Phosphorylation of phosphoprotein phosphatase inhibitor-2 (I-2) in rat fat cells

Fat cells were incubated with 32Pi for 2 h before the [32P]I-2 was immunoprecipitated, subjected to SDS/PAGE, and detected by autoradiography. [32P]I-2 (Mr = 32,000) was not recovered when excess purified I-2 was added with the antiserum or when nonimmune serum was used. Immunoprecipitated I-2 was heat-stable, inhibited phosphatase activity, and could be synergistically phosphorylated by casein kinase II and FA/GSK-3. Several times more [32P]phosphoserine than [32P]phosphothreonine was found in I-2 from 32P-labeled cells. Insulin increased the 32P-content of I-2 by as much as 40%, suggesting that phosphorylation of I-2 might be involved in the effect of insulin on stimulating protein dephosphorylation.     

Growth factor-stimulated protein phosphorylation in 3T3-L1 cells. Evidence for protein kinase C-dependent and -independent pathways

Exposure of serum-deprived 3T3-L1 fibroblasts to phorbol 12-myristate 13-acetate (PMA), synthetic diacylglycerols, platelet-derived growth factor (PDGF), or pituitary fibroblast growth factor (FGF) resulted in stimulated phosphorylation of an acidic, multicomponent, soluble protein of Mr 80,000. Phosphorylation of this protein was promoted to a lesser extent by epidermal growth factor; however, neither insulin nor dibutyryl cAMP was effective. Phosphoamino acid analysis and peptide mapping of the Mr 80,000 32P-protein after exposure of fibroblasts to PDGF revealed identical patterns to those obtained with PMA or diacylglycerols. In contrast to the Mr 80,000 protein, proteins of Mr 22,000 (and pI 4.4) and Mr 31,000 were also phosphorylated in response to insulin as well as to PMA, diacylglycerols, epidermal growth factor, PDGF, and FGF in these cells. Similar findings were noted in fully differentiated 3T3-L1 adipocytes. Preincubation of the cells with high concentrations of active phorbol esters abolished specific [3H]phorbol 12,13-dibutyrate binding, protein kinase C activity, and immunoreactivity and also prevented stimulated phosphorylation of the Mr 80,000 protein by PMA, diacylglycerols, PDGF, or FGF, supporting the contention that this effect was mediated through protein kinase C. The stimulated phosphorylation of the Mr 22,000 and 31,000 proteins in response to PMA was also abolished by such pretreatment. In contrast, the ability of insulin, PDGF, and FGF to promote phosphorylation of the Mr 22,000 and 31,000 proteins was unaffected in the protein kinase C-deficient cells. We conclude that PDGF and FGF may exert some of their effects on these cells through at least two distinct pathways of protein phosphorylation, phorbol ester-like (P) activation of protein kinase C, and an insulin-like (I) pathway exemplified by phosphorylation of the Mr 22,000 and 31,000 proteins.     

Control of glycogen synthase phosphorylation in isolated rat hepatocytes by epinephrine, vasopressin and glucagon

