scholarly journals Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts.

1987 ◽  
Vol 7 (9) ◽  
pp. 3147-3155 ◽  
Author(s):  
E Erikson ◽  
D Stefanovic ◽  
J Blenis ◽  
R L Erikson ◽  
J L Maller
Keyword(s):  
Chicken Embryo ◽  
Kinase Activity ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Cell Types ◽  
Polyclonal Antiserum ◽  
Xenopus Laevis Oocytes ◽  
S6 Kinase ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Ribosomal protein S6 becomes highly phosphorylated during progesterone- or insulin-induced maturation of Xenopus laevis oocytes. We have previously purified an Mr 92,000 protein as one of the major S6 kinases from Xenopus unfertilized eggs. In this paper we confirm by renaturation of activity from a sodium dodecyl sulfate-polyacrylamide gel that this protein is an S6 kinase. This enzyme, termed S6 kinase II (S6 K II), was used for the preparation of polyclonal antiserum. Immunocomplexes formed with the antiserum and purified S6 K II were able to express kinase activity with the same substrate specificity as that of the purified enzyme, including autophosphorylation of S6 K II itself. The antiserum did not react with S6 kinase I, another major S6 kinase present in Xenopus eggs, which is chromatographically distinct from S6 K II. The administration of progesterone to oocytes resulted in a 20- to 25-fold increase in S6 kinase activity in extracts of these cells. Immunocomplex kinase assays done on extracts revealed that anti-S6 K II serum reacted with S6 kinase from progesterone-treated oocytes. This antiserum also reacted with the activated S6 kinase from insulin-stimulated oocytes. In addition, anti-S6 K II serum reacted with activated S6 kinase from chicken embryo fibroblasts stimulated with serum or transformed by Rous sarcoma virus. These results indicate that S6 K II or an antigenically related S6 kinase(s) is subject to regulation by mitogenic stimuli in various cell types.

Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts

1987 ◽  
Vol 7 (9) ◽  
pp. 3147-3155
Author(s):  
E Erikson ◽  
D Stefanovic ◽  
J Blenis ◽  
R L Erikson ◽  
J L Maller
Keyword(s):  
Chicken Embryo ◽  
Kinase Activity ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Cell Types ◽  
Polyclonal Antiserum ◽  
Xenopus Laevis Oocytes ◽  
S6 Kinase ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Ribosomal protein S6 becomes highly phosphorylated during progesterone- or insulin-induced maturation of Xenopus laevis oocytes. We have previously purified an Mr 92,000 protein as one of the major S6 kinases from Xenopus unfertilized eggs. In this paper we confirm by renaturation of activity from a sodium dodecyl sulfate-polyacrylamide gel that this protein is an S6 kinase. This enzyme, termed S6 kinase II (S6 K II), was used for the preparation of polyclonal antiserum. Immunocomplexes formed with the antiserum and purified S6 K II were able to express kinase activity with the same substrate specificity as that of the purified enzyme, including autophosphorylation of S6 K II itself. The antiserum did not react with S6 kinase I, another major S6 kinase present in Xenopus eggs, which is chromatographically distinct from S6 K II. The administration of progesterone to oocytes resulted in a 20- to 25-fold increase in S6 kinase activity in extracts of these cells. Immunocomplex kinase assays done on extracts revealed that anti-S6 K II serum reacted with S6 kinase from progesterone-treated oocytes. This antiserum also reacted with the activated S6 kinase from insulin-stimulated oocytes. In addition, anti-S6 K II serum reacted with activated S6 kinase from chicken embryo fibroblasts stimulated with serum or transformed by Rous sarcoma virus. These results indicate that S6 K II or an antigenically related S6 kinase(s) is subject to regulation by mitogenic stimuli in various cell types.

scholarly journals Two distinct enzymes contribute to biphasic S6 phosphorylation in serum-stimulated chicken embryo fibroblasts.

1990 ◽  
Vol 10 (6) ◽  
pp. 2787-2792 ◽  
Author(s):  
L J Sweet ◽  
D A Alcorta ◽  
R L Erikson
Keyword(s):  
Chicken Embryo ◽  
Kinase Activity ◽  
Chicken Embryo Fibroblast ◽  
Total Serum ◽  
S6 Kinase ◽  
Serum Stimulation ◽  
Ribosomal Protein S6 ◽  
Phosphopeptide Analysis ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Serum stimulation of quiescent chicken embryo fibroblasts resulted in a time-dependent, biphasic activation of S6 kinase activity. Chromatographic fractionation of serum-stimulated cell lysates resolved two distinct S6 kinase activities. Anti-Xenopus S6 kinase II antiserum immunoprecipitated a 90,000-Mr S6 kinase but did not cross-react with a smaller, 65,000-Mr S6 kinase. Phosphopeptide analysis confirmed that the 90,000- and 65,000-Mr proteins were structurally unrelated and established that the 65,000-Mr protein isolated by the current protocol was the same serum-stimulated chicken embryo fibroblast S6 kinase as that previously characterized (J. Blenis, C. J. Kuo, and R. L. Erikson, J. Biol. Chem. 262:14373-14376, 1987). These results demonstrate the contribution of two distinct S6 kinases to total serum-stimulated ribosomal protein S6 phosphorylation.

