepatocytes are derived. The present studies were undertaken to investigate this change in fetal hepatocyte growth regulation. Examination of E19 fetal hepatocyte primary cultures using immunocytochemistry for 5-bromo-2'-deoxyuridine (BrdU) incorporation showed that approximately 80% of these cells traverse S-phase of the cell cycle over the first 48 h in culture. Similarly, 65% of E19 hepatocytes maintained in culture under defined mitogen-free conditions for 24 h showed nuclear expression of proliferating cell nuclear antigen (PCNA). These in vitro findings correlated with a high level of immunoreactive PCNA in immunofluorescent analyses of E19 liver. In contrast, E21 (term) liver showed little immunoreactive PCNA. The in vivo finding was recapitulated by in vitro studies showing that E21 hepatocytes had low levels of BrdU incorporation during the first day in culture and were PCNA negative shortly after isolation. However, within 12 h of plating, E21 hepatocytes showed cytoplasmic staining for PCNA. Although maintained under mitogen-free conditions, PCNA expression progressed synchronously to a nucleolar staining pattern at 24 to 48 h in culture followed by intense, diffuse nuclear staining at 60 h which disappeared by 72 h. This apparently synchronous cell cycle progression was confirmed by studies showing peak BrdU incorporation on the third day in culture. Whereas DNA synthesis by both E19 and E21 hepatocytes was potentiated by transforming growth factor alpha (TGF alpha), considerable mitogen-independent DNA synthesis was seen in hepatocytes from both gestational ages. These results may indicate that fetal hepatocytes come under the influence of an exogenous, in vivo growth inhibitory factor as term approaches and that this effect is relieved when term fetal hepatocytes are cultured.

Developmental regulation of CRD-BP, an RNA-binding protein that stabilizes c-myc mRNA in vitro.

We previously isolated and characterized a coding region determinant-binding protein (CRD-BP) that might regulate c-myc mRNA post-transcriptionally. CRD-BP binds specifically to the coding region of c-myc mRNA and might stabilize c-myc mRNA in vitro by protecting it from endonucleolytic cleavage. Since c-myc abundance is regulated during embryonic development and cell replication, we investigated whether CRD-BP is also regulated in animal tissues. We focused on CRD-BP expression during rat liver development and liver regeneration, because c-myc mRNA is regulated post-transcriptionally in both cases. CRD-BP expression parallels c-myc expression during liver development; the protein is present in fetal and neonatal liver but is absent or in low abundance in adult liver. In contrast, the up-regulation of c-myc mRNA following partial hepatectomy is not accompanied by up-regulation of CRD-BP. To our knowledge, CRD-BP is the first example of a putative mammalian mRNA-binding protein that is abundant in a fetal tissue but either absent from or scarce in adult tissues. Its expression in fetal liver and in transformed cell lines suggests CRD-BP is an oncofetal protein.

A comparative study of the hepatic mitogen-activated protein kinase and Jun-NH2-terminal kinase pathways in the late-gestation fetal rat.

Mitogen-activated protein kinases (MAPKs) and Jun-NH2-terminal kinases (JNKs) are considered members of parallel but separate signal transduction cascades. We have compared the regulation and, indirectly, the role of these two pathways in hepatic development during late gestation in the rat. Our initial experiments showed that the two pathways crossed over to a significant degree. Both could be activated in primary cultures of fetal rat hepatocytes by exposure to transforming growth factor alpha (TGF-alpha), a potent MAPK activator, or tumor necrosis factor alpha (TNF-alpha), a potent JNK activator. Fractionation of fetal hepatocyte lysates showed that the same MAPKs were stimulated by TGF-alpha as by TNF-alpha. In contrast, TGF-alpha activated only one JNK, whereas TNF-alpha stimulated multiple kinases with activity toward Jun. JNKs were found to be active under normal conditions in 19-day fetal liver compared with adult liver, whereas MAPK was not. Moreover, although JNKs could be activated further by intraperitoneal injection of TNF-alpha to the intact 19-day fetus, MAPKs could not be activated in vivo by i.p. injection of epidermal growth factor (EGF), which, like TGF-alpha, acts via the EGF receptor. EGF could not activate fetal hepatic MAPKs even though signal initiation was intact, as indicated by tyrosine phosphorylation of the adaptor protein Shc and Shc/Grb2 complex formation. These results indicate that the JNK-signaling pathway may support fetal hepatocyte proliferation in vivo. In contrast, MAPK signaling is uncoupled, possibly indicating that it is not involved in maintaining hepatocyte proliferation in the late-gestation rat.