Isolated rat hepatocytes were incubated in a medium containing 0.1 mM [32P]phosphate (0.1 mCi/ml) before exposure to epinephrine, glucagon or vasopressin. 32P-labeled glycogen synthase was purified from extracts of control or hormone-treated cells by the use of specific antibodies raised to rabbit skeletal muscle glycogen synthase. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that a single 32P-labeled polypeptide, apparent Mr 88000, was removed specifically by the antibodies and corresponded to glycogen synthase. Similar electrophoretic analysis of CNBr fragments prepared from the immunoprecipitate revealed that 32P was distributed between two fragments, of apparent Mr 14000 (CB-1) and 28000 (CB-2). Epinephrine, vasopressin or glucagon increased the 32P content of the glycogen synthase subunit. CB-2 phosphorylation was increased by all three hormones while CB-1 was most affected by epinephrine and vasopressin. These effects correlated with a decrease in glycogen synthase activity. From studies using rat liver glycogen synthase, purified by conventional methods and phosphorylated in vitro by individual protein kinases, it was found that electrophoretically similar CNBr fragments could be obtained. However, neither cyclic-AMP-dependent protein kinase nor three different Ca2+-dependent enzymes (phosphorylase kinase, calmodulin-dependent protein kinase, and protein kinase C) were effective in phosphorylating CB-2. The protein kinases most effective towards CB-2 were the Ca2+ and cyclic-nucleotide-independent enzymes casein kinase II (PC0.7) and FA/GSK-3. The results demonstrate that rat liver glycogen synthase undergoes multiple phosphorylation in whole cells and that stimulation of cells by glycogenolytic hormones can modify the phosphorylation of at least two distinct sites in the enzyme. The specificity of the hormones, however, cannot be explained simply by the direct action of any known protein kinase dependent on cyclic nucleotide or Ca2+. Therefore, either control of other protein kinases, such as FA/GSK-3, is involved or phosphatase activity is regulated, or both.     

The activation of eukaryotic initiation factor (eIF)2B by growth factors in PC12 cells requires MEK/ERK signalling

Epidermal and nerve growth factors (EGF and NGF) activate protein synthesis and initiation factor eIF2B in rat phaeochromocytoma (PC12) cells. The activation of protein synthesis by EGF or NGF depends upon extracellular regulated kinase kinase (MEK)/extracellular regulated kinase signalling. Here we show that PD98059, an inhibitor of MEK activation, blocks the activation of eIF2B by EGF or NGF. It is known that eIF2B activity can be inhibited by phosphorylation at Ser535 in its epsilon-subunit by glycogen synthase kinase (GSK)-3. We find that inactivation of GSK-3 by EGF or NGF is blocked by PD98059. However, neither EGF nor NGF caused a detectable change in phosphorylation of Ser535 of eIF2Bepsilon. Thus, the EGF- and NGF-induced activation of eIF2B in PC12 cells involves regulatory mechanisms distinct from dephosphorylation of the GSK-3 site.     

Rapid phosphorylation of MAP-2-related cytoplasmic and nuclear Mr 300,000 protein by serine kinases after growth stimulation in quiescent cells

Antibody against brain microtubule-associated protein 2 (MAP-2) immunoprecipitated Mr 300,000 and 80,000 proteins of cultured fibroblasts and kidney cells. These proteins were not appreciably phosphorylated in quiescent cells, but were rapidly phosphorylated after growth stimulation by insulin, epidermal and fibroblast growth factors, transferrin, phorbol ester and diacylglycerol in the presence of Ca2+, in a manner similar to that of MAP-1-related Mr 350,000 protein (J. Cell Biol. 100, 748-753). A Ca2+ ionophore, which is known to make the quiescent cell competent but not to enter into the growth cycle, did not induce the phosphorylation. In a chase experiment, decay half lives of labeled phosphoproteins were 5 h for Mr 350,000 and 300,000 proteins, and 1.5 h for Mr 80,000 protein. On subcellular fractionation, phosphorylated Mr 350,000 and 300,000 proteins were detected first mainly in the cytoplasm and then in the nucleus, while Mr 80,000 phosphoprotein was consistently detected in the cytoplasm. The phosphorylation of these proteins occurred on serine residues after stimulation with various factors. Thus, the phosphorylation of cytoskeleton-associated Mr 350,000 and 300,000 proteins by serine kinases seems to be a common second process after growth stimulation and to link cytoplasmic and intranuclear events.     

Nerve growth factor induces the association of a 130-Kd phosphoprotein with its receptor in PC-12 pheochromocytoma cells.

