Introduction of protein kinase recognition sites into proteins: a review of their preparation, advantages, and applications.

Labeled proteins are used in a variety of applications. This review focuses on methods that utilize genetic engineering to introduce protein kinase recognition sites into proteins. Many protein kinase recognition sites can be introduced into proteins and serve as useful tags for a variety of purposes. The introduction of protein kinase recognition sites into proteins can be achieved without modifying the essential structure or function of the proteins. Because proteins modified by these procedures retain their activity after phosphorylation, they can be used in many applications. The phosphorylatable proteins can be labeled easily to high specific activity with radioisotopes ((32)P, (33)P, or (35)S), or the nonradioactive (31)P can be used. The use of these radioisotopes provides a convenient and safe method for radiolabeling proteins. Moreover, the use of the nonradioactive (31)P with protein tyrosine kinase recognition sites permits the tagging of proteins and their detection with the many anti-phosphotyrosine antibodies available. Overall, the procedure represents a convenient, safe, and efficient method to label proteins for a variety of applications.     

Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes.

Agents known to inphorylation of specific endogenous proteins in intact synaptosomes from rat brain. Synaptosome preparations, preincubated in vitro with 32Pi, incorporated 32P into a variety of specific proteins. Veratridine and high (60 mM) K+, which increase Ca2+ transport across membranes, through a mechanism involving membrane depolarization, as well as the calcium ionophore A23187, each markedly stimulated the incorporation of 32P into two specific proteins (80,000 and 86,000 daltons) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. All three agents failed to stimulate protein phosphorylation in calcium-free medium containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Moreover, the Ca2+-dependent protein phosphorylation could be reversed by the addition of sufficient EGTA to chelate all free extracellular Ca2+. Veratridine, high K+, and A23187 also stimulated 45Ca2+ accumulation by synaptosomes. Tetrodotoxin blocked the stimulation both of protein phosphorylation and of 45Ca2+ accumulation by veratridine but not by high K+ or A23187. Cyclic nucleotides and several putative neurotransmitters were without effect on protein phosphorylation in these intact synaptosome preparations. The absence of any endogenous protein phosphorylation in osmotically shocked synaptosome preparations incubated with 32Pi, and the inability of added [gamma-32P]ATP to serve as a substrate for veratridine-stimulated protein phosphorylation in intact preparations, indicated that the Ca2+-dependent protein phosphorylation occurred within intact subcellular organelles. Fractionation of a crude synaptosome preparation on a discontinuous Ficoll/sucrose flotation gradient indicated that these organelles were synaptosomes rather than mitochondria. The data suggest that conditions which cause an accumulation of calcium by synaptosomes lead to a calcium-dependent increase in phosphorylation of specific endogenous proteins. These phosphoproteins may be involved in the regulation of certain calcium-dependent nerve terminal functions such as neurotransmitter synthesis and release.     

Regulation by lipolytic and antillipolytic compounds of the phosphorylation of specific proteins in isolated intact fat cells.

The effects of various lipolytic and antilipolytic compounds on the phosphorylation of specific proteins, on lipolysis, and on cyclic AMP levels have been studied in isolated intact fat cells of rats. Norepinephrine (NE), adrenocorticotropic hormone (ACTH), 3-isobutyl-1-methylxanthine (IBMX), and monobutyryl cyclic AMP (MBcAMP) each increased the incorporation of [³²P] into three proteins, with apparent molecular weights of approximately 130,000 (protein A), 69,000 (protein B), and 47,000 (protein C), as determined by gel electrophoresis in the presence of sodium dodecyl sulfate (DodSO₄^?). The concentrations of lipolytic agents necessary to obtain a half-maximal increase in phosphorylation of these proteins were similar to the concentrations necessary to obtain a half-maximal stimulation of lipolysis. Propranolol, a β-adrenergic blocking agent, blocked the effects of NE both on protein phosphorylation and on lipolysis, but did not modify the effects of ACTH, IBMX, or MBcAMP on these parameters. When the NE-induced increase in phosphorylation of proteins B and C was maximal, addition of propranolol resulted in a rapid dephosphorylation of these proteins and a rapid cessation of lipolysis; under the same experimental conditions, propranolol had almost no effect on the dephosphorylation of protein A. Concentrations of insulin that prevented or reversed the actions of NE and ACTH on lipolysis also prevented or reversed the NE- and ACTH-induced increase in [³²P] incorporation into proteins B and C. Insulin did not modify the effects of IBMX or MBcAMP either on lipolysis or on [³²P] incorporation into proteins B and C. Insulin increased the incorporation of [³²P] into a protein which, by several criteria, appeared to be protein A. Under a variety of experimental conditions in which lipolytic and antilipolytic hormones were studied, the rate of lipolysis correlated well with the level of phosphorylation of proteins B and C, but not with the level of cyclic AMP.     

