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Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma ligands have recently been shown to induce activation of mitogen-activated protein kinases (MAPKs), which in turn phosphorylate PPARs, thereby affecting transcriptional activity. However, the mechanism for PPAR ligand-dependent MAPK activation is unclear. In the current study, we demonstrate that various PPARalpha (nafenopin) and gamma (ciglitazone and troglitazone) agonists rapidly induced extracellular signal-regulated kinase (Erk) and/or p38 phosphorylation in rat liver epithelial cells (GN4). The selective epidermal growth factor receptor (EGFR) kinase inhibitors, PD153035 and ZD1839 (Iressa), abolished PPARalpha and gamma agonist-dependent Erk activation. Consistent with this, PPAR agonists increased tyrosine autophosphorylation of the EGFR as well as phosphorylation at a putative Src-specific site, Tyr845. Experiments with the Src inhibitor, PP2, and the antioxidant N-acetyl-L-cysteine revealed critical roles for Src and reactive oxygen species as upstream mediators of EGFR transactivation in response to PPAR ligands. Moreover, PPARalpha and gamma ligands increased Src autophosphorylation as well as kinase activity. EGFR phosphorylation, in turn, led to Ras-dependent Erk activation. In contrast, p38 activation by PPARalpha and gamma ligands occurred independently of Src, oxidative stress, the EGFR, and Ras. Interestingly, PPARalpha and gamma agonists caused rapid activation of proline-rich tyrosine kinase or Pyk2; Pyk2 as well as p38 phosphorylation was reduced by intracellular Ca2+ chelation without an observable effect on EGFR and Erk activation, suggesting a possible role for Pyk2 as an upstream activator of p38. In summary, PPARalpha and gamma ligands activate two distinct signaling cascades in GN4 cells leading to MAPK activation.

Mer belongs to the Mer/Axl/Tyro3 receptor tyrosine kinase family, which regulates immune homeostasis in part by triggering monocyte ingestion of apoptotic cells. Mutations in Mer can also cause retinitis pigmentosa, again due to defective phagocytosis of apoptotic material. Although, some functional aspects of Mer have been deciphered, how receptor activation lead to the physiological consequences is not understood. By using yeast two-hybrid assays, we identified the carboxyl-terminal region of the guanine nucleotide-exchange factor (GEF) Vav1 as a Mer-binding partner. Unlike similar (related) receptors, Mer interacted with Vav1 constitutively and independently of phosphotyrosine, yet the site of binding localized to the Vav1 SH2 domain. Mer activation resulted in tyrosine phosphorylation of Vav1 and release from Mer, whereas Vav1 was neither phosphorylated nor released from kinase-dead Mer. Mutation of the Vav1 SH2 domain phosphotyrosine coordinating Arg-696 did not alter Mer/Vav1 constitutive binding or Vav1 tyrosine phosphorylation but did retard Vav1 release from autophosphorylated Mer. Ligand-dependent activation of Mer in human monocytes led to Vav1 release and stimulated GDP replacement by GTP on RhoA family members. This unusual constitutive, SH2 domain-dependent, but phosphotyrosine-independent, interaction and its regulated local release and subsequent activation of Rac1, Cdc42, and RhoA may explain how Mer coordinates precise cytoskeletal changes governing the ingestion of apoptotic material by macrophages and pigmented retinal epithelial cells.

Mer is a member of theAxl/Mer/Tyro3 receptor tyrosine kinase family, a family whose physiological function is not well defined. We constructed a Mer chimera using the epidermal growth factor receptor (EGFR) extracellular and transmembrane domains and the Mer cytoplasmic domain. Stable transfection of the Mer chimera into interleukin 3 (IL-3)-dependent murine 32D cells resulted in ligand-activable surface receptor that tyrosine autophosphorylated, stimulated intracellular signaling, and dramatically reduced apoptosis initiated by IL-3 withdrawal. However, unlike multiple other ectopically expressed receptor tyrosine kinases including full-length EGFR or an EGFR/Axl chimera, the Mer chimera did not stimulate proliferation. Moreover, and in contrast to EGFR,Mer chimera activation induced adherence and cell flattening in the normally suspension-growing 32D cells. TheMer chimera signal also blocked IL-3-dependent proliferation leading to G1/S arrest, dephosphorylation of retinoblastoma protein, and elongation of cellular processes. Unlike other agonists that lead to a slow (4–8 days) ligand-dependent differentiation of 32D cells, the combinedMer and IL-3 signal resulted in differentiated morphology and growth cessation in the first 24 h. Thus the Merchimera blocks apoptosis without stimulating growth and produces cytoskeletal alterations; this outcome is clearly separable from the proliferative signal produced by most receptor tyrosine kinases.

