Proline-directed phosphorylation of human Tau protein.

The primary sequence of the microtubule-associated protein tau contains multiple repeats of the sequence -X-Ser/Thr-Pro-X-, the consensus sequence for the proline-directed protein kinase (p34cdc2/p58cyclin A). When phosphorylated by proline-directed protein kinase in vitro, tau was found to incorporate up to 4.4 mol of phosphate/mol of protein. Isoelectric focusing of the tryptic phosphopeptides demonstrated the presence of five distinct peptides with pI values of approximately 6.9, 6.5, 5.6-5.9, 4.7, and 3.6. Mapping of the tryptic phosphopeptides by high performance liquid chromatography techniques demonstrated three distinct peaks. Data from gas phase sequencing, amino acid analysis, and phosphoamino acid analysis suggest that proline-directed protein kinase phosphorylates tau at four sites. Each site demonstrates the presence of a proline residue on the carboxyl-terminal side of the phosphorylated residue. Two phosphorylation sites are located adjacent to the three-repeat microtubule-binding domain that has been found to be required for the in vivo co-localization of tau protein to microtubules. Two other putative phosphorylation sites are located within the identified epitope of the monoclonal antibody Tau-1. Phosphorylation of these sites altered the immunoreactivity of tau to Tau-1 antibody. Since the neuronal microtubule-associated protein tau is multiply phosphorylated in Alzheimer's disease, and Tau-1 immunoreactivity is similarly reduced in neurofibrillary tangles and enhanced after dephosphorylation, phosphorylation at one or more of these sites may correlate with abnormally phosphorylated sites in tau protein in Alzheimer's disease.     

Tetranectin expression in gastric adenocarcinomas

AIMS: The aim was to analyze the immunohistochemical localization of tetranectin in gastric adenocarcinomas and the adjacent tissues of the wall of the stomach. METHODS AND RESULTS: Forty cases of gastric adenocarcinomas were stained by the indirect immunoperoxidase method. Of the ten cases of mucinous signet ring cell carcinomas 5 showed high, 3 moderate and 2 low tetranectin expression. Of the ten cases of well-differentiated intestinal type adenocarcinomas (ITA) 4 showed moderate regional, 3 low regional and 3 negative tetranectin expression. Of the ten cases of moderately-differentiated ITA 3 showed moderate regional, 4 low regional and 3 negative tetranectin expression. Of the ten cases of poorly-differentiated ITA 4 showed focal low and 6 negative tetranectin expression. Overall, the mucinous signet ring carcinomas showed significantly higher tetranectin expression compared to ITA (chi2 = 3.95, p<0.05). In contrast, no significant relationship was found between tetranectin expression and the degree of differentiation in ITA (chi2 = 2.5, p>0.05). In all cases, the perineoplastic desmoplastic reactive stroma showed high expression of tetranectin intra- and extracellularly. The mast cells and goblet cells in the areas of intestinal metaplasia showed high tetranectin expression. CONCLUSIONS: This study shows that: a) tetranectin is produced and deposited extracellularly in the desmoplastic peritumoral stroma of infiltrating gastric adenocarcinomas; b) tetranectin is more highly expressed by the mucinous signet ring cell carcinomas compared to ITA; and c) the amount of tetranectin produced by the ITA is unrelated with the degree of tumor differentiation.     

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.     

Adrenergic stimulation of membrane protein phosphorylation in human erythrocytes.

Adrenergic modification of membrane protein phosphorylation was studied in intact human erythrocytes. Micromolar norepinephrine increased 32P incorporation into Band 2 by 70%, and into Band 3 by 40%. Phosphorylation levels observed with a series of specific agonists and antagonists suggest that an alpha-adrenergic receptor is involved in this effect. The mechanism of linkage between this receptor and protein phosphorylation does not appear to involve modulation of intracellular concentrations of ATP, cyclic AMP, or cyclic GMP.     

C-reactive protein decreases protein phosphorylation in stimulated human neutrophils

Treatment of human neutrophils with C-reactive protein (CRP) causes a concentration-dependent in the extent of activation of superoxide production and of granule secretion, induced by phorbol-12-myristate-13-acetate (PMA) or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF). The same treatment also causes a significant reduction in the degree of PMA- and fMLF-stimulated phosphorylation of several cell proteins. These include the proteins of 43-47 kDa, whose extent of phosphorylation correlates with the activation of superoxide production and of secretion. Contrary to the effects exerted on protein phosphorylation, CRP does not affect the fMLF-elicited increase in neutrophil cytosolic Ca2+.     