To explore the molecular mechanisms of nerve growth factor (NGF) action, we have attempted to identify proteins that immunoprecipitate with the NGF receptor. An anti-NGF receptor antibody was developed that immunoprecipitated the 75-Kd receptor in PC-12 cells. In [35S]methionine-labeled cells lysed with nonionic detergent, immunoprecipitation with this antireceptor antisera specifically brought down several associated proteins, although prior treatment of cells with NGF produced no apparent change in the distribution of these proteins. However, in vitro phosphorylation assays of the immunoprecipitated complex revealed the presence of a serine kinase that phosphorylated two predominant substrates with Mrs of 60 and 130 Kd. Prior treatment of cells produced no change in the appearance of the 60-Kd phosphoprotein, but NGF did stimulate the appearance of the 130-Kd protein. This effect was observed with as little as 0.1 nM NGF and was maximal at 5 min, but declined thereafter. Prior treatment of cells with NGF did not increase the phosphorylation of enolase added exogenously to the immunoprecipitates, suggesting that this action of NGF may have reflected the hormone-dependent association of the 130-Kd protein with the receptor, rather than activation of a receptor-associated kinase. Thus the association of the NGF 75-Kd receptor with a 130-Kd protein may be involved in signal transduction for the growth factor, although the role of this receptor in the NGF-dependent tyrosine phosphorylation remains unclear.     

Protein kinase C-stimulated phosphorylation in vitro of a Mr 80,000 protein phosphorylated in response to phorbol esters and growth factors in intact fibroblasts. Distinction from protein kinase C and prominence in brain

In previous studies in intact 3T3-L1 fibroblasts and adipocytes, we demonstrated that the phosphorylation state of an acidic, multicomponent Mr 80,000 protein appeared to be a specific and useful marker for the activation state of protein kinase C (Blackshear, P.J., Witters, L.A., Girard, P.R., Kuo, J.F., and Quamo, S.N. (1985) J. Biol. Chem. 260, 13304-13315). In the present studies, we demonstrate that the Mr 80,000 protein from rat adipose tissue was a substrate for protein kinase C in vitro, and co-migrated on two-dimensional gels with the analogous protein from murine 3T3-L1 adipocytes labeled by exposure of intact cells to 32Pi and phorbol 12-myristate 13-acetate. Partial proteolytic maps of the two 32P-proteins were nearly identical, supporting the postulate that the sites phosphorylated by protein kinase C in vitro, and in response to phorbol 12-myristate 13-acetate in vivo, were similar or identical. Despite their similar apparent molecular weights, we were able to distinguish between the Mr 80,000 protein and protein kinase C by several physical criteria. The Mr 80,000 protein kinase C substrate was found in fractions of all rat tissues examined, but was most prominent in rat brain. Phorbol 12-myristate 13-acetate also stimulated phosphorylation of the Mr 80,000 protein in several types of cultured neuronal cells, suggesting a possible role for this protein in cholinergic neurotransmission. The Mr 80,000 protein appears to be a useful marker for protein kinase C activation in a variety of cell types.     

Insulin action inhibits insulin-like growth factor-II (IGF-II) receptor phosphorylation in H-35 hepatoma cells. IGF-II receptors isolated from insulin-treated cells exhibit enhanced in vitro phosphorylation by casein kinase II

