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PubMed1987

Rat liver fructose-1,6-bisphosphatase. Identification of serine 338 as a third major phosphorylation site for cyclic AMP-dependent protein kinase and activity changes associated with multisite phosphorylation in vitro.

Rat liver fructose-1,6-bisphosphatase was phosphorylated with [32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. After digestion with trypsin, two peptides were isolated containing 68 and 32% of the total radioactivity, respectively. The former was found to contain the sequence Ala-Lys-Ser(P)-Arg-Pro-Ser(P)-Leu-Pro. In this fragment, Ser-341, but not Ser-338, had earlier been reported to be a phosphorylation site. The other peptide contained phosphorylated Ser-356. It was demonstrated that all the protein-bound [32P]phosphate was distributed evenly between these three serines in the native enzyme regardless of the degree of phosphorylation. Preservation of the three-dimensional structure, however, was needed to obtain phosphorylation of Ser-356. Peptides containing each phosphorylatable serine residue were sequentially removed by digesting the enzyme with chymotrypsin which cleaved off Ser-356, denaturing it with urea, digesting it further with chymotrypsin, thus removing Ser-341, and finally treating it with trypsin which eliminated the rest of the radioactivity which was bound to Ser-338. Kinetic studies of fructose-1,6-bisphosphatase digested in this manner revealed that phosphorylation of Ser-338 decreased the apparent Km for fructose 1,6-bisphosphatase, whereas phosphorylation of Ser-341 decreased the inhibitory effect of AMP and fructose 2,6-bisphosphatase, Phosphorylation of Ser-356 did not affect these parameters.

Amino Acid Sequence↗
PubMed1986

Phosphorylation of rabbit muscle glycogen synthase by casein/glycogen synthase kinase-1 (CK-1). Stoichiometry and distribution of the phosphorylation sites on the glycogen synthase subunit.

The stoichiometry of the phosphorylation of rabbit muscle glycogen synthase by casein/glycogen synthase kinase-1 (CK-1) depended on the concentration of protein kinase in the assay and reached values of 7-8 mol/mol subunit at high concentrations. Phosphorylation by CK-1 above 4 mol/mol subunit promoted a further decrease of glycogen synthase activity when determined by the low glucose-6-phosphate/high glucose-6-phosphate activity ratio assay. Analysis by limited proteolysis with trypsin and chymotrypsin showed that all of the regions in glycogen synthase phosphorylated by casein/glycogen synthase kinase-2 (CK-2), the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase), FA/glycogen synthase kinase-3 (FA/GSK-3) and phosphorylase b kinase were also phosphorylated by CK-1. Digestion with CNBr of glycogen synthase phosphorylated by CK-1 revealed the presence of the two phosphopeptides also labeled by the other protein kinases, the largest phosphopeptide (CB2) containing more phosphorylation sites for CK-1 than the smallest one (CB1). Three phosphopeptides (CB2-c, CB2-d and CB2-e) were obtained by trypsinization of CB2 phosphorylated by CK-1. None of them coincided with those labeled by A-kinase, a fact that was confirmed by the additivity of the effect of both protein kinases. In contrast, CB2-d comigrated with the peptide phosphorylated by FA/GSK-3, and CB2-e with that labeled by CK-2, whereas CB2-c would correspond to a new phosphopeptide.

Animals↗
PubMed1982

Type II regulatory subunit of cAMP-dependent protein kinase. Phosphorylation by casein kinase II at a site that is also phosphorylated in vivo.