Protein Phosphorylation in Astrocytes Mediated by Protein Kinase C: Comparison with Phosphorylation by Cyclic AMP-Dependent Protein Kinase

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate-shaped, process-bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP-dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of 32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP-dependent mechanisms.     

Protein kinase C-zeta phosphorylates insulin-responsive aminopeptidase in vitro at Ser-80 and Ser-91

Insulin-responsive aminopeptidase (IRAP) colocalizes with glucose transporter type 4 (GLUT4) in adipocytes and is recruited to the plasma membrane in response to insulin. Microinjection of peptides corresponding to the IRAP cytoplasmic domain sequences causes GLUT4 recruitment in adipocytes. Inhibitors of protein kinase C-zeta (PKC-zeta) abolish the insulin-induced GLUT4 recruitment in rat adipocytes. These findings suggest an interesting possibility that PKC-zeta may phosphorylate IRAP, playing a key role in GLUT4/IRAP recruitment. To test this possibility, here we studied the (32)P incorporation into IRAP catalyzed by PKC-zeta in insulin-stimulated cells. There was a small but significant (32)P incorporation into IRAP in rat adipocytes, which was partly abolished upon addition of a PKC-zeta pseudosubstrate, suggesting that PKC-zeta may be responsible in part for the IRAP phosphorylation in adipocytes. PKC-zeta also catalyzed the incorporation of (32)P not only into IRAP in GLUT4 vesicles isolated from rat adipocytes but also into the IRAP cytoplasmic domain inserts in glutathione S-transferase-fusion proteins, demonstrating direct IRAP phosphorylation by PKC-zeta. Reversed-phase HPLC, matrix-assisted laser desorption ionization mass spectrometry, and radiosequencing of the tryptic digests of the (32)P-labeled IRAP fusion proteins identified Ser-80 and Ser-91 as major phosphorylation sites. In GLUT4 vesicles, the (32)P incorporation into IRAP was exclusively localized at a 6.9-kDa tryptic fragment identified as IRAP(76-138) and the (32)P labeling at Ser-80 accounted for 80-90% of the total IRAP labeling, suggesting that Ser-80 is the major phosphorylation site in intact IRAP. These findings are consistent with the possibility that the IRAP cytoplasmic domain phosphorylation by PKC-zeta plays a key role in insulin-induced IRAP or GLUT4 recruitment in adipocytes.     

Endogenous phosphorylation of proteins and phosphatidylinositol in the plasma membranes of a human astrocytoma

Incubation of plasma membrane preparations from several tissues with [gamma-32P]ATP resulted in the phosphorylation of phosphatidylinositol as well as of proteins. The presence of an active phosphatidylinositol kinase in these membranes was indicated by equal or greater incorporation of 32P into phosphatidylinositol phosphate than into proteins. Phosphorylation of endogenous protein and lipid substrates by protein and phosphatidylinositol kinases in the plasma membranes of a human astrocytoma was investigated in detail. Maximal protein phosphorylation required the presence of Nonidet-P40 and phosphatase inhibitors (vanadate or fluoride). The rate of protein phosphorylation was greater with Mg2+ than with Mn2+, and phosphoserine accounted for 60% of the radioactivity incorporated into proteins. In the presence of Mn2+, phosphorylation of tyrosine was increased and was equal to that of serine phosphorylation (40%). With one exception, the overall pattern of phosphorylated proteins was similar with either Mg2+ or Mn2+. Maximal phosphatidylinositol phosphorylation of the astrocytoma plasma membranes also required detergent and phosphatase inhibitors. However, the enzymatic characteristics of lipid phosphorylation differed from those of protein phosphorylation with respect to divalent cation activation, ATP dependence, and sensitivity to inhibition by p-chloromercuriphenyl sulfonate, quercetin, and nucleoside derivatives. These results suggest that phosphorylation of plasma membrane proteins and phosphatidylinositol is catalyzed by different enzymes. The fact that membrane preparations exhibited phosphatidylinositol kinase activity almost 100,000 times greater than that exhibited by the purified tyrosine kinase of ros gene would exclude this and similar oncogene proteins from making a significant contribution to the overall phosphatidylinositol phosphorylation of cell membranes.     