Freshly isolated human monocytes do not express p125FAK but upon adherence to substrata activate the highly related calcium-dependent tyrosine kinase (CADTK), also known as Pyk2, CAKβ, RAFTK, and FAK2. The monocyte CADTK was 5 kDa smaller than protein from epithelial cells; isolation and sequencing of the monocyte CADTK cDNA revealed a predicted 42-amino acid deletion between the two proline-rich domains of the enzyme. The nucleic acid sequence suggests that the deletion is caused by alternative RNA splicing. This species was also found in T and B lymphocytes and appears to be the predominant form of cytoskeletal associated tyrosine kinase in non-neoplastic, circulating, hematopoietic cells. CADTK was not activated when monocytes maintained in suspension were treated with agents that produce an intracellular calcium (thapsigargin) or protein kinase C (phorbol 12-myristate 13-acetate) signal including a chemokine, RANTES, that binds to the HIV co-receptor, CCK5. In contrast, monocyte adherence to tissue culture plastic-stimulated CADTK tyrosine phosphorylation, a process that was enhanced by thapsigargin, phorbol 12-myristate 13-acetate, and RANTES but that was completely blocked by preincubation with cytochalasin D. When compared with plastic, adherence to fibronectin- or collagen-coated surfaces produced only minimal CADTK activation but permitted significant stimulation by added thapsigargin. These data suggest that in a cell type that lacks p125FAK, CADTK plays an early role in post-adherence signaling. Its activation involves two stages, cytoskeletal engagement, which is permissive, and co-stimulatory signals (calcium or protein kinase C) generated by extensive cell surface engagement, agonists, or inflammatory chemokines.

A novel, p125FAK homologue, CADTK, has been detected in neural, epithelial, or hematopoietic cells but not in fibroblasts. We now demonstrate CADTK expression in a mesenchymal cell, rat aortic smooth muscle cells (RSMC). Angiotensin II (Ang II) or platelet-derived growth factor (PDGF-BB and PDGF-AA) markedly stimulated CADTK tyrosine phosphorylation in RSMC but did not affect p125FAK phosphorylation. The PDGF-dependent CADTK tyrosine phosphorylation was slower and more prolonged than that of Ang II, correlating well with the differential effects of these agonists on cytosolic calcium ([Ca2+] i ) signaling. An intracellular calcium chelator inhibited both the rapid and sustained activation of CADTK by Ang II and PDGF. Extracellular calcium chelation inhibited the PDGF-stimulated increase in CADTK tyrosine phosphorylation as well as the sustained (but not the early) activation by Ang II. In contrast, p125FAK tyrosine phosphorylation was maximal in quiescent, adherent RSMC and was not affected by incubation with EGTA. Depletion of protein kinase C activity partially inhibited both the Ang II- and PDGF-induced CADTK tyrosine phosphorylation. Additional results confirm a relation between CADTK and the cytoskeleton. First, the tyrosine phosphorylation of paxillin correlated with activation of CADTK; this increase was inhibited by EGTA. Second, cytochalasin D blocked the PDGF- or Ang II-stimulated tyrosine phosphorylation of CADTK, suggesting a role for the cytoskeleton in agonist-dependent CADTK activation. Third, immunofluorescence analysis of CADTK localization demonstrated actin-like cytoskeleton staining extending into focal contacts. These results suggest that in mesenchymal cells, CADTK is localized to and activated by an actin cytoskeleton-dependent mechanism; a mechanism that is regulated in a calcium and protein kinase C-dependent manner independently of p125FAK.