Insulin caused a rapid, dose-dependent increase in the binding of 125I-insulin-like growth factor-II (IGF-II) to the surface of cultured H-35 hepatoma cells. The [32P]phosphate content of the IGF-II receptors, immunoprecipitated from extracts of H-35 cell monolayers previously incubated with [32P]phosphate for 24 h, was decreased after brief exposure of the cells to insulin. Analysis of tryptic digests of labeled IGF-II receptors by bidimensional peptide mapping revealed that the decrease in the content of [32P]phosphate occurred to varying degrees on three tryptic phosphopeptides. Thin layer electrophoresis of an acid hydrolysate of isolated IGF-II receptors revealed the presence of [32P] phosphoserine and [32P]phosphothreonine. Insulin treatment of cells caused a decrease in the labeled phosphoserine and phosphothreonine content of IGF-II receptors. The ability of a number of highly purified protein kinases (cAMP-dependent protein kinase, protein kinase C, phosphorylase kinase, and casein kinase II) to catalyze the phosphorylation of purified IGF-II receptors was examined. Casein kinase II was the only kinase capable of catalyzing the phosphorylation of the IGF-II receptor on serine and threonine residues under the conditions of our assay. Bidimensional peptide mapping revealed that the kinase catalyzed phosphorylation of the IGF-II receptor on a tryptic phosphopeptide which comigrated with the main tryptic phosphopeptide found in receptors obtained from cells labeled in vivo with [32P]phosphate. IGF-II receptors isolated by immunoadsorption from insulin-treated H-35 cells were phosphorylated in vitro by casein kinase II to a greater extent than the receptors isolated from control cells. Similarly, IGF-II receptors from plasma membranes obtained from insulin-treated adipocytes were phosphorylated by casein kinase II to a greater extent than the receptors from control adipocyte plasma membranes. Thus, the insulin-regulated phosphorylation sites on the IGF-II receptor appear to serve as substrates in vivo for casein kinase II or an enzyme with similar substrate specificity.     

Ca2+-dependent and cAMP-dependent control of nicotinic acetylcholine receptor phosphorylation in muscle cells

Mouse BC3H1 myocytes were incubated with 32Pi before acetylcholine receptors were solubilized, immunoprecipitated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 90% of the 32P found in the receptor was bound to the delta subunit. Two phosphorylation sites in this subunit were resolved by reverse phase high performance liquid chromatography after exhaustive proteolysis of the protein with trypsin. Sites 1 and 2 were phosphorylated to approximately the same level in control cells. The divalent cation ionophore, A23187, increased 32P in site 1 by 40%, but did not affect the 32P content of site 2. In contrast, isoproterenol increased 32P in site 2 by more than 60%, while increasing 32P in site 1 by only 20%. When dephosphorylated receptor was incubated with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase, the delta subunit was phosphorylated to a maximal level of 1.6 phosphates/subunit. Approximately half of the phosphate went into site 2, with the remainder going into a site not phosphorylated in cells. The alpha subunit was phosphorylated more slowly, but phosphorylation of both alpha and delta subunits was blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase. Phosphorylation of the receptor was also observed with preparations of phosphorylase kinase. In this case phosphorylation occurred in the beta subunit and site 1 of the delta subunit, neither of which were phosphorylated by cAMP-dependent protein kinase. The rate of receptor phosphorylation by phosphorylase kinase was slow relative to that catalyzed by cAMP-dependent protein kinase. Therefore, it can not yet be concluded that phosphorylase kinase phosphorylates the beta subunit and the delta subunit site 1 in cells. However, the results strongly support the hypothesis that phosphorylation by cAMP-dependent protein kinase accounts for phosphorylation of the alpha subunit and the delta subunit site 2 in response to elevations in cAMP.     

Phosphorylation of the glucose transporter in rat adipocytes. Identification of the intracellular domain at the carboxyl terminus as a target for phosphorylation in intact-cells and in vitro