A study of the phosphorylated sites on the regulatory subunit of type II cyclic AMP-dependent protein kinase is described. The site of autophosphorylation, previously reported (Takio, K., Walsh, K. A., Neurath, H., Smith, S. B., Krebs, E. G., and Titani, K. (1980) FEBS Lett. 114, 83-88), is found to contain the only residue phosphorylated by the catalytic subunit. Endogenous protein-bound phosphate, present at a second site, is observed in the NH2-terminal region of purified type II regulatory subunit. A serine residue in this same domain of type II regulatory subunit is shown to be phosphorylated in vitro by casein kinase II. The stoichiometry of phosphate incorporation by casein kinase II indicates a single site of phosphorylation that is equivalent to the site containing the endogenous phosphate. The apparent Km and Vmax of casein kinase II for the phosphorylation of purified type II regulatory subunit containing 0.55 molar equivalents of endogenous phosphate are 36 microM and 3.5 mumol/min/mg, respectively. Phosphatase treatment of type II regulatory subunit decreased the determined Km to 13 microM but does not change the Vmax consistent with the suggestion that casein kinase II is phosphorylating a site partially occupied by endogenous phosphate. Comparison of peptide maps of type II regulatory subunit phosphorylated in vitro by casein kinase II with in vivo modified type II regulatory subunit indicates that the site modified in vitro is also phosphorylated in vivo.

Amino Acid Sequence↗
PubMed1995

Lipopolysaccharide stimulates the tyrosine phosphorylation of mitogen-activated protein kinases p44, p42, and p41 in vascular endothelial cells in a soluble CD14-dependent manner. Role of protein tyrosine phosphorylation in lipopolysaccharide-induced stimulation of endothelial cells.

Vascular endothelial cell (EC) injury or activation by LPS plays a critical role in the pathogenesis of Gram-negative meningitis and endotoxic shock. EC do not express membrane CD14, but respond to LPS in a soluble CD14-dependent manner. The signal transduction mechanisms involved in LPS-induced EC responses are largely unknown. We used bovine and human brain microvessel EC (BBMEC, and HBMEC) to study LPS-induced protein tyrosine phosphorylation. LPS rapidly induced the tyrosine phosphorylation of several proteins in BBMEC and HBMEC, which was detectable by 5 to 15 min, reached a maximum by 30 min, and declined by 60 to 90 min. The increase in tyrosine phosphorylation was apparent following stimulation with LPS at 0.1 ng/ml and was dose dependent up to 100 ng/ml. Similar changes in tyrosine phosphorylation were induced by smooth and rough LPS as well as lipid A, but not by the inactive lipid A analogue, Rhodopseudomonas sphaeroides diphosphoryl lipid A. Pretreatment of EC with the tyrosine kinase inhibitor, herbimycin A, inhibited LPS-stimulated protein tyrosine phosphorylation and LPS-mediated lactic dehydrogenase release from BBMEC and IL-6 release from HBMEC in a dose-dependent manner. Three proteins with apparent m.w. of 44, 42, and 41 kDa were predominant among the LPS-induced tyrosine phosphoproteins, and they were identified as mitogen-activated protein kinase isoforms ERK1, ERK2, and p38, respectively. LPS-induced protein tyrosine phosphorylation in HBMEC and BBMEC was soluble CD14 dependent, since pretreatment of these cells with anti-hCD14 mAb inhibited the LPS-induced tyrosine phosphorylation of p44, p42, and p41. Additionally, LPS induced a mobility shift in p44 and p42 mitogen-activated protein kinase isozymes, which was inhibited by herbimycin A pretreatment of the EC. These findings demonstrate for the first time that increased protein tyrosine phosphorylation and activation of mitogen-activated protein kinases occur rapidly after LPS stimulation of EC in the presence of soluble CD14. Our data also suggest that a herbimycin-sensitive step, presumably a tyrosine kinase, is involved in mediating LPS-induced human EC activation and IL-6 secretion.

Amino Acid Sequence↗
PubMed1993

Identification of the sites of phosphorylation in insulin-like growth factor binding protein-1. Regulation of its affinity by phosphorylation of serine 101.

