Phosphorylations of the subcellular matrix in cells transformed by Rous' sarcoma virus

The transforming protein of Rous' sarcoma virus (RSV) is a phosphoprotein of Mr 60 000 (pp60src) which displays protein kinase activity specific for tyrosine residues; pp60src is associated with the plasma membrane and is recovered in the detergent-insoluble material which represents the subcellular matrix of the cell. After phosphorylation of this material of RSV-transformed cells with [gamma-32P]ATP, five phosphoproteins have been detected which are not seen in normal cells. These proteins (Mr = 135 000, 125 000, 75 000, 70 000, 60 000) contain phosphotyrosine. Their phosphorylation is strongly inhibited by anti-pp60src antibodies. In cells transformed by a temperature-sensitive mutant of RSV, these phosphoproteins, present at the permissive temperature, are no longer detected at the non-permissive temperature. It is concluded that these phosphorylations are mediated by pp60src protein kinase activity. This supports a possible role of the phosphorylation of cytoskeletal proteins in the transformation process.     

Modulation of Phosphorylation of a 30-kD Polyribosomal Protein (pp30) by ACTH and Spermine: Comparison with Modulation of Brain Protein Synthesis

Gel electrophoretic separation of proteins phosphorylated in a postmitochondrial supernatant fraction of brain in the presence of spermine or adrenocorticotropin (ACTH) indicated modulation in only one region (30 kD) of the gel. The 30-kD (pp30) protein together with enzyme activity catalyzing its phosphorylation and sensitivity of the phosphorylation to spermine and ACTH were retained in a free polyribosomal fraction of this extract. ACTH(11-24) inhibited phosphorylation at all the spermine or Mg2+ concentrations tested. Structure-activity studies revealed that the inhibitory activity within ACTH(1-24) resides in the sequences ACTH(11-24), (5-18, 17Lys, 18Lys)-NH2, (15-24), (7-16)-NH2, and (1-16)-NH2 and can also be found in certain polylysine fragments. Phosphorylation under conditions suitable for measuring protein synthesis revealed only one phosphoprotein (pp30), sensitive to both ACTH(15-24) and spermine. The possibility of a relationship between modulation of pp30 phosphorylation and modulation of brain cell-free protein synthesis is discussed in relation to the effects of ACTH, spermine, and Mg2+.     

Fat cell protein phosphorylation. Identification of phosphoprotein-2 as ATP-citrate lyase.

We have purified to apparent homogeneity a phosphoprotein from rat adipose tissue which is rapidly phosphorylated in vitro by ATP. The native phosphoprotein has an approximate sedimentation coefficient of 14.8 S. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the protein dissociated into identical subunits of Mr = 128,000. A phosphoprotein with similar properties was also isolated from liver. Purified phosphoproteins from fat cells and liver had ATP-citrate lyase activity and co-migrated on sodium dodecyl sulfate gels with fat cell phosphoprotein-2, the phosphorylation of which is increased by incubating fat cells with insulin. The phosphoamino acid residue of the cells with insulin. The phosphoamino acid residue of the phosphoprotein was identified as tau-phosphohistidine. These evidences suggest that fat cell phosphoprotein-2 is ATP-citrate lyase.     

Polyamine Regulation of Protein Phosphorylation in the Brain of the Tobacco Hornworm, Manduca sexta

An analysis of the effects of polyamines on protein phosphorylation in cytosolic fractions of the pupal brain of Manduca sexta showed that spermine elicited an increase in casein phosphorylation in a dose-dependent manner (maximum three- to fourfold at 2.0 mM), whereas spermidine was less effective and putrescine was without effect. In contrast, with phosvitin as the exogenous substrate, higher doses of polyamines, especially spermine, inhibited phosphorylation. High salt conditions abolished the polyamine response. Cytosol protein kinase activity eluted from DEAE-cellulose at 0.2-0.3 M NaCl. This activity was enhanced in the presence of spermine, and inhibited in the presence of heparin (IC50 approximately equal to 30 ng/ml). The enzyme was characterized by a sedimentation coefficient of 6.5S, and a Stokes radius of 49 A, consistent with a Mr of 130,000. Both GTP (Km, 55 microM) and ATP (Km, 34 microM) were utilized as phosphoryl donors (Vmax for ATP being four-fold higher than that observed for GTP). These results indicate the presence in the insect brain of an enzyme very similar to vertebrate casein kinase II. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated that low concentrations of spermine (100 microM) strongly enhanced the phosphorylation of three high-molecular-weight cytosolic proteins (305,000, 340,000, and 360,000) localized in the insect nervous system.     

Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts.

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp60src as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)--comprising ca. 20, 45, and 35%, respectively, of the total activity--were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.     

In vitro phosphorylation of sea urchin sperm adenylate cyclase by cyclic adenosine monophosphate-dependent protein kinase

Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase.     

Phosphorylation of endogenous proteins of cilia from Paramecium tetraurelia in vitro

Several endogenous substrate proteins of cilia from axenically grown Paramecium tetraurelia were phosphorylated in vitro by inherent protein kinases (PKs). Labeling was stimulated by cAMP and to a lesser extent by cGMP. ATP breakdown was most rapid in cilia and subciliary fractions. Using multiple substrate additions during incubations it was shown that phosphorylation was almost completed within 30 s. Very little dephosphorylation by phosphoprotein phosphatases occurred during 5 min of incubation. Proteins of molecular weight of 103 000 and 46 000 were shown to be particularly associated with axonemal structures of the cilia. No distinct differences in phosphorylation patterns were apparent in ciliary membrane vesicles of low and high buoyant density, which exhibit differential enzyme patterns. cAMP receptor proteins were identified by use of the photoaffinity label 8-azido-[32P]cAMP. Receptor proteins with apparent molecular weights of 43 000, 39 000, 37 000, 31 000 and 30 000 were probably related to the regulatory subunits of cAMP-dependent protein kinases as evidenced by inhibition of incorporation of the photoaffinity label by low concentrations of cAMP. Tagging of a protein of 85 000 molecular weight was specifically inhibited by cGMP, thus in all likelihood it corresponded to a cGMP-dependent protein kinase. Corresponding autophosphorylated protein bands were observed with gamma-[32P]ATP. A functional role for protein phosphorylation in cilia of Paramecium remains to be established.     

Pattern of protein phosphorylation in aortic endothelial cells. Modulation by adenine nucleotides and bradykinin

In bovine aortic endothelial cells, ATP (10-100 microM) and bradykinin (0.1-1.0 microM) enhanced the phosphorylation of two major protein substrates with apparent molecular masses of 95 and 28 kDa. The action of ATP involved P2y purinoceptors. The kinetics were distinct for the two phosphopeptides. The phosphorylation of the 95-kDa protein was rapid (within 30 s) but transient (maintained for only 2 min). This time course agrees with that observed for the increase of the cytosolic Ca2+ level induced by ATP in these cells. Ionophore A23187 (greater than or equal to 100 nM) induced this phosphorylation for a longer period (5-10 min), whereas phorbol 12-myristate 13-acetate (PMA) was completely inactive. The enhancement of the 28-kDa protein phosphorylation was detectable after a 5-min lag and was maintained for at least 20 min. PMA (50 nM) stimulated weakly the phosphorylation of the 28-kDa protein, whereas A23187 (100-300 nM) was even more effective than ATP and bradykinin. The 95-kDa phosphoprotein seems to be related to a 100-kDa substrate of calmodulin-dependent protein kinase III recently identified as elongation factor-2. The 28-kDa protein, which was resolved as three variants in bidimensional gel electrophoresis, appears very similar to a slightly heavier phosphoprotein from thrombin-stimulated human platelets. In addition, bidimensional electrophoresis allowed the detection of at least 10 substrates (from 18 to 46 kDa) whose phosphorylation was enhanced equally well by ATP, bradykinin, and A23187 and only partially by PMA. In conclusion, protein phosphorylation induced by ATP and bradykinin in aortic endothelial cells seems to be catalyzed mostly by Ca2+-dependent kinases, distinct from protein kinase C.     