Phosphorylation of the insulin-regulatable glucose transporter (IRGT) is increased by incubating rat adipocytes with isoproterenol or by incubating microsomal membranes with cAMP-dependent protein kinase. To attempt to locate the sites of phosphorylation, the IRGT (apparent Mr = 46,000) was immunoprecipitated from 32P-labeled adipocytes and cleaved with CNBr or trypsin. Essentially all of the 32P could be recovered in a single CNBr fragment, denoted CB-T (Mr = 8,000), which bound a polyclonal antibody (R820) against a peptide having the sequence of the last 12 amino acids in the COOH terminus of the IRGT. 32P-Labeling of the IRGT was also confined to CB-T when membranes were incubated with [gamma-32P]ATP and cAMP-dependent protein kinase. Isoproterenol increased phosphorylation of CB-T, but insulin was without effect. To resolve phosphorylation sites further, IRGT from 32P-labeled cells was subjected to exhaustive proteolysis with trypsin. Samples were applied to a C-18 column, and 32P-labeled fragments were resolved into three peak fractions by elution with an increasing gradient of acetonitrile. [32P]Phosphoserine was the only phosphoamino acid detected in any of the peaks. Peak III contained approximately 80% of the 32P and was increased by isoproterenol. Almost all of the 32P introduced by cAMP-dependent protein kinase in vitro eluted in Peak III. In all cases, the 32P-labeled species in Peak III were quantitatively immunoprecipitated by R820. Digesting the peptide(s) in Peak III with V8 protease generated a single peak of 32P which eluted at lower acetonitrile than Peak III and contained 32P-labeled species that did not interact with R820. Automated Edman degradation indicated that the serine residue in Peak III phosphorylated by cAMP-dependent protein kinase was the 3rd or 4th residue from the NH2 terminus of the peptide. These findings indicate that phosphorylation of the IRGT is restricted to the presumed intracellular domain at the COOH terminus and that Ser488 is a major site phosphorylated both by cAMP-dependent protein kinase in vitro and in response to isoproterenol in vivo.     

Stimulation of a MR 80,000 protein phosphorylation by EGF in EGF receptor-hyperproducing human tumor cells

NA and Ca9-22 cells derived from squamous cell carcinomas of the tongue possess a large number of epidermal growth factor (EGF) receptors (2.0 X 10(6) and 1.3 X 10(6) receptors/cell, respectively). In these cell lines, EGF stimulated receptor autophosphorylation and phosphatidylinositol (PI) turnover. Furthermore, EGF enhanced the phosphorylation of an acidic protein of Mr 80,000. Phosphorylation of this protein was also stimulated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a phorbol ester tumor promoter, and was mainly at serine residues. Phosphopeptide mapping using protease V8 or trypsin indicated that Mr 80,000 proteins isolated from the EGF- and TPA-treated cells were identical. The Mr 80,000 protein was present mainly in the cytosol, but it became closely associated with the membrane as a phosphorylated form upon EGF or TPA stimulation. These results suggest that the EGF-stimulated phosphorylation of the Mr 80,000 acidic phosphoprotein in EGF receptor-hyperproducing tumor cells is mediated through the activation of PI turnover and protein kinase C.     

Ecto-Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Abstract: The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [γ-³²P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with ³²P_i. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P_i. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-α-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75–87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg²⁺, implicating this activity in the previously demonstrated regulation of Ca²⁺-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10–100 μM) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis.     

Multiple phosphorylation of rabbit skeletal muscle glycogen synthase. Evidence for interactions among phosphorylation sites and the resolution of electrophoretically distinct forms of the subunit

Phosphorylation of rabbit skeletal muscle glycogen synthase by a cyclic nucleotide and Ca2+-independent protein kinase, PC0.7, caused the enzyme to be a better substrate for phosphorylation by another cyclic nucleotide and Ca2+-independent protein kinase, FA/GSK-3. In contrast, phosphorylation by the combination of FA/GSK-3 and cyclic AMP-dependent protein kinase led to less phosphorylation than predicted from the individual actions of the protein kinases. These results are explained in part by the existence of cooperative interactions among the phosphorylation sites of glycogen synthase. Phosphorylation by FA/GSK-3 also correlated with a reduction in the electrophoretic mobility, in the presence of sodium dodecyl sulfate, of the glycogen synthase subunit from an apparent molecular weight of 85,000-86,000 to values of 88,000 and ultimately 90,000. The synergistic phosphorylation by PC0.7 and FA/GSK-3 was associated with an increased formation of the species of reduced electrophoretic mobility. The effects on subunit mobility were also reflected in the behavior of a larger phosphorylated CNBr fragment of glycogen synthase, CB-2, which gave apparent molecular weights of 22,000-27,000 depending on its phosphorylation state.     