Similar to insulin, osmotic shock of 3T3L1 adipocytes stimulated an increase in glucose transport activity and translocation of GLUT4 protein from intracellularly localized vesicles to the plasma membrane. The docking/fusion of GLUT4 vesicles with the plasma membrane appeared to utilize a similar mechanism, since expression of a dominant interfering mutant of syntaxin-4 prevented both insulin- and osmotic shock-induced GLUT4 translocation. However, although the insulin stimulation of GLUT4 translocation and glucose transport activity was completely inhibited by wortmannin, activation by osmotic shock was wortmannin-insensitive. Furthermore, insulin stimulated the phosphorylation and activation of the Akt kinase, whereas osmotic shock was completely without effect. Surprisingly, treatment of cells with the tyrosine kinase inhibitor, genistein, or microinjection of phosphotyrosine antibody prevented both the insulin- and osmotic shock-stimulated translocation of GLUT4. In addition, osmotic shock induced the tyrosine phosphorylation of several discrete proteins including Cbl, p130(cas), and the recently identified soluble tyrosine kinase, calcium-dependent tyrosine kinase (CADTK). In contrast, insulin had no effect on CADTK but stimulated the tyrosine phosphorylation of Cbl and the tyrosine dephosphorylation of pp125(FAK) and p130(cas). These data demonstrate that the osmotic shock stimulation of GLUT4 translocation in adipocytes occurs through a novel tyrosine kinase pathway that is independent of both the phosphatidylinositol 3-kinase and the Akt kinase.

In rat liver epithelial cells (GN4), angiotensin II (Ang II) and thapsigargin stimulate a novel calcium-dependent tyrosine kinase (CADTK) also known as PYK2, CAKbeta, or RAFTK. Activation of CADTK by a thapsigargin-dependent increase in intracellular calcium failed to stimulate the extracellular signal-regulated protein kinase pathway but was well correlated with a 30-50-fold activation of c-Jun N-terminal kinase (JNK). In contrast, Ang II, which increased both protein kinase C (PKC) activity and intracellular calcium, stimulated extracellular signal-regulated protein kinase but produced a smaller, less sustained, JNK activation than thapsigargin. 12-O-Tetradecanoylphorbol 13-acetate (TPA), which slowly activated CADTK, did not stimulate JNK. These findings suggest either that CADTK is not involved in JNK activation or PKC activation inhibits the CADTK to JNK pathway. A 1-min TPA pretreatment of GN4 cells inhibited thapsigargin-dependent JNK activation by 80-90%. In contrast, TPA did not inhibit the50-fold JNK activation effected by anisomycin or UV. The consequence of PKC-dependent JNK inhibition was reflected in c-Jun and c-Fos mRNA induction following treatment with thapsigargin and Ang II. Thapsigargin, which only minimally induced c-Fos, produced a much greater and more prolonged c-Jun response than Ang II. Elevation of another intracellular second messenger, cAMP, for 5-15 min also inhibited calcium-dependent JNK activation by approximately 80-90% but likewise had no effect on the stress-dependent JNK pathway. In summary, two pathways stimulate JNK in cells expressing CADTK, a calcium-dependent pathway modifiable by PKC and cAMP-dependent protein kinase and a stress-activated pathway independent of CADTK, PKC, and cAMP-dependent protein kinase; the inhibition by PKC can ultimately alter gene expression initiated by a calcium signal.

Many G protein-coupled receptors (e.g. that of angiotensin II) activate phospholipase Cβ, initially increasing intracellular calcium and activating protein kinase C. In the WB and GN4 rat liver epithelial cell lines, agonist-induced calcium signals also stimulate tyrosine phosphorylation and subsequently increase the activity of c-Jun N-terminal kinase (JNK). We have now purified the major calcium-dependent tyrosine kinase (CADTK), and by peptide and nucleic acid sequencing identified it as a rat homologue of human PYK2. CADTK/PYK2 is most closely related to p125FAK and both enzymes are expressed in WB and GN4 cells. Angiotensin II, which only slightly increases p125FAK tyrosine phosphorylation in GN4 cells, substantially increased CADTK tyrosine autophosphorylation and kinase activity. Agonists for other G protein-coupled receptors (e.g. LPA), or those increasing intracellular calcium (thapsigargin), also stimulated CADTK. In comparing the two rat liver cell lines, GN4 cells exhibited ∼ 5-fold greater angiotensin II- and thapsigargin-dependent CADTK activation than WB cells. Although maximal JNK activation by stress-dependent pathways (e.g. UV and anisomycin) was equivalent in the two cell lines, calcium-dependent JNK activation was 5-fold greater in GN4, correlating with CADTK activation. In contrast to JNK, the thapsigargin-dependent calcium signal did not activate mitogen-activated protein kinase and Ang II-dependent mitogen-activated protein kinase activation was not correlated with CADTK activation. Finally, while some stress-dependent activators of the JNK pathway (NaCl and sorbitol) stimulated CADTK, others (anisomycin, UV, and TNFα) did not. In summary, cells expressing CADTK/PYK2 appear to have two alternative JNK activation pathways: one stress-activated and the other calcium-dependent.