Cyclic nucleotide- and calcium-independent phosphorylation of proteins in rat brain polyribosome: Effects of ACTH,spermine, and hemin

The incorporation of [-³²P]ATP into proteins of rat brain polyribosomes was studied in vitro. The effects of cyclic nucleotides, calcium, hemin, ACTH, GTP, and spermine were examined. The incorporation of phosphate into proteins increased with time and phosphatase activity was very low; thus, the extent of phosphorylation was predominantly a reflection of protein kinase activity. Phosphorylation of proteins was not sensitive to Ca²⁺ in the presence or absence of either calmodulin or phosphatidylserine. Phosphorylation was also unaffected by cyclic nucleotides in the absence of exogenous enzymes. However, addition of a cMAP-dependent protein kinase together with cAMP resulted in a stimulation of the incorporation of phosphate into 4 phosphoproteins (pp70, pp58, pp43, and pp32); phosphorylation of pp32 was completely dependent on the addition of the kinase. ACTH (1–24), (11–24), and spermine inhibited the endogenous phosphorylation of one protein band (pp30). The phosphorylation of this 30 kD band was also selectively increased by hemin (5 M). Higher concentrations of hemin exerted an inhibitory effect on the majority of the phosphoproteins. Protein phosphatase activity was not influenced by ACTH or spermine. The specific inhibition of pp30 phosphorylation by ACTH or spermine is most probably explained by an interaction with a cyclic nucleotide- and Ca²⁺-independent protein kinase.     

Characterization of the pp70 protein phosphorylation in the extract of rice young panicle

The endogenous phosphorylation pattern of the extract prepared from rice young panicle has been examined. The extract was phosphorylated in vitro by incubating with [gamma-32p] ATP, and then analyzed by SDS-PAGE and autoradiography. At least eight major phosphoproteins with apparent molecular weights of 70, 60, 52, 40, 33, 30, 20, 16 and 15 kd were detected in the crude extract of rice young panicle. The pp70 which represents the major phosphoprotein in the crude extract of young panicle of rice is a cytosolic protein. Photoaffinity labeling with 8-azido-ATP revealed that a major protein with molecular weight 42,000 dalton but not pp70 can be specifically bound ATP. This suggests that pp70 is not a protein kinase itself, but a substrate of protein kinase. The in vitro phosphorylation of the pp70 occurs at a serine or threonine residue and is time and temperature dependent. The phosphorylation of pp70 does not depend by exogenous Ca++ or cAMP, suggesting that pp70 is a major substrate of an Ca++ or cAMP independent protein kinase in rice young panicle.     

Intracellular pH regulates bovine sperm motility and protein phosphorylation

Bovine sperm in neat caudal epididymal fluid become motile in response to either pH elevation or dilution of the fluid. Buffers containing permeant weak acids at physiologic concentrations are able to mimic these effects of caudal fluid. These observations lead to the hypothesis that a pH-dependent epididymal fluid quiescence factor regulates bovine sperm motility by modulating sperm intracellular pH (pHi). Here we report that sperm pHi, measured with the fluorescent pH probe carboxyfluorescein, increases by approximately 0.4 units in response to either of these motility-initiating manipulations. At least 26 discrete phosphoprotein bands are distinguishable by sodium dodecylsulfate-polyacrylamide gel electrophoresis after incubation of intact caudal sperm with 32PO4. A prominent phosphoprotein, with Mr approximately 255,000 (pp255) and a relatively high specific radioactivity, is reversibly dephosphorylated in response to elevations in pHi that initiate sperm motility. Unlike most of the sperm phosphoproteins, the extraction of pp255 requires reducing agents. This phosphoprotein cosediments with the sperm heads but not the tail, midpiece, soluble, or plasma membrane fractions. No other pHi-dependent phosphorylation changes are apparent in gels of whole sperm extracts. However, subcellular fractionation allows the detection of increased phosphorylation of two plasma membrane phosphoproteins (Mr approximately 105,000 and 97,000) and decreased phosphorylation of another plasma membrane phosphoprotein (Mr approximately 120,000) in response to increasing pHi. This is the first report describing changes in endogenous phosphoproteins from intact motile and nonmotile bovine sperm that are regulated by pHi.     