Alterations in tubulin immunoreactivity; relation to secondary structure

Blood sinusoidal plasma membrane subfractions were isolated from normal mouse liver in the presence of the proteinase inhibitors PhMeSO2F and iodoacetamide. They were purified from smooth microsomal and Golgi vesicle contaminants. The phosphorylation reaction was studied at 33 degrees C, in the presence of 2 mM MnCl2. Addition of epidermal growth factor (EGF) to the preparations stimulated 32P incorporation from [gamma-32P]ATP or [gamma-32P]GTP essentially into one 170 000 Mr protein. Some incorporation was observed in a minor 120 000-Mr component which appears to be a degradation product of the 170 000-Mr component. No EGF-dependent phosphorylation of other membrane proteins or various exogenous proteins could be detected in vitro. The dephosphorylation of the 170 000-Mr component was observed after 4 min of incubation at 33 degrees C. This dephosphorylation reaction was inhibited by addition of 5 mM p-nitrophenyl phosphate but not by addition of micromolar Zn2+, Be2+ or orthovanadate. The 170 000-Mr protein specifically bound 125I-labeled EGF and thus appeared to be the hepatic EGF receptor. The EGF stimulatable kinase activity considerably enhances incorporation of 32P into tyrosine residues of the 170 000-Mr EGF receptor at 33 degrees C. Tryptic peptide maps of the 32P-labeled 170 000-Mr protein revealed a multiplicity of phosphorylated sites. Seven 32P-labeled phosphopeptides were observed after EGF stimulation, three of them being largely prominent. Tryptic peptide maps of the 170 000-Mr protein after it was covalently linked to 125I-labeled EGF showed only one 125I-labeled peptide, the migration of which appeared different from that of 32P-labeled phosphopeptides. These findings were confirmed by V8 protease unidimensional peptide mapping of the 170 000-Mr protein, labeled with 32P or 125I-EGF.     

Tyrosine hydroxylase in rat brain dopaminergic nerve terminals. Multiple-site phosphorylation in vivo and in synaptosomes.

Tyrosine hydroxylase, which catalyzes the initial step in catecholamine biosynthesis, is phosphorylated at serines 8, 19, 31, and 40 in intact pheochromocytoma (PC12) cells (Haycock, J.W. (1990) J. Biol. Chem. 265, 11682-11691). After 32Pi labeling of rat corpus striata in vivo or rat corpus striatal synaptosomes, 32P incorporation into tyrosine hydroxylase occurred predominantly at serines 19, 31, and 40. Electrical stimulation (30 Hz, 20 min) of the medial forebrain bundle (containing the afferent dopaminergic fibers) increased 32P incorporation into each of the three sites. Brief depolarization of the synaptosomes with elevated [K+]o (20-60 mM, 5-30 s) or veratridine (50 microM, 2 min) produced a selective increase in 32P incorporation into Ser19. Phorbol 12,13-dibutyrate (1 microM, 5 min) increased 32P incorporation into Ser31, and cAMP-acting agents such as forskolin (10 microM, 5 min) increased 32P incorporation into Ser40. In contrast, 32P incorporation into Ser8, which was usually detectable but very low, was not regulated either in vivo or in situ by any of the activators of signal transduction pathways. In synaptosomes, the only treatment found to increase Ser8 phosphorylation was okadaic acid (a protein phosphatase inhibitor), which increased 32P incorporation into all four phosphorylation sites. Thus, three different signal transduction systems appear to mediate the physiological regulation of tyrosine hydroxylase phosphorylation at three different sites.     

Protein phosphorylation in normal and neoplastic development. Phosphorylation of proteins endogenous to foetal tissues and tumours.