Two distinct enzymes contribute to biphasic S6 phosphorylation in serum-stimulated chicken embryo fibroblasts

1990 ◽  
Vol 10 (6) ◽  
pp. 2787-2792
Author(s):  
L J Sweet ◽  
D A Alcorta ◽  
R L Erikson
Keyword(s):  
Chicken Embryo ◽  
Kinase Activity ◽  
Chicken Embryo Fibroblast ◽  
Total Serum ◽  
S6 Kinase ◽  
Serum Stimulation ◽  
Ribosomal Protein S6 ◽  
Phosphopeptide Analysis ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Serum stimulation of quiescent chicken embryo fibroblasts resulted in a time-dependent, biphasic activation of S6 kinase activity. Chromatographic fractionation of serum-stimulated cell lysates resolved two distinct S6 kinase activities. Anti-Xenopus S6 kinase II antiserum immunoprecipitated a 90,000-Mr S6 kinase but did not cross-react with a smaller, 65,000-Mr S6 kinase. Phosphopeptide analysis confirmed that the 90,000- and 65,000-Mr proteins were structurally unrelated and established that the 65,000-Mr protein isolated by the current protocol was the same serum-stimulated chicken embryo fibroblast S6 kinase as that previously characterized (J. Blenis, C. J. Kuo, and R. L. Erikson, J. Biol. Chem. 262:14373-14376, 1987). These results demonstrate the contribution of two distinct S6 kinases to total serum-stimulated ribosomal protein S6 phosphorylation.

Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts

1983 ◽  
Vol 3 (3) ◽  
pp. 380-390
Author(s):  
K D Nakamura ◽  
R Martinez ◽  
M J Weber
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Chicken Embryo ◽  
Sodium Dodecyl ◽  
Biological Response ◽  
Egf Receptors ◽  
Phosphorylation Of Proteins ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts ◽  
Stimulation Of

We found that stimulation of density-inhibited chicken embryo fibroblasts with serum, epidermal growth factor (EGF), platelet-derived growth factor, (PDGF), or multiplication-stimulating activity (MSA) leads to an increase in tyrosine phosphorylation of proteins in the region of Mr 40,000 (40K) to 42K. The increase in tyrosine phosphorylation after serum or EGF stimulation was transient, reaching a maximum at about 5 min and then declining. By fine-resolution analysis of proteins separated on sodium dodecyl sulfate-polyacrylamide gels, we found that after EGF stimulation, the major increase in phosphotyrosine content was in a 42K Mr protein, with a smaller increase in a 40K Mr protein. The increased phosphorylation in the 40K to 42K Mr region accounted for almost all of the increase in phosphotyrosine observed in these cells. These phosphotyrosine-containing proteins were different from the major phosphotyrosine-containing protein of Rous sarcoma virus-transformed chicken embryo fibroblasts, which migrates at an approximate Mr of 36K. Increased tyrosine phosphorylation of proteins of similar Mr was found in 3T3 cells treated with EGF, but not in NR-6 cells, which lack detectable EGF receptors. It is possible that the 40K to 42K Mr phosphotyrosine-containing proteins are involved in the integration of the biological response to a number of different growth factors.

Identification of mitogen-responsive ribosomal protein S6 kinase pp90rsk, a homolog of Xenopus S6 kinase II, in chicken embryo fibroblasts

1990 ◽  
Vol 10 (5) ◽  
pp. 2413-2417
Author(s):  
L J Sweet ◽  
D A Alcorta ◽  
S W Jones ◽  
E Erikson ◽  
R L Erikson
Keyword(s):  
Ribosomal Protein ◽  
Immune Complex ◽  
Chicken Embryo ◽  
Chicken Embryo Fibroblast ◽  
S6 Kinase ◽  
Ribosomal Protein S6 ◽  
Embryo Fibroblasts ◽  
Ribosomal S6 Kinase ◽  
Chicken Embryo Fibroblasts ◽  
Ribosomal Protein S6 Kinase

Antiserum raised against recombinant Xenopus ribosomal protein S6 kinase (rsk) was used to identify a 90,000-Mr ribosomal S6 kinase, pp90rsk, in chicken embryo fibroblasts. Adding serum to cells stimulated the phosphorylation of pp90rsk on serine and threonine residues and increased the activity of S6 kinase measured in immune complex assays. Xenopus S6 kinase II and chicken embryo fibroblast pp90rsk had nearly identical phosphopeptide maps.

scholarly journals Biological and biochemical properties of the c-src+ gene product overexpressed in chicken embryo fibroblasts.