Effect of growth hormone on protein phosphorylation in isolated rat hepatocytes

Hepatocytes from male rats were incubated with [32P]Pi for 40 min at 37 degrees C, thereby equilibrating the cellular ATP pool with 32P. Subsequent exposure to bovine growth hormone for 10 additional min did not change the specific activity of cellular [gamma-32P]ATP. Two-dimensional gel electrophoresis or chromatofocusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to fractionate phosphoproteins solubilized from control or hormone-stimulated cells. Stimulation of hepatocytes with 5 nM growth hormone for 10 min at 37 degrees C affected the phosphorylation of a number of proteins including an Mr 46,000 species of pI 4.7 whose phosphorylation was augmented (2.65 +/- 0.50)-fold. A significant fraction of the maximal effect of growth hormone on phosphorylation of the Mr 46,000 species was elicited by 1-5% receptor occupancy. Bovine growth hormone, which binds to somatogenic receptors with great specificity, or recombinant human growth hormone, which is not contaminated with other hormones, affected phosphorylation of hepatic proteins similarly. The Mr 46,000 phosphoprotein was isolated in a fraction enriched in cytosol after centrifugation of cellular homogenates. Phosphorylation of the Mr 46,000 phosphoprotein was also increased (1.75 +/- 0.35)-fold and (2.15 +/- 0.50)-fold by insulin and glucagon, respectively. These observations are consistent with the possibility that selective changes in the phosphorylation state of cellular proteins may mediate growth hormone actions in cells.     

Evidence for calcium enhanced phosphorylation of pyruvate kinase by pancreatic islets

Summary Pancreatic islet cytosol contains a calcium-calmodulin dependent protein kinase that can mediate the phosphorylation of an endogenous protein that has an Mr of 57 000, as well as exogenous muscle pyruvate kinase (subunit Mr, 57000). EGTA and trifluoperazine decreased the phosphorylation. Alkaline inactivation of pyruvate kinase made it a better substrate for the kinase. As in rat islet cytosol, rabbit islet cytosol catalyzed the phosphorylation of a 57 000 Mr protein in the presence of calcium and calmodulin. This phosphoprotein was immunoprecipitated with anti-pyruvate kinase antibody. This is consistent with the idea that the 57 000 Mr phosphoprotein in islet cytosol is the subunit of pyruvate kinase. The paper following this paper shows that the kinetic and immunologic properties of the islet pyruvae kinase indicate it is the M₂ isoenzyme and that its phosphorylation does not affect its catalytic activity.     

The effects of glucagon, phenylephrine and insulin on the phosphorylation of cytoplasmic, mitochondrial and membrane-bound proteins of intact liver cells from starved rats.

1. The effects of glucagon, insulin and phenylephrine on the phosphorylation of cytoplasmic, mitochondrial and membrane proteins were studied in intact hepatocytes from 24 h-starved rats incubated with [32P]Pi. A rapid cell-fractionation technique was used, followed by radioautography of the proteins separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. Glucagon consistently caused a significant increase in the phosphorylation of four readily separable cytoplasmic phosphoproteins, of Mr 93000, 50000, 46000 and 20000, and a decrease in phosphorylation of a phosphoprotein of Mr 22000. Phosphorylation of the protein of Mr 46000 was also enhanced by both phenylephrine and insulin, and that of Mr 93000 by phenylephrine. 3. The phosphoprotein of Mr 22000 was not precipitated by boiling for 5 min, and had a mobility identical with that of similar protein whose phosphorylation is enhanced in the adipocyte by insulin [Belsham & Denton (1980) Biochem. Soc. Trans. 8, 382-383]. 4. Glucagon, but not phenylephrine or insulin, enhanced the phosphorylation of a mitochondrial protein of Mr 35000 and of four plasma- or microsomal-membrane proteins of Mr 50000, 30000, 23000 and 19000. 5. Mitochondria from glucagon-treated animals or hepatocytes phosphorylated a protein of Mr 30000 when incubated in vitro with [32P]Pi and ADP. Phosphorylation of this protein did not occur with mitochondria from control, phenylephrine- or insulin-treated cells. 6. The significance of these hormonally induced changes in protein phosphorylation is discussed.     