The abilities of proteins endogenous to normal and neoplastic tissues to serve as substrates in a protein-phosphorylation reaction in vitro were compared. After the tissue extracts were incubated with [gamma-32P]ATP, the phosphorylated proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the dried gels were subjected to radioautography. Considerable incorporation of 32P into a protein of mol.wt. 135000 was observed with extracts from foetal tissues and tumours, but only minimal incorporation into this protein occurred when extracts from adult tissues were used. The ability of this protein to become phosphorylated in vitro may be related to cell proliferation. When ascites cells were incubated with [32P]Pi, one of the major phosphoproteins migrated on sodium dodecyl suphate/polyacrylamide gels at mol.wt. 135000, suggesting that this protein can be phosphorylated both in intact cells and broken-cell preparations. A protein of mol.wt. 87000 was highly phosphorylatable in extracts from solid tumours, but was not phosphorylated in extracts from ascites tumours, foetal or adult tissues. The phosphorylation pattern of these two proteins can thus distinguish solid neoplasms and normal adult tissues from ascites tumours and from foetal tissues. A protein of mol.wt. 49000, which was the most labelled protein in adult tissues, was also one of the major phosphoproteins in foetal and neoplastic tissues. Numerous mechanisms are postulated to explain how the extent of 32P incorporation into a protein could vary as a function of biological state.     

Co-ordinated changes in the cyclic AMP signalling system and the phosphorylation of two nuclear proteins of Mr 130,000 and 110,000 during proliferative stimulation of the rat parotid gland by isoprenaline. Possible identity of the two proteins with pp135 and nucleolin.

Parotid glands were stimulated to growth by repeated injection of the beta-agonist isoprenaline into rats. Incubation of intact parotid-gland lobules with [32P]Pi and subsequent analysis of nuclear proteins revealed in the stimulated glands an increased 32P incorporation into two acid-soluble non-histone proteins with apparent Mr values of 110,000 and 130,000 (p110 and p130). After a single injection of isoprenaline, leading to a biphasic increase in DNA synthesis (maximum at 24 h), the same two proteins showed a transiently increased 32P incorporation at 17 h after injection. At this time point at the onset of DNA synthesis the total activity of soluble cyclic AMP-dependent protein kinase decreased. No change in p110/p130 phosphorylation was observed at 0.3 h after stimulation, a time of maximal stimulation of secretion. Administration of the beta-antagonist propranolol 8 h after the injection of isoprenaline suppressed the increase in DNA synthesis, the preceding changes in the concentration of cyclic AMP and in the activity of cyclic AMP-dependent protein kinase, as well as the increased phosphorylation of p110 and p130. Cross-reactivity of p110 and p130 with specific antisera against two nucleolar phosphoproteins of similar molecular mass (nucleolin and pp135), as well as their localization in a nucleolar cell fraction, indicated a possible identity of p110 and p130 with these two proteins. Our results suggest that nucleolin and pp135 are nuclear target proteins of cyclic AMP in the cyclic AMP-influenced regulation of the transition of cells from the G1 to the S phase.     

Multiple signaling pathways in bovine chromaffin cells regulate tyrosine hydroxylase phosphorylation at Ser19, Ser31, and Ser40