In rat liver epithelial cell lines (WB or GN4), angiotensin II (Ang II) stimulates cytosolic tyrosine kinase activity, in part, through a calcium-dependent mechanism. In other cell types, selected hormones that activate Gi- or Gq-coupled receptors stimulate the soluble tyrosine kinase, p125FAK. Immunoprecipitation of p125FAK from Ang II-activated GN4 cells demonstrated a doubling of p125FAK kinase activity. However, an additional Ang II-activated tyrosine kinase (or kinases) representing the majority of the total activity was detected when the remaining cell lysate, immunodepleted of p125FAK, was reimmunoprecipitated with an anti-phosphotyrosine antibody. Cytochalasin D pretreatment blocks G-protein receptor-dependent tyrosine phosphorylation in Swiss 3T3 cells. While cytochalasin D decreased the Tyr(P) content of 65-75-kDa substrates in Ang II-treated GN4 cells, it did not diminish tyrosine phosphorylation of 115-130-kDa substrates, again suggesting activation of at least two tyrosine kinase pathways in GN4 cells. To search for additional Ang II-activated enzymes, we used molecular techniques to identify 20 tyrosine kinase sequences in these cell lines. None was the major cytosolic enzyme activated by Ang II. Specifically, JAK2, which had been shown by others to be stimulated by Ang II in smooth muscle cells, was not activated by Ang II in GN4 cells. Finally, we purified Tyr(P)-containing tyrosine kinases from Ang II-treated cells, using anti-Tyr(P) and ATP affinity resins; 80% of the tyrosine kinase activity migrated as a single 115-120-kDa tyrosine-phosphorylated protein immunologically distinct from p125FAK. In summary, Ang II activates at least two separate tyrosine kinases in rat liver epithelial cells; p125FAK and a presumably novel, cytosolic 115-120-kDa protein referred to as the calcium-dependent tyrosine kinase.

We have recently shown that changes in tyrosine phosphorylation of a 130-kDa protein(s) (pp130) may be involved in integrin signaling (Kornberg, L., Earp, H.S., Turner, C., Prokop, and Juliano, R. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8392-8396). One component of the pp130 protein complex reacts with an antibody generated against p125fak, which is a focal contact-associated tyrosine kinase (Schaller, M.D., Borgman, C. A., Cobb, B. S., Vines, R. R., Reynolds, A. B., and Parsons, J. T. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5192-5196). Both antibody-mediated integrin clustering and adhesion of KB cells to fibronectin leads to increased tyrosine phosphorylation of p125fak. The phosphorylation of p125fak is coincident with adhesion of cells to fibronectin and is maximal prior to cell spreading. Tyrosine phosphorylation of p125fak is induced when KB cells are allowed to adhere to fibronectin, collagen type IV, or laminin, but is not induced on polylysine. When KB cells are subjected to indirect immunofluorescence microscopy, p125fak colocalizes with talin in focal contacts. These data provide additional evidence that tyrosine kinases are involved in integrin signaling.

In order to determine whether distinct platelet-derived growth factor (PDGF) receptors (alpha and beta) can modulate the activity of one another, PDGF isoform (AA, BB, and AB)-stimulated changes in Ca2+i were monitored by digitized video microscopy in single cells upon sequential addition of PDGF isoforms. In Balb/c 3T3 fibroblasts, all PDGF isoforms were capable of stimulating increases in Ca2+i of 200-600% above basal levels, although with different potencies: BB greater than or equal to AB greater than AA. All cells were BB-PDGF-responsive, but only 74% of cells examined responded to AA-PDGF. The Ca2+i response elicited by BB-PDGF was inhibited by 60-75% in cells stimulated 10 min earlier with the AA isoform. The half-life of this inhibition was 22 min. In cells in which the alpha receptor was down-regulated by prolonged incubation with AA-PDGF, BB-induced Ca2+i responses were not inhibited. Pretreatment of cells with phorbol ester did not inhibit BB-PDGF-induced increases in Ca2+i, yet down-regulation of PKC activity prevented the AA-PDGF inhibition of BB-PDGF-induced Ca2+i responses. An increase in Ca2+i induced by AlF(4-)-stimulated IP3 generation did not inhibit a subsequent BB-PDGF Ca2+i response; however, attenuation of AA-PDGF-induced extracellular Ca2+ influx with EGTA prevented the inhibition of BB-PDGF-induced Ca2+i increases. Readdition of Ca2+ to the medium after removal of EGTA restored the inhibition of the BB-PDGF Ca2+i response. The inhibition of the BB-PDGF Ca2+i response by AA-PDGF was not caused by inhibition of PDGF receptor tyrosine autophosphorylation, which was unchanged after pretreatment with AA-PDGF. These results demonstrate: (a) that only a subpopulation of cells possess a functional alpha receptor-mediated response as assessed by AA-PDGF-induced increases in Ca2+i, whereas all cells possess the beta receptor-mediated responses; and (b) AA-PDGF and its associated alpha receptor can modulate the activity of the beta receptor through a mechanism that is dependent upon Ca(2+)-influx which may be controlled in part by PKC activation.