Lymphocyte subpopulations of regional lymph nodes in human colon and gastric adenocarcinomas

 In order to study the host immune response to tumours, previous knowledge of the cellular composition of regional draining lymph nodes is necessary. Enlarged regional lymph nodes are a common finding in colon and gastric adenocarcinomas. We have studied the cellular composition of normal non-reactive and of regional draining lymph nodes of colon and gastric adenocarcinomas. In normal non-reactive lymph nodes, T lymphocytes (CD2⁺, CD7⁺) constituted the largest fraction of the lymphoreticular cells. These lymphocytes were mainly CD4⁺, and there were more cells expressing the CD45RA isoform of the CD45 antigen than CD45RO. Reactive lymph nodes presented a decreased proportion of CD4⁺ CD45RA⁺ cells and an increased number of B cells. Although most of the T cells in the reactive nodes were CD4⁺ CD45RO⁺, their proportion was similar to that found in normal non-reactive nodes. We studied the presence of the molecules CD28 and CD80 involved in the processes of interaction and activation of T and B lymphocytes. The CD28 molecule was found in all the T lymphocytes, while the CD80 molecule was weakly expressed on the B lymphocyte membrane. Received: 4 January 1996 / Accepted: 28 May 1996     

Immunohistochemical localization of sodium-potassium ATPase in human normal stomach and gastric adenocarcinomas

We studied the expression pattern of Na, K-ATPase beta 1 subunit in human normal stomachs and in gastric adenocarcinomas by using anti-Na, K-ATPase beta 1 subunit-specific monoclonal antibody. Tissue samples were processed in formalin solution or in a cold acetic acid-ethanol solution, routinely processed, embedded in paraffin, and an immunoperoxidase method for Na, K-ATPase beta 1 subunit was performed. After antigen retrieval using a steamer in citrate buffer (pH 6.0), tissue sections initially fixed in cold acetic acid-ethanol showed intense immunoreactivity with the antibody at the lateral or basolateral cytoplasmic membrane of normal gastric epithelial cells, at the cytoplasmic membrane of gastric carcinoma cells according to the level of differentiation, and at the cytoplasmic membrane and in the cytoplasm of Schwann cells and neurons in the mesenteric plexus of the gastric wall. Acetic acid-ethanol and paraffin embedding is a useful method for the investigation of the immunohistochemical localization of Na, K-ATPase in normal and diseased tissues.     

Calcium-phospholipid enhanced protein phosphorylation in human placenta

Calcium-activated, phospholipid-dependent protein phosphorylation has not been studied in placenta. Human placental cytosol was subjected to an endogenous protein phosphorylation assay using [gamma-32P]ATP in the presence of calcium and phosphatidylserine. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When compared to basal levels, calcium (10(-6) M) in combination with phosphatidylserine (50 micrograms/ml) significantly enhanced (P less than 0.01) 32P incorporation into phosphoproteins having mol wt 47,000, 43,000, and 37,000. Half-maximal 32P incorporation was observed with 3.5 X 10(-7) M Ca2+ in the presence of phosphatidylserine (50 micrograms/ml). The effect of phosphatidylserine was biphasic. In the presence of Ca 10(-6) M, 32P incorporation increased to a maximum at 70 micrograms/ml of phosphatidylserine. The increase was suppressed at 150 micrograms/ml. Tetracaine caused a dose-dependent inhibition of calcium-activated, phospholipid-dependent enhancement of the three phosphorproteins. Calcium in the absence of phospholipid enhanced the phosphorylation of a protein of 98,000 mol wt. Phosphatidylserine suppressed this enhancement. Calmodulin (10(-6) M) had no detectable effect upon phosphorylation beyond that of calcium alone, but the calmodulin inhibitor R-24571 specificlly inhibited the calcium-stimulated 98,000 mol wt phosphoprotein. Calcium-activated, phospholipid-dependent phosphoproteins are present in human placental cytosol; whether calcium-activated, calmodulin-dependent phosphoproteins also are present remains a question.     