Calcium-independent phospholipid/diolein-dependent phosphorylation of a soluble ovarian Mr 80,000 substrate protein: biochemical characteristics.

Soluble ovarian extracts were incubated with protein kinase effectors in the presence of [gamma 32P]ATP and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Autoradiograms revealed phosphorylation of an ovarian Mr = 80,000 substrate in the presence of EGTA ([ethylenebis(oxyethylenenitrilo)]tetraacetic acid), phosphatidylserine and 1,2-diolein. In contrast to a classical response pattern to C-kinase effectors, the ovarian Mr = 80,000 phosphorylation was inhibited by 2 x 10(-7) M or greater free Ca2+. The ovarian Mr = 80,000 substrate was distinguished from the myristoylated acidic Mr = 80,000 C-kinase substrate of brain tissue on the basis of heat stability and phosphorylative response to effectors. Phosphorylation of the exogenous substrate myelin basic protein by DEAE-resolved ovarian kinase showed the variant effector dependence, maximal in the presence of EGTA, phosphatidylserine and 1,2-diolein. Finally, the effect of Ca2+ on ovarian Mr = 80,000 [32P]phosphate content could not be accounted for by post-phosphorylation activities, or by DEAE-resolvable or hydroxylapatite-resolvable inhibitory activities.     

NGF activates the phosphorylation of MAP1B by GSK3beta through the TrkA receptor and not the p75(NTR) receptor

We have recently shown that nerve growth factor (NGF) induces the phosphorylation of the microtubule-associated protein 1B (MAP1B) by activating the serine/threonine kinase glycogen synthase kinase 3beta (GSK3beta) in a spatio-temporal pattern in PC12 cells that correlates tightly with neurite growth. PC12 cells express two types of membrane receptor for NGF: TrkA receptors and p75NTR receptors, and it was not clear from our studies which receptor was responsible. We show here that brain-derived neurotrophic factor, which activates p75NTR but not TrkA receptors, does not stimulate GSK3beta phosphorylation of MAP1B in PC12 cells. Similarly, NGF fails to activate GSK3beta phosphorylation of MAP1B in PC12 cells that lack TrkA receptors but express p75NTR receptors (PC12 nnr). Chick ciliary ganglion neurons in culture lack TrkA receptors but express p75NTR and also fail to show NGF-dependent GSK3beta phosphorylation of MAP1B, whereas in rat superior cervical ganglion neurons in culture, NGF activation of TrkA receptors elicits GSK3beta phosphorylation of MAP1B. Finally, inhibition of TrkA receptor tyrosine kinase activity in PC12 cells and superior cervical ganglion neurons with K252a potently and dose-dependently inhibits neurite elongation while concomitantly blocking GSK3beta phosphorylation of MAP1B. These results suggest that the activation of GSK3beta by NGF is mediated through the TrkA tyrosine kinase receptor and not through p75NTR receptors.     

Phosphorylation of the erbB gene product from an avian erythroblastosis virus-transformed chick fibroblast cell line

Polyclonal antiserum prepared against the human epidermal growth factor receptor immunoprecipitated four proteins of Mr = 66,000, 68,000, 74,000, and 82,000 from avian erythroblastosis virus-transformed chick embryo fibroblasts (cell line AEV-C23) which seemed to be related to the erbB gene product. The Mr = 66,000 and 68,000 proteins chased into the Mr = 74,000 and 82,000 proteins in pulse-chase experiments. The Mr = 68,000 and 82,000 proteins were found to be phosphorylated primarily on serine and threonine residues and contained minor amounts of phosphotyrosine. Tryptic peptide analysis of these phosphoproteins revealed several major peptides, and treatment of cells with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate resulted in the appearance of an additional phosphopeptide. 12-O-Tetradecanoyl-phorbol-13-acetate also inhibited growth of AEV-C23 cells in soft agar and in monolayer culture. In vitro phosphorylation of Mr = 68,000 and 74,000 proteins in immunoprecipitates occurred on tyrosine with lesser amounts of phosphoserine and phosphothreonine detected.     