Intact bovine adrenal medullary chromaffin cells were preincubated with³²PO₄, and the multiplesite phosphorylation of tyrosine hydroxylase (TH) was studied. Up to eight³²P-labeled peptides were produced by tryptic hydrolysis of TH; however, all of the tryptic phosphopeptides were derived from four phosphorylation sites—Ser⁸, Ser¹⁹, Ser³¹ and Ser⁴⁰. In situ regulation of³²P incorporation into the latter three sites was demonstrated with a diverse set of pharmacological agents.³²P incorporation into Ser¹⁹ was preferentially increased by brief exposures to depolarizing secretagogues. Longer treatments also increased Ser³¹ and Ser⁴⁰ phosphorylation. Nicotine, muscarine and vasoactive intestinal polypeptide—reflecting cholinergic and non-cholinergic components of sympatho-adrenal transmission—each produced different patterns of multiple-site phosphorylation of TH. Nicotine, bradykinin and histamine increased³²P incorporation at each of the three sites whereas muscarine, angiotensin II, endothelin III, prostaglandin E1, GABA and ATP selectively increased Ser³¹ phosphorylation. Nerve growth factor did not influence TH phosphorylation in chromaffin cells from adult adrenal glands but selectively increased Ser³¹ phosphorylation in chromaffin cells isolated from calf adrenal glands.³²P incorporation into Ser⁴⁰ was selectively increased by forskolin and other cAMP-acting agents whereas vasoactive intestinal polypeptide increased Ser³¹ and Ser⁴⁰ phosphorylation. Thus, the phosphorylation of TH in bovine chromaffin cells appears to be regulated at three sites by three separate intracellular signaling pathways—Ser¹⁹ via Ca²⁺/calmodulin-dependent protein kinase II; Ser³¹ via ERK (MAP2 kinases); and Ser⁴⁰ via cAMP-dependent protein kinase. These signaling pathways, as well as the extracellular signals that were effective in stimulating them, are similar to those previously described for TH in rat pheochromocytoma cells. However, several of the pharmacological agents produced different patterns of multiple-site TH phosphorylation in the bovine chromaffin cells. These differences between tissues could be accounted for by differences in the coupling/access between the extracellular signal transduction systems and the intracellular signaling pathways as opposed to differences in the intracellular signaling pathwaysper se.Special issue dedicated to Dr. Paul Greengard     

Neuromodulator-Mediated Phosphorylation of Specific Proteins in a Neurotumor Hybrid Cell Line (NCB-20)

Mouse neuroblastoma X embryonic Chinese hamster brain explant hybrid cell line (NCB-20) forms functional synapses when intracellular cyclic AMP levels are elevated for a prolonged period of time. NCB-20 cells were labeled with [32P]orthophosphate under conditions where 2-chloroadenosine gave maximum increases of 32P incorporation into tyrosine hydroxylase in nerve growth factor dibutyryl cyclic AMP-differentiated PC12 (pheochromocytoma) cells. When NCB-20 cells were exposed to activators [5-hydroxytryptamine (5-HT), prostaglandin E1, or forskolin], resulting in activation of cyclic AMP-dependent protein kinase, increased 32P incorporation into two major proteins [130 kilodaltons (kDa) and 90 kDa] occurred. 5-HT (in the presence of phosphodiesterase inhibitor, isobutylmethylxanthine) gave a three- to fourfold increase, and forskolin a four- to sevenfold increase in 32P incorporation into the 90-kDa protein. [D-Ala2,D-Leu5]-enkephalin, which decreased cyclic AMP levels and reversed the 2-chloroadenosine-stimulated phosphorylation of tyrosine hydroxylase in differentiated PC12 cells, also reversed the stimulation of phosphorylation of the 90-kDa protein in NCB-20 cells. Pretreatment of NCB-20 cells with a calcium ionophore, A23187, gave increased phosphorylation of the 90- and 130-kDa proteins, but phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (tumor promoting agent), cell depolarization with high K+, or pretreatment with dibutyryl cyclic GMP had no effect on phosphorylation of these proteins. In contrast, phosphorylation of an 80-kDa protein was decreased by forskolin, but increased following activation of the calcium/phospholipid-dependent kinase with tumor promoting agent. Neither the 90-kDa nor the 80-kDa protein showed any immunological cross-reactivity with synapsin, a major synaptic protein known to be phosphorylated by cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase, but not calcium/phospholipid-dependent protein kinase. This suggests that in NCB-20 cells, several unique proteins can be phosphorylated by cyclic AMP-dependent protein kinase in response to hormonal elevation of cyclic AMP levels. In contrast, an 80-kDa protein is the primary substrate for calcium/phospholipid-dependent protein kinase, and its phosphorylation is inhibited by agents that elevate cyclic AMP levels and thereby activate cyclic AMP-dependent protein kinase.     

Evidence for the participation of cytosolic protein kinases in membrane phosphorylation in intact erythrocytes.