A comparison of epidermal growth factor receptor-mediated mitogenic signaling in response to transforming growth factor alpha and epidermal growth factor in cultured fetal rat hepatocytes.

We compared the ability of Epidermal Growth Factor (EGF) and Transforming Growth Factor alpha (TGF alpha) to transduce a mitogenic signal via their common receptor, the EGF receptor, in primary cultures of fetal rat hepatocytes. Mitogenic potency, measured as DNA synthesis, was similar in response to EGF and TGF alpha although signal initiation, measured as EGF receptor tyrosine phosphorylation, was more than 3-fold higher in response to EGF compared to TGF alpha. Downstream signal transduction events including Shc tyrosine phosphorylation, Shc/Grb2 complex formation and MAP kinase activation were similar in response to EGF and TGF alpha, thus indicating a dissociation between potency for receptor activation versus signal propagation. These data suggest that TGF alpha may preferentially activate an EGF receptor population linked to the Ras/MAP kinase pathway. In contrast, EGF shows no such selectivity, thereby reducing the mitogenic potency of EGF relative to its ability to activate the EGF receptor.

Developmental changes in the activity and cellular localization of hepatic casein kinase II in the rat.

The activity and cellular localization of hepatic casein kinase II (CKII) was examined during late fetal development in the rat. Cultured fetal hepatocytes displayed constitutive CKII activity which was not further activated by growth factor exposure. Similarly, fetal liver CKII showed approximately fivefold greater activity than adult liver. The fetal hepatic activity was, to a large degree, localized to a nuclear fraction. Postnuclear cytosol preparations from fetal and adult liver showed similar CKII activity. In all cases, FPLC ion exchange chromatography followed by Western immunoblotting showed that immunoreactive CKII coincided with kinase activity. However, parallel determinations of CKII activity and immunoreactive CKII levels showed a higher (five- to sixfold) CKII specific activity in nuclear extracts compared to cytosol. In summary, fetal hepatic CKII demonstrates coincident nuclear localization and activation. We hypothesize that the regulation of hepatic CKII is relevant to the mitogen-independent proliferation displayed by fetal rat hepatocytes.

In vitro and in vivo regulation of hepatic mitogen-activated protein kinases in fetal rats.

We have studied the role of mitogen-activated protein (MAP) kinases in fetal hepatocyte growth in vitro and in vivo. With myelin basic protein (MBP) as the phosphate acceptor, kinase activity in cultured fetal hepatocyte lysates increased fourfold after exposure to transforming growth factor-alpha (TGF-alpha) for 10 min. This TGF-alpha-responsive MBP kinase activity was accounted for by five distinct MAP kinase isoforms detected by Western immunoblotting. All had negligible activity in cultured fetal hepatocytes under basal conditions. Treatment of fetal hepatocytes with hepatocyte growth factor led to activation of the predominant isoforms, relative molecular weight (M(r)) = 42,000 and 44,000 in a manner indistinguishable from TGF-alpha, whereas insulin had no effect. All five of the immunoreactive MAP kinases were present in both fetal and adult liver homogenates. The M(r) = 42,000 and 44,000 isoforms were only minimally activated in vivo. We conclude that the mitogen-independent growth exhibited by fetal hepatocytes in primary culture is not associated with tonic activation of the MAP kinase system. Our data support the possibility that fetal hepatic growth may be, in part, independent of the action of growth factors as mediated via the MAP kinase system.