Insulin stimulates tyrosine phosphorylation of its receptor beta-subunit in intact rat hepatocytes.

We studied the phosphorylation of the beta subunit of the insulin receptor in intact freshly isolated rat hepatocytes, labelled with [32P]Pi. Insulin receptors partially purified by wheat-germ agglutinin chromatography were immunoprecipitated with either antibodies to insulin receptor or antibodies to phosphotyrosine. Receptors derived from cells incubated in the absence of insulin contained only phosphoserine. Addition of insulin to hepatocytes led to a dose-dependent increase in receptor beta-subunit phosphorylation, with half-maximal stimulation being observed at 2 nM-insulin. Incubation of cells with 100 nM-insulin showed that, within 1 min of exposure to the hormone, maximal receptor phosphorylation occurred, which was followed by a slight decrease and then a plateau. This insulin-induced stimulation of its receptor phosphorylation was largely accounted for by phosphorylation on tyrosine residues. Sequential immunoprecipitation of receptor with anti-phosphotyrosine antibodies and with anti-receptor antibodies, and phosphoamino acid analysis of the immunoprecipitated receptors, revealed that receptors that failed to undergo tyrosine phosphorylation were phosphorylated on serine residues. The demonstration of a functional hormone-sensitive insulin-receptor kinase in normal cells strongly supports a role for this receptor enzymic activity in mediating biological effects of insulin.     

Bombesin, diacylglycerols, and phorbol esters rapidly stimulate the phosphorylation of an Mr = 80,000 protein kinase C substrate in permeabilized 3T3 cells. Effect of guanine nucleotides

We have used digitonin permeabilization to study the mechanism of bombesin-induced activation of protein kinase C in Swiss 3T3 cells. Protein kinase C-mediated phosphorylations in permeabilized cells were identified using phorbol esters and diacylglycerols. Addition of phorbol 12,13-dibutyrate (PDBu) in the presence of [gamma-32P]ATP and digitonin caused a marked and rapid time- and dose-dependent increase in the phosphorylation of an Mr 80,000 cellular protein (maximum stimulation = 12.6 +/- 1.6-fold after 1 min, EC50 = 27 nM). 1-oleoyl-2-acetylglycerol substituted for PDBu in stimulating the phosphorylation of Mr 80,000 protein (EC50 = 13 microM). Bombesin also caused a striking increase in the phosphorylation of Mr 80,000 protein with a time course similar to that observed with PDBu. This phosphorylation was mimicked by mammalian bombesin-like peptides and blocked by the bombesin antagonists [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P and [Leu13 psi (CH2NH)Leu14]bombesin. Down-regulation of protein kinase C in intact cells by prolonged exposure to PDBu prevented Mr 80,000 protein phosphorylation upon subsequent bombesin addition in digitonin-permeabilized cells. Comigration on one- and two-dimensional gel electrophoresis and phosphopeptide mapping confirmed that the Mr 80,000 protein phosphorylated in permeabilized cells was indistinguishable from the Mr 80,000 protein which is the major protein kinase C substrate in intact cells. The GDP analogue guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) caused a 70% inhibition of the bombesin-induced phosphorylation of Mr 80,000 protein but had no effect on the phosphorylation induced by PDBu. Bombesin stimulated Mr 80,000 protein phosphorylation in permeabilized cells in a dose-dependent manner (EC50 = 4 nM), and GDP beta S shifted the bombesin dose response curve to higher bombesin concentrations (EC50 = 14 nM). These results demonstrate for the first time a growth factor receptor-mediated activation of protein kinase C in permeabilized cells and provide functional evidence for the involvement of a G protein in the transmembrane signaling pathway that mediates the stimulation of protein kinase C by bombesin in Swiss 3T3 cells.