Molecular medicine of gastric adenocarcinomas

Upper gastrointestinal (GI) tract malignancies, including carcinomas of the esophagus, the GI junction and the stomach, are among the most common cancers worldwide. Overall survival is poor because many patients present with locally advanced and often incurable disease. This review focuses on the pathogenesis of adenocarcinomas of the stomach. It summarises recent findings on genetic predisposition to gastric cancer, in particular in relation to germline mutations in the E-cadherin gene. It also describes the molecular basis of sporadic gastric cancer, including alterations in oncogenes, tumour suppressor genes and growth factors, and discusses how these findings might be used in the clinic for improved diagnosis and therapy.     

Detection of chromogranin A in human gastric adenocarcinomas using a sensitive immunohistochemical technique

Neuroendocrine cells are often disclosed in human gastric adenocarcinomas and may be recognised by their immunoreactivity towards chromogranin A. However, in dedifferentiated neuroendocrine tumour cells, the chromogranin A content may be reduced making it difficult to detect with conventional immunohistochemical methods. We therefore used a sensitive signal amplification technique in order to evaluate chromogranin A immunoreactivity and thus neuroendocrine differentiation in 40 gastric adenocarcinomas.Neuroendocrine cells were visualised by means of a monoclonal chromogranin A antibody and the avidin–biotin peroxidase complex technique, without and with addition of tyramide signal amplification. Double immunohistochemistry towards chromogranin A and Ki-67 were used to disclose proliferation in the neoplastic cells.A marked increase in the number of carcinomas containing chromogranin A-immunoreactive neoplastic cells was noted when applying the tyramide signal amplification technique. In addition, the number of immunoreactive cells within each tumour increased, and in some cases almost all the neoplastic cells became immunoreactive. Chromogranin A-immunoreactive tumour cells showing signs of proliferation were found in the majority of these carcinomas.In conclusion, we have disclosed widespread immunoreactivity towards chromogranin A in a proportion of gastric adenocarcinomas when enhancing the signal with tyramide signal amplification. Neuroendocrine differentiation is thus a common finding in gastric carcinomas when using sensitive methods.     

Involvement of protein serine and threonine phosphorylation in human sperm capacitation.

The involvement of serine and threonine phosphorylation in human sperm capacitation was investigated. Anti-phosphoserine monoclonal antibody (mAb) recognized six protein bands in the 43-55-kDa, 94 +/- 2-kDa, 110-kDa, and 190-kDa molecular regions, in addition to a faint band each in the 18-kDa and 35-kDa regions. Anti-phosphothreonine mAb recognized protein bands in six similar regions, except that the 18-kDa, 35-kDa, and 94 +/- 2-kDa protein bands were sharper and thicker, and an additional band was observed in the 110-kDa molecular region. In the 43-55-kDa molecular region, there was a well-characterized glycoprotein, designated fertilization antigen, that showed a further increase in serine/threonine phosphorylation after exposure to solubilized human zona pellucida. In a cell-free in vitro kinase assay carried out on beads or in solution, four to eight proteins belonging to similar molecular regions, namely 20 +/- 2 kDa, 43-55 kDa, 94 +/- 2 kDa, and 110 +/- 10 kDa, as well as in 80 +/- 4 and 210 +/- 10 kDa regions, were phosphorylated at dual residues (serine/tyrosine and threonine/tyrosine). Capacitation increased the intensity of serine/threonine phosphorylation per sperm cell, increased the number of sperm cells that were phosphorylated, and induced a subcellular shift in the serine/threonine-specific fluorescence. These findings indicate that protein serine/threonine phosphorylation is involved and may have a physiological role in sperm capacitation.     

Modulation of protein tyrosine phosphorylation in gastric mucosa during re-epithelization processes