Characterization of maturation-activated histone H1 and ribosomal S6 kinases in sea star oocytes

DEAE-Sephacel chromatography of cytosolic extracts from sea star oocytes resolved at least two distinct peaks of maturation-activated protein kinase activity, each of which catalyzed the phosphorylation of histone H1, ribosomal protein S6, and Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (RRLSSLRA), a synthetic peptide based on the sequence of a phosphorylation site in the latter protein. The first peak (elution conductivity approximately equal to 6 mmho) contained the major activated kinase with respect to the phosphorylation of histone H1, and the second peak (elution conductivity approximately equal to 10.5 mmho) contained the major activated kinase with respect to the phosphorylation of S6 and RRLSSLRA. These kinase activities were barely detectable in extracts from immature oocytes. The major stimulated histone H1 kinase exhibited an apparent Mr of approximately 90 000 on Sephacryl S-300 but eluted from TSK-400 with an apparent Mr of approximately 10 000. After DEAE-Sephacel fractionation, this kinase was shown to utilize both ATP (apparent Km approximately equal to 45 microM) and GTP (apparent Km approximately equal to 10 microM), although the Vmax was 8-fold higher with ATP than with GTP. The enzyme phosphorylated histone H1 with an apparent Km approximately equal to 50 micrograms/mL. Its properties resembled those of the growth-associated histone kinase. The major stimulated RRLSSLRA kinase had an apparent Mr of approximately 84 000 on Sephacryl S-300 and approximately 40 000 on TSK-400. After DEAE-Sephacel chromatography, this kinase selectively utilized ATP (apparent Km approximately equal to 25 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor, but not insulin, stimulates tyrosine phosphorylation of an endogenous protein of Mr 95,000 in triton extracts of human placental syncytiotrophoblast membranes.

1. Triton extracts of syncytiotrophoblast membranes were incubated with [gamma-32P]ATP, MgCl2 and MnCl2. Addition of epidermal growth factor (EGF) resulted in increased phosphorylation not only of the EGF receptor and a Mr-35,000 protein as previously described, but also a protein of Mr 95,000 on both tyrosine and serine residues. In addition, a small increase in the phosphorylation of a protein of Mr 105,000 was observed. Spermine had a similar effect on the phosphorylation of the Mr-95,000 protein, without affecting the phosphorylation of the other proteins. In the absence of MnCl2, the effect of spermine on the phosphorylation of Mr-95,000 protein was still evident, whereas that of EGF was greatly diminished. 2. The Mr-95,000 protein bound poorly to wheat-germ-lectin-Sepharose and was not precipitated by antisera specific for insulin and EGF receptors. The protein continued to exhibit serine and tyrosine phosphorylation on addition of [gamma-32P]ATP, MgCl2 and MnCl2 to a glycoprotein-depleted fraction prepared by chromatography on wheat-germ-lectin-Sepharose. The extent of phosphorylation was no longer increased by spermine or EGF, but was inhibited by heparin. 3. It is suggested that the Mr-95,000 protein not only is a possible direct substrate for the EGF-receptor (but not the insulin receptor) tyrosine kinase but is a substrate for other endogenous kinases, including a protein tyrosine kinase which is probably not a glycoprotein, and a protein serine kinase with properties similar to those of casein kinase II.     

Polyamines stimulate endogenous protein phosphorylation in thyroid cytosol

The polyamine, spermine (1-5 mM), when added to rat thyroid cytosol, increases the phosphorylation of a 107 kDa protein 4-fold as analyzed by sodium dodecyl sulfate polyacrylamide gradient gel electrophoresis (SDS-PAGE) and autoradiography; spermidine was less effective and putrescine was without effect. Sodium chloride, when tested at equivalent ionic strengths (4-40 mM), did not reproduce the effects of spermine. In addition to stimulating the phosphorylation of a 107 kDa protein, spermine had an apparent biphasic effect on the phosphorylation of 88 and 65 kDa proteins; maximum stimulation of approximately 60-70% was observed at 0.5-2 mM. Both basal and spermine-stimulated protein phosphorylation patterns were identical whether [gamma-32P] ATP or [gamma-32P] GTP was used as phosphate donors. Heparin (1 microgram/ml) reduced spermine-stimulated phosphorylation of the 107 kDa protein by 64%. Phosphorylation of a 107 kDa protein was not restricted to rat thyroid as spermine was found to augment the phosphorylation of 107 kDa protein(s) in mouse and beef thyroid cytosol preparations.     