Affinity-purified polyclonal antibodies, raised against two synthetic peptides corresponding to the R domain and the C terminus of the human cystic fibrosis transmembrane conductance regulator (CFTR), were used to characterize and localize the protein in human epithelial cells. Employing an immunoblotting technique that ensures efficient detection of large hydrophobic proteins, both antibodies recognized and approximately 180-kDa protein in cell lysates and isolated membranes of airway epithelial cells from normal and cystic fibrosis (CF) patients and of T84 colon carcinoma cells. Reactivity with the anti-C terminus antibody, but not with the anti-R domain antibody, was eliminated by limited carboxypeptidase Y digestion. When normal CFTR cDNA was overexpressed via a retroviral vector in CF or normal airway epithelial cells or in mouse fibroblasts, the protein produced had an apparent molecular mass of about 180 kDa. The CFTR expressed in insect (Sf9) cells by a baculovirus vector had a molecular mass of about 140 kDa, probably representing a nonglycosylated form. The CFTR in epithelial cells appears to exist in several forms. N-glycosidase treatment of T84 cell membranes reduces the apparent molecular mass of the major CFTR band from 180 kDa to 140 kDa, but a fraction of the T84 cell CFTR could not be deglycosylated, and the CFTR in airway epithelial cell membranes could not be deglycosylated either. Moreover, wheat germ agglutinin absorbs the majority of the CFTR from detergent-solubilized T84 cell membranes but not from airway cell membranes. The CFTR in all epithelial cell types was found to be an integral membrane protein not solubilized by high salt or lithium diiodosalicylate treatment. Sucrose density gradient fractionation of crude membranes prepared from the airway epithelial cells, previously surface-labeled by enzymatic galactosidation, showed a plasma membrane localization for both the normal CFTR and the CFTR carrying the Phe508 deletion (delta F 508). The CFTR in all cases co-localized with the Na+, K(+)-ATPase and the plasma membrane calcium ATPase, while the endoplasmic reticulum calcium ATPase and mitochondrial membrane markers were enriched at higher sucrose densities. Thus, the CFTR appears to be localized in the plasma membrane both in normal and delta F 508 CF epithelial cells.

Pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiated the mitogenic response to insulin-like growth factor I (IGF-I) (Tramontano, D., Moses, A. C., Veneziani, B. M., and Ingbar, S. H. (1988) Endocrinology 122, 127-132; Takahashi, S.-I., Conti, M., and Van Wyk, J. J. (1990) Endocrinology 126, 736-745). The present study was undertaken to determine whether this synergism between TSH and IGF-I in FRTL-5 cells was correlated with changes in tyrosine phosphorylation of intracellular proteins. Tyrosine phosphorylation in intact cells was determined by gel electrophoresis and immunoblotting using monospecific anti-phosphotyrosine antibodies. Cells were preincubated for up to 24 h with TSH, dibutyryl cAMP, forskolin, or cholera toxin and then incubated for an additional 1 min in the absence or presence of IGF-I. As reported by others, IGF-I rapidly increased tyrosine phosphorylation of a 175-kDa protein as well as a less intense band of 90-100 kDa. Pretreatment for 6-12 h with either TSH or other agents that elevate intracellular cAMP potentiated the IGF-I-dependent tyrosine phosphorylation of the 175-kDa substrate by 3-5-fold. Since TSH did not increase IGF receptor number of kinase activity, the effect of TSH is assumed to be exerted at a step distal to IGF receptor tyrosine kinase. Surprisingly, IGF-I-independent tyrosine phosphorylation was also increased by pretreatment with TSH. When intact cells were analyzed TSH produced a time- and concentration-dependent increase in tyrosine phosphorylation of a prominent 120-125-kDa substrate and less prominent 100- and 80-kDa substrates. Assays using Triton X-100-soluble extracts incubated with MgCl2, ATP, and orthovanadate demonstrated that TSH pretreatment increased tyrosine phosphorylation over that observed in untreated cells. In this cell-free assay, TSH pretreatment enhanced the phosphorylation of multiple substrates. These studies suggest that a cAMP stimulus that initiates a trophic effect can be propagated indirectly through multiple pathways including enhancement of tyrosine phosphorylation.