Evidence for a direct hepatotrophic role for insulin in the fetal rat: implications for the impaired hepatic growth seen in fetal growth retardation.

Perturbations of fetal growth produce parallel but disproportionate changes in fetal liver growth that correlate with circulating fetal insulin concentration. We have studied the effects of insulin and two hepatotrophic factors, transforming growth factor-alpha (TGF alpha) and hepatocyte growth factor (HGF), on DNA synthesis by fetal and adult rat hepatocytes in primary culture. Using serum-free Minimum Essential Medium, fetal hepatocytes synthesized DNA without growth factors, unlike adult hepatocytes. Insulin augmented fetal hepatocyte DNA synthesis after 16-24 h in culture. In contrast, TGF alpha or HGF maximally stimulated fetal hepatocyte DNA synthesis after 40 h in culture. Insulin and TGF alpha were not synergistic in stimulating fetal hepatocyte DNA synthesis, but were synergistic in their action on adult hepatocytes. Brief (10-min) exposure of fetal hepatocytes to TGF alpha or HGF, but not insulin, activated mitogen-activated protein kinases 4-fold. Prolonged (24-h) exposure to TGF alpha or HGF abolished the ability of either to activate mitogen-activated protein kinases, whereas insulin had no effect. Maternal fasting for 48 h before isolation and culturing of fetal hepatocytes abolished the in vitro stimulation of DNA synthesis by insulin without affecting TGF alpha action. We conclude that insulin has growth-promoting actions on fetal hepatocytes that are distinct and independent from those of TGF alpha of HGF.

Insulin receptor tyrosine kinase domain auto-dephosphorylation.

We have observed dephosphorylation of the soluble, 48 kDa insulin receptor tyrosine kinase domain following its tyrosine autophosphorylation. Dephosphorylation was associated with generation of inorganic phosphate, thereby making catalysis by reversal of the kinase reaction unlikely. The kinase domain preparations could not be shown to contain detectable, contaminating protein tyrosine phosphatase activity. In addition, dephosphorylation was insensitive to protein phosphatase inhibitors. However, it was blocked by the kinase inhibitor staurosporine. These results are consistent with insulin receptor kinase domain auto-dephosphorylation via catalysis involving the kinase itself. These findings raise the possibility of a novel mechanism for termination of the insulin receptor signal.

Differential regulation of multiple hepatic protein tyrosine phosphatases in alloxan diabetic rats.

The involvement of tyrosine phosphorylation in insulin action led us to hypothesize that increased activity of protein tyrosine phosphatases (PTPases) might contribute to insulin resistance in alloxan diabetes in the rat. Hepatic PTPase activity was measured using two artificial substrates phosphorylated on tyrosine: reduced, carboxyamidomethylated, and maleylated lysozyme (P-Tyr-RCML) and myelin basic protein (P-Tyr-MBP), as well as an autophosphorylated 48-kD insulin receptor tyrosine kinase domain (P-Tyr-IRKD). Rats that were made alloxan diabetic exhibited a significant increase in hepatic membrane (detergent-soluble) PTPase activity measured with P-Tyr-MBP, without a change in activity measured with P-Tyr-RCML or the P-Tyr-IRKD. The PTPase active with P-Tyr-MBP behaved as a high molecular weight peak during gel filtration chromatography. Characterization of this enzyme indicated it shared properties with CD45, the prototype for a class of transmembrane, receptor-like PTPases. Our results indicate that alloxan diabetes in the rat is associated with an increase in the activity of a large, membrane-associated PTPase which accounts for only a small proportion of insulin receptor tyrosine dephosphorylation. Nonetheless, increased activity of this PTPase may oppose tyrosine kinase-mediated insulin signal transmission, thus contributing to insulin resistance.