The effects of adenosine 3':5'-monophosphate (cyclic AMP) on the phosphorylation of membrane proteins in intact rabbit and human erythrocytes were investigated. The addition of cyclic AMP to intact human or rabbit erythrocytes results in an increase in the incorporation of ortho[32P]phosphate into several membrane protein components which are known to serve as substrates for the cyclic-AMP-dependent protein kinases. Thus this increase in protein phsophorylation is probably due to the activation of either soluble or membrane-bound cyclic-AMP-dependent protein kinases. Incubation of human erythrocytes in the presence of ortho [32P]phosphate and cyclic AMP also leads to the phosphorylation of a membrane protein component, band 7, which has not been previously detected in the autophosphorylation of isolated ghosts. Since rabbit erythrocyte membranes do not contain any cyclic-AMP-dependent protein kinase, the results suggest that cytoplasmic kinases also play a role in the phosphorylation of membrane proteins in intact cells.     

Stimulation of tyrosine-specific protein phosphorylation and phosphatidylinositol phosphorylation by orthovanadate in rat liver plasma membrane

Orthovanadate stimulated the incorporation of 32P from [gamma-32P]ATP by Triton X-100-solubilized rat liver plasma membrane into endogenous, trichloroacetic acid-precipitable materials as well as added (Glu4:Tyr1) copolymers. Extraction of incubation mixture with chloroform-methanol-HCl revealed that the increase in 32P incorporation by vanadate was predominantly into endogenous phospholipids. [32P]Phosphatidylinositol 4-phosphate (PtdIns-4-P) was identified by thin-layer chromatography as the major phosphorylated product of vanadate stimulation, which also resulted in elevated 32P, predominantly in P-Tyr in endogenous membrane proteins. Vanadate effects on protein tyrosine and phosphatidylinositol phosphorylation were concomitant and exhibited similar sensitivity. These effects of vanadate were enhanced by the presence of either dithiothreitol or NAD(P)H. Phosphatidylinositol phosphorylation could also be stimulated by a substrate of and inhibited by a synthetic inhibitory copolymer of tyrosine kinase. These results suggest that vanadate, an oxygen radical producer, stimulates a tyrosine kinase-PtdIns kinase coupled system much like those described for a number of growth factors and oncogene encoded products.     

Receptor-mediated phosphorylation of spermatozoan proteins

These studies are the first to report egg peptide-mediated stimulation of protein phosphorylation in spermatozoa. Speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) or resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2) stimulated the incorporation of 32P into various proteins of isolated spermatozoan membranes in the presence, but not absence, of GTP. The Mr of three of the phosphorylated proteins were 52,000, 75,000, and 100,000. GTP gamma S (guanosine 5'-O-(3-thiotriphosphate] but not GDP beta S (guanosine 5'-O-(2-thiodiphosphate] or GMP-PNP (guanylyl imidodiphosphate) also supported the peptide-mediated stimulation of protein phosphorylation. The peptides markedly stimulated guanylate cyclase activity, and GTP gamma S or GTP but not GMP-PNP served as effective substrates for the enzyme. The accumulation of cyclic AMP was not stimulated by the peptides. Subsequently, it was shown that added cyclic GMP or cyclic AMP increased 32P incorporation into the same membrane proteins as those observed in the presence of peptide and GTP. The amount of cyclic GMP (up to 3 microM) formed by membranes in the presence of peptide and 100 microM GTP equated with the amount of added cyclic GMP required to increase the 32P content of a Mr 75,000 protein selected for further study. 32P-Peptide maps of the Mr 75,000 protein indicated that the same domains were phosphorylated in response to cyclic nucleotides or to egg peptide and GTP. Intact cells were subsequently incubated with 32P to determine if the radiolabeled proteins observed in isolated membranes also would be obtained in intact cells. The 32P contents of proteins of Mr 52,000, 75,000, and 100,000 were significantly increased by the addition of resact. Peptide maps confirmed that the increased 32P incorporation obtained in a Mr 75,000 protein of isolated membranes occurred on the same protein domains as the 32P found on the Mr 75,000 protein of intact cells. These results suggest that a GTP or GTP gamma S requirement for peptide-mediated protein phosphorylation in spermatozoan membranes is mainly due to the enhanced formation of cyclic GMP, and it therefore is likely that peptide-induced elevations of cyclic nucleotide concentrations in spermatozoa are responsible for the specific increases in 32P associated with at least three sperm proteins, all apparently localized on the plasma membrane.     

Modification of fos proteins: phosphorylation of c-fos, but not v-fos, is stimulated by 12-tetradecanoyl-phorbol-13-acetate and serum.