Serine phosphorylation of insulin-like growth factor binding protein-1 (IGFBP-1) has been shown to alter its affinity for the insulin-like growth factors (IGF-I and IGF-II) and to modify its capacity to modulate cellular responses to the IGFs. Because of this, we determined the sites of serine phosphorylation. Purification of 32P-labeled IGFBP-1 was followed by digestion with trypsin and endoproteinase Glu-C and radiosequencing of labeled peptides. Three serines were found to be phosphorylated, with Ser101, Ser119, and Ser169 containing 70%, 5%, and 25% of the incorporated 32P, respectively. A mutated IGFBP-1, substituting alanine for serine at positions 98 and 101, was expressed in CHO cells. On nondenaturing gels, the wild type protein migrated as five isoforms (one non-phosphorylated and four phosphorylated). However, in the mutated protein, the most rapidly migrating band (a phosphorylated form) was not present. The cells containing the mutated cDNA incorporated 60% less 32P into immunoprecipitable IGFBP-1. The mutated protein had a 3-fold reduction in affinity for IGF-I compared to the wild type protein. We conclude that Ser101 represents the major site of phosphorylation containing 63% of the total 32P incorporated and that phosphorylation of Ser101 is important for maintenance of high affinity binding for this growth factor.

Alanine↗
PubMed1994

Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis.

Human cdc25C protein, a specific tyrosine phosphatase that activates the p34cdc2 protein kinase at mitosis, is itself a phosphoprotein that shows increased phosphorylation during the G2-M transition. In vitro, cdc25C protein is substantially phosphorylated by purified p34cdc2-cyclin B protein kinase. Of seven putative phosphorylation sites for p34cdc2 protein kinase present in human cdc25C, five are phosphorylated by p34cdc2 protein kinase in vitro, as assessed by tryptic phosphopeptide mapping and peptide sequencing. These same sites are also phosphorylated in vivo during the G2-M transition in normal mammalian fibroblasts and have been precisely mapped. The cdc25C phosphorylated in vitro by p34cdc2 protein kinase exhibits a 2-3-fold higher activity than the nonphosphorylated cdc25C, as assayed by activation of inactive cdc2 prokinase. Microinjection of purified cdc25C proteins into living fibroblasts reveals that only the phosphorylated form of cdc25 is highly effective in activating G2 cells into premature prophase in a manner similar to microinjection of purified active p34cdc2 protein kinase. Together these data show that multisite phosphorylation of cdc25C by p34cdc2-cyclin B protein kinase occurs at the G2-M transition and is sufficient to induce the autoamplification of cdc2/M-phase promoting factor necessary to drive somatic mammalian cells into mitosis.

Amino Acid Sequence↗
PubMed1994

Protein tyrosine phosphorylation induced by lysophosphatidic acid in Rat-1 fibroblasts. Evidence that phosphorylation of map kinase is mediated by the Gi-p21ras pathway.

Lysophosphatidic acid (LPA) is a platelet-derived phospholipid that serves as a mitogen for fibroblasts. LPA activates its own G protein-coupled receptor(s) leading to stimulation of phospholipase C and inhibition of adenylate cyclase. Furthermore, LPA rapidly activates p21ras through a pertussis toxin-sensitive pathway. In this study, we have examined LPA-induced protein tyrosine phosphorylation in Rat-1 fibroblasts. LPA action was compared with that of endothelin, which is a stronger activator of phospholipase C than LPA but fails to activate p21ras and to stimulate DNA synthesis in these cells. LPA and, more effectively, endothelin rapidly stimulate tyrosine phosphorylation of proteins of 110-130, 95, and 65-75 kDa. The effect of LPA is dose- and time-dependent, being half-maximal at 3-30 nM and peaking after 2-5 min. Among the 110-130-kDa group of phosphotyrosyl proteins is the 125-kDa "focal adhesion kinase" (p125FAK) but not the 120-kDa p21ras GTPase-activating protein. Furthermore, LPA, like epidermal growth factor, causes tyrosine phosphorylation and activation of the p42/p44 mitogen-activated protein (MAP) kinases, paralleling p21ras activation. In contrast, endothelin fails to phosphorylate MAP kinase. Treatment of the cells with pertussis toxin blocks LPA-induced MAP kinase phosphorylation without affecting the other tyrosine phosphorylations. The kinase inhibitor staurosporine (1 microM) blocks LPA-induced, but not epidermal growth factor-induced, activation of p21ras and MAP kinase, consistent with an intermediate protein kinase linking the LPA receptor to p21ras activation. The results support a model in which LPA-induced phosphorylation of MAP kinase is mediated by p21ras, and tyrosine phosphorylation of the other substrates, including p125FAK, is associated with phospholipase C activation.