Persistence of impaired insulin secretion in infant rhesus monkeys that had been hyperinsulinemic in utero.

Chronic in utero hyperinsulinemia in the fetal rhesus monkey produces a number of changes in the fetus that are similar to those found in the human infant of the diabetic mother, including macrosomia, selective organomegaly, and altered insulin secretion during the neonatal period. The chronically hyperinsulinemic fetal rhesus model has been used to test the hypothesis that the effects of chronic hyperinsulinemia persist beyond the neonatal period into later life and may, in part, be responsible for the increased prevalence of impaired glucose tolerance or diabetes found in the human infant of the diabetic mother. We report that infant rhesus monkeys that had plasma insulin concentrations of approximately 10 times basal levels (2176 +/- 808 pmol compared to 172 +/- 101 pmol) exhibited reduced insulin secretion during the first 5 months of life. The integrated incremental change in plasma insulin and immunoreactive C-peptide (IRCP) concentration was significantly reduced by approximately 50% in response to i.v. glucose, arginine, and tolbutamide when given at 3, 4, and 5 months of age. The response to glucagon at 2 months of age was equivocal with a significantly reduced insulin response but without the corresponding IRCP reduction. There was no difference between groups in insulin sensitivity as measured at 6 months of age by an i.v. insulin tolerance test. The glucagon and glucose tolerance tests were repeated annually in both groups until the animals were 3 yr of age with no differences in insulin or IRCP secretion being observed. We conclude that chronic in utero euglycemic hyperinsulinemia results in impaired insulin secretion that persists beyond the neonatal period.

Protein tyrosine phosphatase activation during nerve growth factor-induced neuronal differentiation of PC12 cells.

We have studied the role of protein tyrosine phosphatases (PTPases) during neuronal differentiation of PC12 cells. Nerve growth factor (NGF), a well-characterized differentiating agent for these cells, led to a decrease in DNA synthesis within 24 h. This was accompanied by a 2- to 3-fold increase in the activity of PTPases, measured as the dephosphorylation of polyacidic or polybasic substrates phosphorylated on tyrosine. PTPase activation was independent of cell density and proportional to NGF concentration, with a half-maximal effect occurring at 0.35 nM. High-performance liquid chromatography size exclusion chromatography revealed that PTPases with molecular masses of 550, 300, and 60 kilodaltons were activated in response to NGF. Additional studies showed that the presence of NGF made PC12 cells refractory to the mitogenic effect of epidermal growth factor. Our data indicate that NGF-induced neuronal differentiation and growth arrest in PC12 cells are associated with activation of several PTPases. We speculate that PTPase activation in response to NGF may inhibit the mitogenic actions of other growth factors.

Impaired insulin secretion after intravenous glucose in neonatal rhesus monkeys that had been chronically hyperinsulinemic in utero.

Chronic hyperinsulinemia in the fetal rhesus monkey results in fetal macrosomia without change in fetal plasma glucose concentration. After 18 days of hyperinsulinemia, fetuses were delivered by cesarean section, at which time experimental animals had significantly (P less than 0.05) elevated umbilical artery plasma insulin concentrations of 2039 +/- 854 pM compared with 129 +/- 72 pM. Plasma immunoreactive C peptide (IRCP) was significantly reduced to 39 +/- 17 pM compared with 286 +/- 134 pM. Eight hours after the insulin-delivering pumps were removed, plasma glucose, insulin, and IRCP were the same in both the experimental and control groups. At this time, 0.5 g glucose/kg was given intravenously and insulin and IRCP secretion was measured over a 1-hr period. The secretion, as assessed by integrating the incremental response of both insulin and IRCP, was significantly (P less than 0.05) lower by 80% in the experimental animals compared with the controls. Our data show that experimentally produced in utero euglycemic hyperinsulinemia in the fetal rhesus monkey produces a defect in the glucose-mediated insulin secretory mechanism that is detectable in the neonatal period even when hyperinsulinemia is no longer present. This study provides more support for the concept that fuel/hormone-mediated fetal teratogenesis may explain some of the fetopathy of the infant of the diabetic mother.