We have investigated the covalent modification of the proteins encoded by the murine fos proto-oncogene (c-fos) and that of the corresponding gene product of FBJ murine osteosarcoma virus (v-fos). Both proteins are posttranslationally processed in the cell, resulting in forms with lower electrophoretic mobilities than that of the initial translation product on sodium dodecyl sulfate-polyacrylamide gels. Treatment with alkaline phosphatase indicates that most, if not all, of this electrophoretic shift is due to phosphoesterification of both proteins. These phosphoryl groups stoichiometrically modify the v-fos and c-fos proteins on serine residues and turn over rapidly in vivo in the presence of protein kinase inhibitors (half-life, less than 15 min). Direct quantitative comparison of steady-state labeling studies with L-[35S]methionine and [32P]phosphate reveals that the c-fos protein is four- to fivefold more highly phosphorylated than the v-fos protein is. Comparison of tryptic fragments from [32P]phosphate-labeled proteins indicates that although the two proteins have several tryptic phosphopeptides in common, the c-fos protein contains unique major tryptic phosphopeptides that the v-fos protein lacks. These unique sites of c-fos phosphorylation have been tentatively localized to the carboxy-terminal 20 amino acid residues of the protein. Phosphorylation of the c-fos protein, but not the v-fos protein, can be stimulated at least fivefold in vivo by the addition of either 12-tetradecanoyl-phorbol-13-acetate or serum. This increase in the steady-state degree of phosphorylation of c-fos appears to be independent of protein kinase C since phosphorylation is Ca2+ and diacylglycerol independent. The possible role of phosphorylation of these proteins in cellular transformation is discussed.     

Studies on insulin-stimulated phosphorylation of acetyl-CoA carboxylase, ATP citrate lyase and other proteins in rat epididymal adipose tissue. Evidence for activation of a cyclic AMP-independent protein kinase.

Protein kinase activity in high-speed supernatant fractions prepared from rat epididymal adipose tissue previously incubated in the absence or presence of insulin was investigated by following the incorporation of 32P from [gamma-32P]ATP into phosphoproteins separated by sodium dodecyl sulphate/polyacrylamide-gel electro-phoresis. Incorporation of 32P into several endogenous proteins in the supernatant fractions from insulin-treated tissue was significantly increased. These included acetyl-CoA carboxylase and ATP citrate lyase (which exhibit increased phosphorylation within fat-cells exposed to insulin), together with two unknown proteins of subunit Mr 78000 and 43000. The protein kinase activity increased by insulin was distinct from cyclic AMP-dependent protein kinase, was not dependent on Ca2+ and was not appreciably affected by dialysis or gel filtration. The rate of phosphorylation of added purified fat-cell acetyl-CoA carboxylase and ATP citrate lyase was also increased by 60-90% in high-speed-supernatant fractions prepared from insulin-treated tissue. No evidence for any persistent changes in phosphoprotein phosphatase activity was found. It is concluded that insulin action on acetyl-CoA carboxylase, ATP citrate lyase and other intracellular proteins exhibiting increased phosphorylation involves an increase in cyclic AMP-independent protein kinase activity in the cytoplasm. The possibility that the increase reflects translocation from the plasma membrane, perhaps after phosphorylation by the protein tyrosine kinase associated with insulin receptors, is discussed.     

Actions of insulin, epinephrine, and dibutyryl cyclic adenosine 5'-monophosphate on fat cell protein phosphorylations. Cyclic adenosine 5'-monophosphate dependent and independent mechanisms.