Adenylate Cyclase Toxin↗
PubMed1993

Erythropoietin stimulates the tyrosine phosphorylation of Shc and its association with Grb2 and a 145-Kd tyrosine phosphorylated protein.

Although the erythropoietin receptor (EpR) lacks a tyrosine kinase consensus sequence within its proline-rich intracellular domain, addition of its ligand to Ep-responsive cells stimulates the rapid and transient tyrosine phosphorylation of a number of cellular proteins. The characterization of these phosphorylatable substrates, which include 5 major phosphoproteins with molecular masses of approximately 145, 130, 97, 72, and 56 Kd is an essential step in understanding the signal transduction pathways used by Ep. Recently, we and others have shown that the major 72-Kd tyrosine phosphorylated protein is the EpR itself. We now report, using both murine DA-3 and human MO7E cell lines engineered to express high levels of biologically responsive EpRs (and designated DA-ER and MO7-ER, respectively), that the major 56-Kd tyrosine phosphorylated protein is the recently identified SH2-containing protein, p52shc. Interestingly, in Ep-stimulated cells, anti-Shc antibodies coprecipitate the major 145-Kd tyrosine phosphorylated protein in both DA-ER and MO7-ER cells. Tyrosine phosphorylation of both proteins is detectable within 30 seconds of incubation with Ep at 37 degrees C, reaches a maximum between 2 and 5 minutes, and declines by 30 minutes. In addition, tyrosine phosphorylated Shc appears capable of associating with the activated EpR, but this could only be shown in MO7-ER cells. Lastly, as has been shown previously with the tyrosine kinase containing receptors for epidermal growth factor, platelet derived growth factor, and insulin, activation of the EpR leads to the association of p52shc with the 25-Kd polypeptide, Grb2. Taken together, our data suggest that the previously reported increases in rasGTP observed with Ep result, in part, from the tyrosine phosphorylation of Shc and its association with Grb2 and/or a tyrosine phosphorylated 145-Kd protein.

Adaptor Proteins, Signal Transducing↗
PubMed1986

Analysis of smooth muscle myosin phosphorylation with native pyrophosphate gels and its application to studies on myosin phosphorylation in contracting smooth muscle.

Gizzard smooth muscle myosin, the 20,000 Mr light chain (L20) of which had been phosphorylated in vitro with a calmodulin-myosin light chain kinase system, was separated into 5 isolated bands in a pyrophosphate polyacrylamide gel. Their mobilities were in the following order: myosin with 2 unphosphorylated L20 (GM) less than myosin with 1 unphosphorylated and 1 mono-phosphorylated L20 (GMP1) less than myosin with 2 mono-phosphorylated L20 (GMP2) less than myosin with 1 mono-phosphorylated and 1 di-phosphorylated L20 (GMP3) less than myosin with 2 di-phosphorylated L20 (GMP4). We used this pyrophosphate polyacrylamide gel electrophoresis to analyze the phosphorylated state of taenia coli smooth muscle during K+-induced contraction. During the initial 2 min contraction, phosphorylated forms corresponding to GMP1 and GMP2 were detected in addition to the unphosphorylated form.

Animals↗
PubMed1999

CD28 ligation induces rapid tyrosine phosphorylation of the linker molecule LAT in the absence of Syk and ZAP-70 tyrosine phosphorylation.