To study the activity of the epidermal growth factor (EGF) receptor during EGF-directed internalization, liver epithelial cells were exposed to EGF at 37 degrees C for various periods of time, washed, and homogenized at 0 degrees C. EGF receptor autophosphorylation was assessed in homogenates using [gamma-32P]ATP. Autophosphorylation was stimulated 3- to 6-fold in homogenates of cells incubated with EGF (100 ng/ml) for 15 min but was at or below basal levels in homogenates of cells treated with EGF for 2.5-5 min. This was surprising because immunoblotting revealed that EGF receptor phosphotyrosine (P-Tyr) content in intact cells was near maximal from 30 s to 5 min after EGF treatment. Excess EGF (1 microgram/ml), added after homogenization but prior to the assay, increased autophosphorylation in homogenates of cells that had not been treated with EGF, but failed to increase activity in homogenates of cells treated with EGF in culture for 2.5-5 min. Suppression of tyrosine phosphorylation of an exogenous kinase substrate was also observed at times paralleling the suppression of EGF receptor autophosphorylation. The transient suppression of receptor autophosphorylation in the cell-free assay was not explained by persistent occupation of autophosphorylation sites by phosphate added in the intact cells. The sites were greater than 80% dephosphorylated during the homogenization. Additionally phosphatase inhibition that prevented the normal loss of EGF receptor P-Tyr in intact cells at 15 min did not affect the pattern of early (2.5-5 min) suppression and later (15 min) stimulation of autophosphorylation measured in the cell-free assay. The suppression was not explained by activation of protein kinase C in that depletion of greater than 95% of cellular protein kinase C activity by an 18-h incubation of cells with 10 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) did not affect the early suppression of autophosphorylation in EGF-treated cells. Moreover, under the conditions tested, activation of protein kinase C by short-term treatment (0.5-10 min) with TPA or angiotensin II did not appreciably alter subsequent autophosphorylation in the cell-free assay. In contrast, a 30 degrees C preincubation of homogenates from cells with suppressed EGF receptor autophosphorylation led to the recovery of the ability of EGF to stimulate EGF receptor autophosphorylation. These results suggest that a rapid reversible protein kinase C-independent process prevents detection of EGF receptor kinase activity during an early phase of EGF-dependent receptor internalization.

Epidermal growth factor (EGF) stimulated the rapid accumulation of inositol trisphosphate in WB cells, a continuous line of rat hepatic epithelial cells. Since we previously had shown that EGF stimulates EGF receptor synthesis in these cells, we tested whether hormones that stimulate PtdIns(4,5)P2 hydrolysis would increase EGF receptor protein synthesis and mRNA levels. Epinephrine, angiotensin II, and [Arg8]vasopressin activate phospholipase C in WB cells as evidenced by the accumulation of the inositol phosphates, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. A 3-4-h treatment with each hormone also increased the rate of EGF receptor protein synthesis by 3-6-fold as assessed by immunoprecipitation of EGF receptor from [35S]methionine-labeled cells. Northern blot analyses of WB cell EGF receptor mRNA levels revealed that agents linked to the phosphoinositide signaling system increased receptor mRNA content within 1-2 h. A maximal increase of 3-7-fold was observed after a 3-h exposure to EGF and hormones. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C also stimulated EGF receptor synthesis. Pretreatment of WB cells for 18 h with high concentrations of TPA "down-regulated" protein kinase C and blocked TPA-directed EGF receptor mRNA synthesis. In contrast, the effect of EGF on EGF receptor mRNA levels was not significantly decreased by TPA pretreatment. Epinephrine-induced increases in EGF receptor mRNA were reduced from 4- to 2-fold. Similarly, 18 h TPA pretreatment abolished the effect of TPA on EGF receptor protein synthesis but did not affect EGF-dependent EGF receptor protein synthesis. The 18-h TPA pretreatment diminished by 30-50% the induction of receptor protein synthesis by epinephrine or angiotensin II. We conclude that in WB cells EGF receptor synthesis can be regulated by EGF and other hormones that stimulate PtdIns(4,5)P2 hydrolysis. In these cells, EGF receptor synthesis appears to be regulated by several mechanism: one pathway is dependent upon EGF receptor activation and can operate independently of protein kinase C activation; another pathway is correlated with PtdIns(4,5)P2 hydrolysis and is dependent, at least in part, upon protein kinase C activation.