Endogenous and hormone-induced protein (polypeptide) phosphorylations were studied in isolated rat fat cells, in fat pads, and in subcellular fractions obtained from fat tissue under different physiological conditions. Insulin (25-100 muU/ml) increased the incorporation of 32P into two proteins: insulin-phosphorylated proteins (IPP 140 and IPP 50; similar to 140,000 and 50,000 daltons, respectively). Epinephrine (10(-7)-10(-6) M) increased the incorporation of 32P into another protein: epinephrine-phosphorylated protein (EPP 60-65; similar to 60,000-65,000 daltons). Endogenous IPP 140 phosphorylation in fat cells obtained from fasted and refed rats was similar to that of insulin in normal cells. Studies of insulin and epinephrine interactions showed that insulin increased IPP 140 phosphorylation even in the presence of epinephrine or lithium (25 mM times 10(-3) M). dibutyryl cyclic AMP (5 times 10(-4) M) markedly stimulated EPP 60-65 phosphorylation, but neither epinephrine (10(-7)-10(-6) M) nor dibutyryl cyclic AMP reproduced insulin's phosphorylation of APP 140. Lithium inhibited both endogenous and epinephrine-stimulate EPP 60-65 phosphorylation, but did not inhibit that induced by dibutyryl cyclic AMP. These findings suggest that insulin stimulated a specific, cyclic AMP independent protein kinase for IPP 140 phosphorylation. Cell-free extracts from insulin-treated fat tissue catalyzed the specific transfer of 32P from ATP to IPP 140 more rapidly than control extracts. No differences in the total receptor protein or total protein kinase activity using [gamma(-32P]ATP were noted between insulin-treated and control preparations. IPP 140 may be either (a) an insulin-sensitive protein kinase (phosphotransferase) or (b) a protein whose function is regulated by an insulin-sensitive protein kinase or phosphatase.     

Protein phosphorylation in chick kidney. Response to parathyroid hormone, cyclic AMP, calcium, and phosphatidylserine

The regulation of endogenous protein phosphorylation by parathyroid hormone (PTH) was investigated using confluent monolayer cultures of chick kidney cells. Homogenates and subcellular fractions of PTH (bovine 1-34)-treated cells were subjected to an endogenous protein phosphorylation assay using ((gamma- 32P]ATP in the presence or absence of 2.0 microM cAMP or 0.5 mM Ca2+ with 25 micrograms/ml of phosphatidylserine and reactions terminated with sodium dodecyl sulfate. In other experiments, cultures were incubated in a phosphate-free 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline containing 50 muCi/ml of [32P]PO4 and incubations were terminated with sodium dodecyl sulfate. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Cyclic AMP stimulated 32P incorporation into proteins having molecular weights of 17,000, 22,000, 35,000, 42,000, 54,000, 75,000, 80,000, 120,000, and 143,000. Calcium-phosphatidylserine stimulated the phosphorylation of proteins of 20,000, 52,000, 58,000, 60,000, and 143,000. The protein phosphorylation patterns in cultured kidney cells and freshly isolated kidney tissue were quite similar. Treatment of cultured cells with 5-50 ng/ml of PTH resulted in stimulated phosphorylation of the 35,000 and 42,000 dalton proteins as assessed by endogenous phosphorylation in homogenates. In intact cells incubated with [32P]PO4, PTH stimulated most noticeably the phosphorylation of the 35,000-dalton protein. Based on studies with cultured and fresh kidney cells, the majority of the substrate proteins for cAMP and calcium-dependent protein kinases were located in the cytoplasm with the exception of the 42,000-dalton protein which was located in the brush-border-plasma membrane fraction. The cytoplasmic cAMP-dependent protein kinase activity was responsible for the majority of PTH-stimulated protein phosphorylation.     

GRP94 hyperglycosylation and phosphorylation in Sf21 cells

GRP94 is an inducible resident endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) glycoprotein that functions as a protein chaperone and Ca(2+) regulator. GRP94 has been reported to be a substrate for protein kinase CK2 in vitro, although its phosphorylation in intact cells remains unreported. In Sf21 insect cells, overexpression of canine GRP94 led to the appearance of a multiplet of three or more molecular-mass isoforms which was reduced to a single mobility form following treatment of cells with tunicamycin, suggesting stable accumulations of consecutively modified protein. Metabolic labeling of Sf21 cells with (32)P(i) led to a constitutive phosphorylation of GRP94 which, based upon phosphopeptide mapping, occurred specifically on CK2-sensitive sites. Among the GRP94 multiplet, however, only the lowest mobility form of GRP94 was phosphorylated, even though in vitro phosphorylation of GRP94 by CK2 led to phosphorylation of all glycosylated forms. The (32)P(i) incorporation into GRP94 indicated a slow turnover of phosphate incorporation that was unaffected by inhibition of biosynthesis, resulting in a steady-state level of phospho-GRP94 on CK2 sites. These data support a role for protein kinase CK2 in the cell biology for GRP94 and other resident ER/SR proteins that may occur in ER compartments.