CD28 is a T cell surface molecule that is important for T cell activation. CD28-triggered T cell stimulation involves protein tyrosine phosphorylation, a process that is critical for CD28 function. Recently, a linker molecule has been identified as LAT (Linker for Activation of T cells). Studies involving LAT mutants and reconstitution experiments strongly implicate LAT in playing a critical role in T cell activation. We show in the present report that CD28 ligation induces tyrosine phosphorylation of LAT. CD28-induced tyrosine phosphorylation of LAT was rapid, as it was apparent within 1 min of CD28 ligation, reached a peak by 5 min, and declined thereafter. Previous studies implicated the protein tyrosine kinases ZAP-70 and Syk in the TCR-induced tyrosine phosphorylation of LAT. Here, tyrosine phosphorylation of Syk and ZAP-70 was detected after TCR but not after CD28 ligation. Thus, CD28 ligation appears to induce tyrosine phosphorylation of LAT by mechanisms that are independent of ZAP-70 and Syk. The concurrent ligation of CD28 and TCR increased tyrosine phosphorylation of LAT. These results implicate LAT in CD28 signal transduction pathways and in the co-stimulatory process in T cells.

Adaptor Proteins, Signal Transducing↗
PubMed1990

Phosphorylation of microtubule-associated protein tau: identification of the site for Ca2(+)-calmodulin dependent kinase and relationship with tau phosphorylation in Alzheimer tangles.

The microtubule array in neuronal cells undergoes extensive growth, dynamics and rearrangements during neurite outgrowth. While little is known about how these changes are regulated, microtubule-associated proteins (MAPs) including tau protein are likely to perform an important role. Tau is one of the MAPs in mammalian brain. When isolated it is usually a mixture of several isoforms containing between 341 and 441 residues that arise from alternative splicing. Tau can be phosphorylated by several protein kinases. Phosphorylation at certain sites results in major structural and functional changes, as seen by changes in electrophoretic mobility, interaction with microtubules, molecular length and elasticity. Here we show that the sites of phosphorylation by four kinases (PKA, PKC, CK and CaMK) all lie in the C-terminal microtubule-binding half of tau, but only the phosphorylation by CaM kinase shows the pronounced shift in electrophoretic mobility characteristic for tau from Alzheimer neurofibrillary tangles. By using a combination of limited proteolysis, protein sequencing and protein engineering we show that a single phosphorylation site is responsible for this shift, located at Ser 405 in the C-terminal tail of the protein outside the region of internal repeats. Phosphorylation at this site not only reduces the electrophoretic mobility of tau, it also makes the protein long and stiff, as shown earlier. The site is likely to be phosphorylated in tau from Alzheimer neurofibrillary tangles.

Alzheimer Disease↗
PubMed1991

DNA-dependent phosphorylation of histone H2A.X during nucleosome assembly in Xenopus laevis oocytes: involvement of protein phosphorylation in nucleosome spacing.

ATP is required for physiological nucleosome alignment in chromatin reconstituted from high-speed nuclear supernatants of Xenopus laevis oocytes. Here we show that during in vitro nucleosome assembly the histone variant H2A.X becomes phosphorylated upon transfer onto DNA, a process which is also observed in vivo. Histone H2A.X phosphorylation increases in the early phase of the assembly reaction, reaching a steady state after approximately 16 min and is maintained with a half-life of the phosphate groups of approximately 2 h. After 6 h, the overall phosphorylation state of H2A.X is reduced, indicating that the phosphorylation-dephosphorylation ratio decreases considerably over time. Addition of alkaline phosphatase leads to a persistently lowered state of H2A.X phosphorylation, in contrast to other nuclear phosphoproteins which undergo rapid rephosphorylation. This suggests that H2A.X phosphorylation is a unique step in the histone-to-DNA transfer process. Selective inhibition of DNA-dependent phosphorylation of H2A.X and of other proteins causes a loss of the physiological 180 bp spacing.

Animals↗
PubMed2006

Myosin binding protein C is differentially phosphorylated upon myocardial stunning in canine and rat hearts-- evidence for novel phosphorylation sites.