Exposure of a nontransformed, continuous line of epithelial cells derived from rat liver (WB cells) to epidermal growth factor, angiotensin II, [Arg8]vasopressin, and epinephrine resulted in rapid accumulation of the inositol phosphates (InsP) InsP1, InsP2, and InsP3. Although short-term (5-60 min) pretreatment of WB cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly attenuated InsP accumulation in response to all agonists, the inhibitory effects on the InsP response were lost after 2 h incubation with PMA; and, with extended (6-24 h) preincubation, a time-dependent potentiation of the InsP response to angiotensin II, epidermal growth factor and [Arg8]vasopressin was observed. The InsP response during a 15-min challenge with angiotensin II in cells pretreated for 18 h with 600 nM and 10 microM PMA was increased by 2-3-fold and 4-6-fold, respectively. Long-term (18 h) treatment with 600 nM and 10 microM PMA caused a similar 90-100% loss of measurable Ca2+/phospholipid-dependent enzyme (protein kinase C) activity in cytosolic and soluble particulate fractions. The effects of long-term PMA pretreatment do not represent a general enhancement of hormone responsiveness since the InsP response to epinephrine was not affected. In control cells, the InsP response to angiotensin II and epinephrine desensitized very rapidly. Long-term pretreatment with PMA greatly reduced the contribution of agonist-induced desensitization to the angiotensin II response; in contrast, the extent of desensitization occurring during incubation of WB cells with epinephrine was unaltered by long-term treatment with PMA suggesting that an additional mechanism may be involved in alpha 1-adrenergic receptor desensitization. No PMA-induced change in resting levels of [3H]phosphoinositides or the metabolism of exogenous [3H]inositol 1,4,5-trisphosphate by WB homogenates occurred. Stimulation of InsP formation in intact cells by NaF and activation of phospholipase C by GTP gamma S in membranes both were unaltered by short-term or long-term PMA pretreatment. These data are consistent with the idea that following long-term treatment of WB cells with PMA, the occurrence of agonist-induced desensitization of receptors linked to the phosphoinositide/Ca2+ signaling system is reduced, apparently at least in part due to the loss of contribution of a negative feedback regulatory role of protein kinase C.

Exposure of A431 human epidermoid carcinoma cells to epidermal growth factor (EGF), bradykinin, and histamine resulted in a time- and concentration-dependent accumulation of the inositol phosphates (InsP) inositol monophosphate, inositol bisphosphate, and inositol trisphosphate (InsP3). Maximal concentrations of EGF (316 ng/ml; approximately 50 nM), bradykinin (1 microM), and histamine (1 mM) resulted in 3-, 6-, and 3-fold increases, respectively, in the amounts of inositol phosphates formed over a 10-min period. The K0.5 values for stimulation were approximately 10 nM, 3 nM, and 10 microM for EGF, bradykinin, and histamine, respectively. EGF and bradykinin stimulated the rapid accumulation of the two isomers of InsP3, Ins(1,3,4)P3, and Ins(1,4,5)P3 as determined by high performance liquid chromatography analysis; maximal accumulation of Ins(1,4,5)P3 occurred within 15 s. EGF and bradykinin also stimulated a rapid (maximal levels attained within 30 s after addition of hormone) and a sustained 4- and 6-fold rise, respectively, in cytosolic free Ca2+ levels as measured by Fura-2 fluorescence. EGF and bradykinin also produced a rapid, although transient, 3- and 5-fold increase, respectively, in cytosolic free Ca2+ after chelation of extracellular Ca2+ with 3 mM EGTA. These data are consistent with the idea that EGF elevates intracellular Ca2+ levels in A431 cells, at least in part, as a result of the rapid formation of Ins(1,4,5)P3 and the consequential release of Ca2+ from intracellular stores.