AIM: To investigate the role of protein tyrosine phosphorylation in gastric wound formation and repair following ulceration.METHODS: Gastric lesions were induced in rats using restraint cold stress. To investigate the effect of oxidative and nitrosative cell stress on tyrosine phosphorylation during wound repair, total activity of protein tyrosine kinase (PTK), protein tyrosine phosphatase (PTP), antioxidant enzymes, nitric oxide synthase (NOS), 2’,5’-oligoadenylate synthetase, hydroxyl radical and zinc levels were assayed in parallel.RESULTS: Ulcer provocation induced an immediate decrease in tyrosine kinase (40% in plasma membranes and 56% in cytosol, P < 0.05) and phosphatase activity (threefold in plasma membranes and 3.3-fold in cytosol), followed by 2.3-2.4-fold decrease (P < 0.05) in protein phosphotyrosine content in the gastric mucosa. Ulceration induced no immediate change in superoxide dismutase (SOD) activity, 30% increase (P < 0.05) in catalase activity, 2.3-fold inhibition (P < 0.05) of glutathione peroxidase, 3.3-fold increase (P < 0.05) in hydroxyl radical content, and 2.3-fold decrease (P < 0.05) in zinc level in gastric mucosa. NOS activity was three times higher in gastric mucosa cells after cold stress. Following ulceration, PTK activity increased in plasma membranes and reached a maximum on day 4 after stress (twofold increase, P < 0.05), but remained inhibited (1.6-3-fold decrease on days 3, 4 and 5, P < 0.05) in the cytosol. Tyrosine phosphatases remained inhibited both in membranes and cytosol (1.5-2.4-fold, P < 0.05). NOS activity remained increased on days 1, 2 and 3 (3.8-, 2.6-, 2.2-fold, respectively, P < 0.05). Activity of SOD increased 1.6 times (P < 0.05) days 4 and 5 after stress. Catalase activity normalized after day 2. Glutathione peroxidase activity and zinc level decreased (3.3- and 2-fold, respectively, P < 0.05) on the last day. Activity of 2’,5’-oligoadenylate synthethase increased 2.8-fold (P < 0.05) at the beginning, and 1.6-2.3-fold (P < 0.05) during ulcer recuperation, and normalized on day 5, consistent with slowing of inflammation processes.CONCLUSION: These studies show diverse changes in total tyrosine kinase activity in gastric mucosa during the recovery process. Oxidative and nitrosative stress during lesion formation might lead to the observed reduction in tyrosine phosphorylation during ulceration.     

Differential regulation of basic protein phosphorylation by calcium phospholipid and cyclic-AMP-dependent protein kinases

Myelin basic protein, an 80-kilodalton (kDa) protein in rat oligodendrocytes, and an 80-kDa basic protein in neuroblastoma x neonatal Chinese hamster brain explant hybrids were phosphorylated extensively when the cells were treated with either phorbol esters (TPA) or diacylglycerols (e.g., oleyoyl-acetylglycerol). TPA-stimulated phosphorylation was inhibited by pre-incubation with 50 microM psychosine (galactosyl-sphingosine), confirming that it is mediated through the phospholipid-dependent protein kinase C (PK-C). Surprisingly, phosphorylation of these proteins was inhibited by incubation of cells with agents which result in activation of cyclic-AMP-dependent protein kinase (dibutyryl cyclic AMP or forskolin). In contrast, phosphorylation of other nonbasic proteins, for example, the oligodendrocyte-specific 2',3'-cyclic nucleotide phosphohydrolase, was stimulated under these conditions (Vartanian et al.: Proceedings of the National Academy of Sciences of the United States of America 85:939, 1988). The possible role of cyclic AMP in activating specific phosphatases or restricting the availability of diacylglycerol for PK-C activation is discussed.     

Chemoattractant-induced tyrosine phosphorylation and activation of microtubule-associated protein kinase in human neutrophils.

Activation of human neutrophils by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) induces tyrosine phosphorylation of several polypeptides, including a prominent band of approximately 41 kDa. A polypeptide of identical electrophoretic mobility was recognized by a monoclonal antibody raised against a sequence corresponding to amino acids 325-345 of ERK-1, one of a family of mitogen-activated protein (MAP) kinases. To establish the possible identity of these polypeptides, extracts from control and fMLP-treated cells were immunoprecipitated with immobilized antiphosphotyrosine antibodies. Reactivity with anti-ERK-1 antibodies was observed only in the precipitate of chemoattractant-stimulated cells. These data imply that a MAP kinase constitutes at least part of the tyrosine-phosphorylated 41-kDa polypeptide. By using an in vitro renaturation assay, treatment of intact cells with fMLP was found to stimulate several protein kinases, including one of approximately 41 kDa. Renaturation of samples immunoprecipitated with antiphosphotyrosine antibodies revealed the presence of an active protein kinase in chemoattractant-stimulated, but not in control cells. The immunoprecipitated kinase comigrated with the 41-kDa tyrosine phosphorylated polypeptide and the anti-ERK-1 reactive band. We conclude that a MAP kinase closely related or identical to ERK-1 is tyrosine phosphorylated and activated when human neutrophils are stimulated by chemotactic peptides. The rapid phosphorylation of this kinase, which is apparent within seconds, is compatible with a role in the activation of the respiratory burst and/or other neutrophil responses.     