Myocardial stunning is the transient cardiac dysfunction that follows brief episodes of ischemia and reperfusion without associated myocardial necrosis. Currently, there is limited knowledge about its cellular and biochemical mechanisms. In order to better understand the underlying mechanisms of contractile dysfunction associated with the stunning, comprehensive proteomic studies using 2-D DIGE were performed using a regional stunning model in canine heart. Cardiac myosin binding protein C (cMyBP-C), a regulatory myofilament protein associated with the thick filament, and nebulette, a thin filament associated protein, were differentially expressed. Phosphoprotein specific staining indicated both protein changes were due to phosphorylation. Subsequent phosphorylation mapping of canine cMyBP-C using IMAC and MS/MS identified five phosphorylation sites, including three novel sites. In order to further evaluate this finding in a different model, cMyBP-C phosphorylation was examined in a rat model of global stunning. In the rat model, stunning was associated with increased phosphorylation of cMyBP-C at a critical calcium/calmodulin-dependent kinase II site, and the increased phosphorylation was largely inhibited when stunning was prevented by either ischemic preconditioning or reperfusion in the presence of low-calcium buffer. These data indicate cMyBP-C phosphorylation plays an important role in myocardial stunning.

Amino Acid Sequence↗
PubMed1995

Enzymatic deimination of glycogen phosphorylase and a peptide of the phosphorylation site: identification of modification and roles in phosphorylation and activity.

The functional role of arginine residues in glycogen phosphorylase b was probed by enzymatic modification by using peptidylarginine deiminase, which converts arginine residues to citrulline. A peptide with sequence LysArgLysGlnIleSerValArgGlyLeu, corresponding to the phosphorylation site of serine-14 in phosphorylase, was a substrate for the deiminase. Although both arginine residues could be converted to citrulline, modification of arginine-16 occurred more rapidly than modification of arginine-10. Previous studies have implicated a role for arginine, notably arginine-16, in determining phosphorylase kinase activity with the peptide. Deimination altered the phosphorylation of the peptide. Monodeimination of the peptide at arginine-16 slowed down the phosphorylation reaction, but did not diminish the total amount of phosphorylation that could be obtained. Deimination of both arginines produced a peptide that could not be phosphorylated. Modification of phosphorylase b resulted in activation or inactivation of enzyme activity depending on the extent of reaction with peptidylarginine deiminase. A low level of deiminase causes inactivation initially, but after prolonged incubation activation occurs. With high level of deiminase only activation can be observed. Because changes in activity are seen only at subsaturating AMP concentrations (50-100 microM), inactivation and activation are likely due to changes in affinity of the enzyme for AMP. The protein modified with the high level of deiminase has multiple sites of deimination. Arginine 16 was established as a major site of modification. Only protein modified with the high level of deiminase showed modification of arginine-16 and effects on the phosphorylation of phosphorylation b. As with the modified peptide substrate, the reaction was slower with modified phosphorylase in comparison with native phosphorylase b. The results show the importance of the guanidino group of arginine-16 of the protein substrate in modulating the phosphorylase kinase reaction.

Amino Acid Sequence↗
PubMed1996

Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase.

We have critically analyzed current methodologies for distinguishing histidine and serine phosphorylated residues in proteins and report a simple technique that assures a reliable discrimination. Electro-transfer of a phosphorylated enzyme to Immobilon membranes and its treatment at pH 1 and 14 in buffers containing 5% methanol allows unambiguous distinction between serine/threonine and histidine phosphorylation (O-phosphomonoesters and phosphoramide, respectively) since under these conditions only one type of residue is dephosphorylated. The addition of 5% methanol to all buffers was indispensable to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the catalytic phosphate transfer activity of nucleoside diphosphate kinase (NDP kinase). Nonetheless, a significant degree of autophosphorylation on other residues has been reported by several laboratories, and the hypothesis has been advanced that this nonhistidine phosphorylation may play an important role in NDP kinase cellular function, signaling the suppression of metastasis in the case of human NDP kinase A. Using this improved method, we show that human, Escherichia coli and Candida albicans NDP kinases are only autophosphorylated on histidine residues. In addition, we present evidence that the presence of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe that the technique will be generally useful in histidine phosphorylation screenings.

Candida albicans↗
PubMed1994

In human neutrophils the binding to immunocomplexes induces the tyrosine phosphorylation of Fc gamma RII but this phosphorylation is not an essential signal for Fc-mediated phagocytosis.