Heterogeneity of hepatic protein tyrosine phosphatases.

We have identified multiple members of the protein tyrosine phosphatase family in three subcellular compartments from rat liver; membrane, cytoskeleton and cytosol. Characterization based on substrate specificity, size, and reactivity with an anti-peptide antiserum against human placental PTP1B indicate the presence of at least three PTPases in Triton X-100 extracts of particulate membranes. Of these, one of 600 kDa possesses characteristics of a transmembrane, receptor-like enzyme. A fourth particulate PTPase (70 kDa) represents a distinct cytoskeletal PTPase. Cytosol contains one main PTPase species which was detected as a 41 kDa protein in Western immunoblots. These data indicate the existence of multiple hepatic PTPases whose differences in structure and subcellular localization may reflect functional heterogeneity.

Hepatic insulin and EGF receptor phosphorylation and dephosphorylation in fetal rats.

Hepatic insulin receptor and epidermal growth factor (EGF) receptor phosphorylation and dephosphorylation were studied in normal and growth-retarded fetal rats. Insulin receptor autophosphorylation at a subsaturating ATP concentration (0.5 microM) increased by 10-fold from day 17 to 21 of gestation and decreased by 50% in term growth-retarded fetuses of fasted mothers. In vitro kinase activation at 0.5 mM ATP did not change with gestation or maternal fasting. EGF receptor autophosphorylation increased in parallel with receptor number with advancing gestation and did not change with maternal fasting. Protein tyrosine phosphatases (PTPases), which might attenuate receptor signaling in livers from growth-retarded fetuses, were measured using polybasic and polyacidic artificial substrates as well as the insulin receptor kinase domain. Fetal membrane PTPase activities were twofold higher than in the adult and declined with advancing gestation. However, activities were similar in normal and growth-retarded fetuses. We conclude that decreased hepatic growth in growth-retarded fetuses may involve decreased insulin receptor tyrosine kinase activation in vivo, as indicated by diminished receptor autophosphorylation at subsaturating ATP concentrations. Changes in EGF receptor kinase activity and PTPases could not be implicated based on our in vitro findings.

Hepatic protein tyrosine phosphatases in the rat.

Regulation of cell growth and metabolism by protein tyrosine phosphorylation involves dephosphorylation via the action of protein tyrosine phosphatases (PTPases). We have characterized the membrane PTPases in rat liver, monitoring their activity by measuring the dephosphorylation of P-Tyr-reduced, carboxyamidomethylated and maleylated lysozyme (P-Tyr-RCML) and P-Tyr-myelin basic protein (P-Tyr-MBP). Separation of membrane PTPases by poly (L-lysine) chromatography yielded three peaks of PTPase, termed I, II and III. PTPases I and II were most active with P-Tyr-RCML, whereas PTPase III showed greater activity with P-Tyr-MBP than with P-Tyr-RCML (ratio of activities 4:1). Separation of membrane proteins by gel-filtration chromatography yielded two peaks of activity. Based on substrate specificity, sensitivity to inhibitors and requirement for thiol-containing compounds, the activity peak with an Mr of approximately 400,000 corresponded to PTPase III, whereas that with an Mr of approx. 40,000 contained PTPases I and II. All three PTPases dephosphorylated epidermal growth factor receptors and insulin receptors, but only PTPases I and II were active with P-Tyr-asialoglycoprotein receptors. Although none of the above characteristics distinguished between PTPases I and II, only PTPase I reacted in a Western immunoblotting procedure with anti-peptide antibodies directed towards human placental PTPase. We conclude that the membrane fraction from rat liver contains at least three distinct PTPases.