Rat liver regeneration is regulated by a humoral signal that includes insulin and a sustained elevation in glucagon. The intracellular response is characterized by a rise in cAMP as well as altered cGMP metabolism, i.e. increased particulate guanylate cyclase activity. To evaluate the role of hormones in the regulation of guanylate cyclase during liver regeneration, the enzyme activity of primary cultures of rat hepatocytes was examined. Hepatocytes were maintained for 22 h in medium containing various combinations of insulin, glucagon, and cAMP. The cells were then harvested and homogenized and the guanylate cyclase activity was assessed in vitro. Hepatocytes maintained in 100 nM insulin exhibited a 42% (p < 0.001) increase in guanylate cyclase activity when compared to cells cultured in medium alone. Incubation with glucagon (100 nM) produced a 52% (p < 0.01) rise. In the presence of insulin (100 nM), culturing with as little as 5 nM glucagon resulted in increased activity, and 100 nM glucagon produced a 161% (p < 0.001) rise above cultures maintained in insulin alone. Thus, the combination of the two hormones produced an effect that was significantly (p < 0.01) greater than additive. Dibutyryl cAMP and 8-bromoadenosine 3':5'-monophosphoric acid were at least as effective as glucagon; the enzyme activity of cells maintained in 5 microM N6,02'-dibutyryl adenosine 3':5'-monophosphoric acid and 100 nM insulin was 243% (p < 0.001) above those in insulin alone. The findings suggest that the activity of hepatocyte guanylate cyclase is regulated by a synergistic action of insulin and glucagon and that positive interactions between the two cyclic nucleotide second messenger systems exist.

In isolated hepatic membranes, epidermal growth factor (EGF) and the polar solvent dimethyl sulfoxide (Me2SO) selectively stimulated the phosphorylation of the 170,000-dalton EGF receptor (p170) by 13.6 +/- 2.0- and 10.9 +/- 1.1-fold, respectively. The stimulation by maximally effective concentrations of the two substances was similar in rapidity of onset (less than 30 s at 0 degree C), time course of phosphorylation, and tyrosine residue specificity. These maximal effects were not additive when the substances were combined, indicating that the same kinase/substrate combination is activated by each. The lectin concanavalin A, which inhibits EGF receptor binding, blocked the effect of EGF but not Me2SO. In membranes solubilized with Triton X-100, EGF stimulated p170 phosphorylation by 40- to 55-fold. Me2SO also stimulated phosphorylation, indicating that it acts directly on the protein. However, the effect of the solvent was reduced by half. Additionally, Me2SO blocks the effect of EGF in the solubilized preparation. A room temperature preincubation after addition of either substance was necessary for maximal stimulation of p170 phosphorylation in solubilized membranes. With EGF, 30-40 min was necessary; with Me2SO, only 10 min was required. Thus, a secondary process appears to be involved in EGF receptor/kinase activation.

Epidermal growth factor (EGF) activates the intrinsic tyrosine-specific protein kinase of its receptor (EGF-R). We studied the effect of EGF-dependent EGF-R internalization on receptor autophosphorylation and on the appearance of tyrosine phosphoproteins in rat liver epithelial cells. Peak receptor autophosphorylation activity (3- to 6-fold over basal) was found in homogenates of EGF-treated cells at times when the majority of receptors (greater than 90%) had been internalized but not yet degraded (15 to 30 min). Stimulated activity persisted for at least 2 h if EGF-R degradation was blocked by methylamine or 18 degrees C incubation. Detection of stimulated autophosphorylation in homogenates of cells treated with EGF in culture required detergent in the assay. Detergent was not necessary to detect stimulated autophosphorylation when EGF was added directly to homogenates of untreated cells. Immunoblots using antibodies against phosphotyrosine (p-Tyr) demonstrated that EGF treatment of intact cells increased the p-Tyr content of at least seven proteins (EGF-R, 115, 100, 75, 66, 57, and 52 kDa) within 5 s. Incubation of intact cells with EGF at 0 degrees C to prevent endocytosis still resulted in tyrosine phosphorylation of these seven proteins. In contrast, several substrates (120, 78, and 38 kDa) showed delayed increases (45-90 s) in tyrosine phosphorylation at 37 degrees C; their phosphorylation was even slower at 18 degrees C and did not occur at 0 degrees C. In cells incubated with EGF at 18 degrees C or in the presence of methylamine, EGF-R p-Tyr in the intact cell was lost by 2 h even though receptor was not degraded and still exhibited enhanced autophosphorylation in the homogenate assay. These findings suggest that tyrosine phosphorylation in response to EGF occurs predominantly during the initial stages of endocytosis and is mediated for the most part by ligand-receptor complexes at the cell surface. A subset of phosphorylations may require intracellular movement.