The isolation and analysis of the luminal plasma membrane of calf urinary bladder epithelium.

The luminal plasma membrane of calf urinary bladder epithelium (urothelium) has been isolated by a method designed to preserve enzymic activity as well as structural integrity. The yield was about 80 micrograms per calf bladder. Low levels of 5' nucleotidase, Mg2+-ATPase and (Na+ + K+)-ATPase activities were found in the luminal membrane fraction. Cerebroside was the major lipid present and dodecyl sulphate gel electrophoresis revealed a complex protein and glycoprotein composition in the whole membrane. A membrane fraction consisting of only the plaque areas was shown to have a simpler protein composition with major polypeptides of apparent Mr 12 000 and 22 000. These may associate to form a 30 000 apparent Mr complex which could represent the individual 'particles' of the dodecameric subunits seen by electron microscopy in the plaque regions.     

An endogenous substrate for the insulin receptor-associated tyrosine kinase

Insulin binding to its receptor stimulates a tyrosine-specific protein kinase. This enzyme phosphorylates the insulin receptor, as well as a variety of exogenous substrates in vitro. In the present studies, we have identified an endogenous substrate for the insulin receptor-associated kinase. We studied insulin-stimulated protein phosphorylation in partially purified insulin receptor preparations from the livers of dexamethasone-treated rats. In this cell-free system, insulin stimulated the phosphorylation of its own receptor as well as of a phosphoprotein of apparent Mr = 120,000 (termed pp120). pp120 was not immunoprecipitated by three anti-receptor antisera, nor was the receptor immunoprecipitated by antisera raised against pp120, suggesting that pp120 is not antigenically related or tightly bound to the insulin receptor. Dose-response curves for receptor and pp120 phosphorylation stimulated by pork insulin were essentially identical, and showed the appropriate specificity (insulin much greater than proinsulin) for a receptor-mediated event. Phosphoamino acid analysis revealed that insulin stimulated the incorporation of 32P predominantly into tyrosine residues of pp120. Casein, an artificial substrate for the insulin receptor kinase, competed with pp120 for insulin-stimulated phosphorylation. Phosphorylation of pp120 was rapid (half-maximal effect within 2 min at 24 degrees C) and, like receptor phosphorylation, was supported with Mn2+ or Mg2+ as divalent cation and ATP as the phosphate donor. While receptor autophosphorylation and artificial substrate phosphorylation were not altered by prior treatment of the rats with dexamethasone, insulin-stimulated pp120 phosphorylation was enhanced in preparations derived from dexamethasone-treated rats, suggesting an alteration of pp120, not the receptor, as a result of dexamethasone-treatment. Further studies of this newly identified endogenous substrate may help clarify the physiologic role of the insulin receptor-associated kinase.     

Spermine-enhanced protein phosphorylation in human placenta

Polyamines are known to have a role in cell proliferation, differentiation, and protein synthesis. During pregnancy, major changes in polyamine levels occur in maternal serum, amniotic fluid, and placental tissue. Polyamine-activated phosphorylation has recently been proposed as a mechanism by which polyamines may regulate metabolic processes in target tissues. Polyamine-activated protein phosphorylation has not been studied in placenta. Homogenate membrane and cytosol fractions from human placenta were subjected to an endogenous protein phosphorylation assay using [gamma-32P]ATP in the presence and absence of the polyamines, spermine and spermidine, and the diamine, putrescine. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When compared to basal levels, spermine (10(-3) M) significantly (P less than 0.001) stimulated 32P incorporation into phosphoproteins having molecular weights of 55,000 and 105,000. At this concentration spermidine and putrescine failed to stimulate phosphorylation. Half-maximal 32P incorporation was observed with 3.7 +/- 1.25 X 10(-4) M spermine. Polylysine enhanced the phosphorylation of phosphoproteins of the same molecular weight as those enhanced by spermine. Heparin and high Mg2+ inhibited spermine-induced phosphorylation. cAMP and Ca2+ did not stimulate phosphorylation of the spermine-dependent phosphoproteins. Spermine, however, acted as an antagonist for cAMP-dependent phosphorylation of a Mr 45,000 phosphoprotein.