Differential phosphorylation of SNAP-25 in vivo by protein kinase C and protein kinase A

SNAP-25 is a key protein required for the fusion of synaptic vesicles with the plasma membrane during exocytosis. This study establishes that SNAP-25 is differentially phosphorylated by protein kinase C and protein kinase A in neuroendocrine PC12 cells. Using phosphopeptide mapping and site-directed mutagenesis we identified both Thr138 and Ser187 as the targets of SNAP-25 phosphorylation by protein kinase C and Thr138 as the exclusive site of SNAP-25 phosphorylation by protein kinase A in vivo. Finally, despite published data to the contrary, we demonstrate that stimulation of regulated exocytosis under physiological conditions is independent of a measurable increase in SNAP-25 phosphorylation in PC12 cells.     

Vasopressin dependent tyrosine phosphorylation of a 38 kDa protein in human platelets.

Arginine vasopressin administration (10(-10)-10(-6) M) to isolated human platelets induces an increase in the specific immunoblotting of a 38 kDa protein revealed by a phosphotyrosine antibody. This signal is biphasic with maximal stimulation within one minute. Neither forskolin (10(-5) M) nor phorbol ester (10(-6) M) produces a similar 38 kDa signal. The specific immunoblotted signals are competitively abolished by 1 mM phosphotyrosine but not phosphoserine or phosphothreonine. Electrophoretic separation at pH 3.5 of the acid hydrolysates of the 38 kDa proteins reveals a vasopressin dependent increase in levels of phosphotyrosine as well as phosphoserine and phosphothreonine. The 38 kDa phosphorylation is also induced by the specific arginine vasopressin V1 receptor agonist (Phe2Orn8Vastocina) and blocked by the V1 receptor antagonist [desGly(NH2)d(CH2)5Tyr(Me) AVPb]. These observations suggest that arginine vasopressin signal transduction may be associated with the tyrosine phosphorylation of a 38 kDa protein.     

Differential effects of chlorpromazine on secretion, protein phosphorylation and phosphoinositide metabolism in stimulated platelets.

Increasing concentrations of chlorpromazine (30-500 microM) caused a progressive lysis of gel-filtered platelets, as monitored by the extracellular appearance of cytoplasmic ([14C]adenine-labelled) adenine nucleotides. The chlorpromazine-induced lysis was markedly enhanced by thrombin and phorbol ester, and complete cytolysis was found at chlorpromazine concentrations of 100 microM and above in the presence of thrombin. At non-lytic concentrations, chlorpromazine caused a dramatic increase in the thrombin- or phorbol ester-mediated incorporation of 32P into phosphatidylinositol 4-phosphate and, to a lesser extent, into phosphatidylinositol 4,5-bisphosphate in platelets pulse-labelled with [32P]Pi. Chlorpromazine alone also caused an incorporation of 32P into the phosphoinositides. Non-lytic concentrations of chlorpromazine had no effect on the phosphorylation of the 47 kDa protein (regarded as the substrate for protein kinase C), but markedly inhibited the accompanying secretion of ATP + ADP and beta-hexosaminidase when platelets were incubated with 0.17 microM-phorbol ester or 0.1-0.2 unit of thrombin/ml. At lower concentrations of thrombin, chlorpromazine did not inhibit, but slightly enhanced, secretion. A protein of 82 kDa was phosphorylated during the interaction of platelets with thrombin and phorbol ester, and this phosphorylation was enhanced by chlorpromazine (non-lytic). These results suggest that the previously reported inhibition of protein kinase C by chlorpromazine is probably non-specific and due to cytolysis. However, since non-lytic concentrations of chlorpromazine inhibit secretion, but not protein kinase C, in platelets, activation of protein kinase C is not involved in the stimulation-secretion coupling, or chlorpromazine acts at a step after kinase activation. Possible mechanisms of this inhibition by chlorpromazine are discussed in the light of its effect on phosphoinositide metabolism and protein phosphorylation.     

Adrenergic stimulation of membrane protein phosphorylation in human erythrocytes

Adrenergic modification of membrane protein phosphorylation was studied in intact human erythrocytes. Micromolar norepinephrine increased ³²P incorporation into Band 2 by 70%, and into Band 3 by 40%. Phosphorylation levels observed with a series of specific agonists and antagonists suggest that an α-adrenergic receptor is involved in this effect. The mechanism of linkage between this receptor and protein phosphorylation does not appear to involve modulation of intracellular concentrations of ATP, cyclic AMP, or cyclic GMP.