It has been recently suggested that protein tyrosine phosphorylation is involved in Fc gamma Rs-mediated signalling. In this paper we have investigated if in human neutrophils a tyrosine phosphorylation of Fc gamma RII takes places after the binding with immunocomplexes and if this phosphorylation plays a role in phagocytic signal. The immunoprecipitation with mAb anti-Fc gamma RII of lysates of neutrophils challenged in suspension with insoluble immunocomplexes (IIC) or sheep erythrocytes opsonized with IgG (E-IgG), followed by immunoblotting with anti-phosphotyrosine antibody, demonstrated that Fc gamma RII was tyrosine phosphorylated. When neutrophils were pretreated with different doses of tyrosine kinase inhibitors, genistein or erbstatin, the phosphorylation of Fc gamma RII induced by IIC or E-IgG was dose dependently inhibited. In these conditions both genistein and erbstatin failed to inhibit the phagocytosis of E-IgG. These results demonstrated that in human neutrophils in suspension the binding to Fc of IgG induces a tyrosine phosphorylation of Fc gamma RII but this phosphorylation is not an essential signal for phagocytosis of IgG-opsonized particles.

Antigen-Antibody Complex↗
PubMed1981

The soluble, cyclic AMP-stimulated protein kinase catalyses the phosphorylation of different membrane proteins from those which are phosphorylated by the membrane bound enzyme.

Soluble, cyclic AMP stimulated protein kinase (type a1 protein kinase) stimulated the phosphorylation of membrane proteins in preparations from a wide variety of tissues. This kinase does not, however, appear to catalyse the phosphorylation of the same membrane proteins which are phosphorylated by the intrinsic protein kinase activity of the preparations. This was demonstrated in two ways: 1. If preparations of membrane fragments are incubated with [gamma 32P] ATP in the absence of type a1 protein kinase, until all membrane bound protein acceptor sites are saturated with phosphate addition of type a1 protein kinase causes the incorporation of more phosphate into the membrane proteins. 2. The nature of the membrane proteins phosphorylated by type a1 protein kinase and the intrinsic protein kinase of the preparation was compared by SDS gel electrophoresis. Membrane preparations were phosphorylated with [gamma 32P] ATP in the absence of added type a1 protein kinase and with [gamma 33P] ATP in the presence of the enzyme. Aliquots of the two samples were then mixed, separated by SDS gel electrophoresis, and the distribution of radioactivity measured. A comparison of the distribution of protein bound 33P and 32P made it quite clear that type a1 protein kinase catalysed the phosphorylation of different membrane proteins from those which are phosphorylated by the intrinsic protein kinase activity of the preparations.

Animals↗
PubMed1993

Neurofilaments in rat and cat spinal cord; a comparative immunocytochemical study of phosphorylated and non-phosphorylated subunits.

Neurofilament immunoreactivity was examined in spinal cords of rats and cats with antibodies to all three subunits (68 kD, 155 kD and 200 kD) and to different phosphorylation states of 200 kD. NFHP-, an antibody against non-phosphorylated 200 kD, labelled all rat neuronal perikarya but failed to label cat neurofilaments. In both species, the perikarya and processes of motoneurones were immunoreactive for all three subunits but most dorsal horn neuronal perikarya were not immunoreactive for 68 kD and 155 kD. Motoneuronal perikarya and proximal processes showed filamentous labelling for 68 kD but not for 155 kD in the rat, while in neither species did these show labelling with RT97, an antibody against a highly phosphorylated form of 200 kD; immunoreactivity for 200 kD was present in both filamentous (probably partially phosphorylated) and non-filamentous (non-phosphorylated) forms, but in dorsal horn neurones only the latter was present. Interpretations consistent with this data are: in rat and possibly also cat, motoneuronal neurofilaments consist of a 68 kD backbone with partially phosphorylated 200 kD sidearms, with both 155 kD and 200 kD (nonphosphorylated) subunits in a non-filamentous form; this neurofilament becomes more highly phosphorylated along the proximal processes. The dorsal horn neurones probably contain 200 kD in a non-filamentous form but may lack the other subunits.

Animals↗