1989 ◽  
Vol 9 (8) ◽  
pp. 3332-3341 ◽  
Author(s):  
J B Levy ◽  
J S Brugge
Keyword(s):  
Protein Kinase ◽  
Chicken Embryo ◽  
Kinase Activity ◽  
Biochemical Properties ◽  
Serine Phosphorylation ◽  
Protein Kinase Activity ◽  
Independent Manner ◽  
Amino Terminal ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

The c-src protein isolated from neuronal cells (pp60c-src+) displays a higher level of protein kinase activity than does pp60c-src from nonneural tissues. There are two structural alterations present in the amino-terminal half of pp60c-src+ expressed in neurons which could contribute to the enhanced activity of this form of pp60c-src: (i) a hexapeptide insert located at amino acid 114 of avian pp60c-src+ and (ii) a novel site(s) of serine phosphorylation. We characterized pp60c-src+ expressed in a nonneuronal cell type to identify factors that regulate the activity of the c-src+ protein and the importance of the neuronal environment on this regulation. The c-src+ protein overexpressed in chicken embryo fibroblasts (CEFs) displayed higher kinase activity than did pp60c-src. The major sites of phosphorylation of the c-src+ protein were Ser-17 and Tyr-527. The unique site(s) of serine phosphorylation originally identified in pp60c-src+ expressed in neurons was not detected in the c-src+ protein overexpressed in CEFs. Therefore, the hexapeptide insert is sufficient to cause an elevation in the tyrosine protein kinase activity of pp60c-src+. Our data also indicate that CEFs infected with the Rous sarcoma virus (RSV)c-src+ display phenotypic changes that distinguish them from cultures producing pp60c-src and that pp60c-src+-expressing cells are better able to grow in an anchorage-independent manner. The level of total cellular tyrosine phosphorylation in RSVc-src+-infected cultures was moderately higher than the level observed in cultures infected with RSVc-src. This level was not as pronounced as that observed in cells infected with RSVv-src or oncogenic variants of RSVc-src. Thus, pp60c-src+ could be considered a partially activated c-src variant protein much like other c-src proteins that contain mutations in the amino-terminal domain.

scholarly journals Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts.

1983 ◽  
Vol 3 (3) ◽  
pp. 380-390 ◽  
Author(s):  
K D Nakamura ◽  
R Martinez ◽  
M J Weber
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Chicken Embryo ◽  
Sodium Dodecyl ◽  
Biological Response ◽  
Egf Receptors ◽  
Phosphorylation Of Proteins ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts ◽  
Stimulation Of

We found that stimulation of density-inhibited chicken embryo fibroblasts with serum, epidermal growth factor (EGF), platelet-derived growth factor, (PDGF), or multiplication-stimulating activity (MSA) leads to an increase in tyrosine phosphorylation of proteins in the region of Mr 40,000 (40K) to 42K. The increase in tyrosine phosphorylation after serum or EGF stimulation was transient, reaching a maximum at about 5 min and then declining. By fine-resolution analysis of proteins separated on sodium dodecyl sulfate-polyacrylamide gels, we found that after EGF stimulation, the major increase in phosphotyrosine content was in a 42K Mr protein, with a smaller increase in a 40K Mr protein. The increased phosphorylation in the 40K to 42K Mr region accounted for almost all of the increase in phosphotyrosine observed in these cells. These phosphotyrosine-containing proteins were different from the major phosphotyrosine-containing protein of Rous sarcoma virus-transformed chicken embryo fibroblasts, which migrates at an approximate Mr of 36K. Increased tyrosine phosphorylation of proteins of similar Mr was found in 3T3 cells treated with EGF, but not in NR-6 cells, which lack detectable EGF receptors. It is possible that the 40K to 42K Mr phosphotyrosine-containing proteins are involved in the integration of the biological response to a number of different growth factors.

scholarly journals The actin of muscle and fibroblasts

1976 ◽  
Vol 155 (2) ◽  
pp. 297-301 ◽  
Author(s):  
P J. Anderson
Keyword(s):  
Chicken Embryo ◽  
Cell Types ◽  
Amino Acid Sequences ◽  
Terminal Region ◽  
Breast Muscle ◽  
Chicken Breast ◽  
Double Labelling ◽  
Muscle Actin ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

The isolation and quantification of an 18-residue peptide from the N-terminal region of chicken actin was used to quantify the amount of actin in acetone-dried powders of chicken breast muscle and chicken-embryo fibroblasts. Either isotope dilution or double labelling can be used for peptide quantification. About 17% of the protein of chicken breast muscle was estimated to be actin. However, only 0.25% of the protein of chicken-embryo fibroblasts was determined to be actin by quantification of this peptide. The actin content of fibroblasts may be low or the amino acid sequences of muscle and fibroblast actin may differ in the N-terminal region. The methodology used can be extended to examine whether other regions of muscle actin sequence are present in fibroblasts or other cell types.

Hematopoietic lineage-specific heterogeneity in the 5'-terminal region of the chicken proto-myb transcript

1989 ◽  
Vol 9 (9) ◽  
pp. 3771-3776
Author(s):  
W K Kim ◽  
M A Baluda
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cell ◽  
Chicken Embryo ◽  
Cell Types ◽  
Terminal Region ◽  
The Other ◽  
Bursa Of Fabricius ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Comparison of the nucleotide sequence of the upstream c-myb exon UE3 with the sequences of a thymus c-myb cDNA and of a B-lymphoma c-myb cDNA suggested the existence of T- and B-cell-specific heterogeneity in the 5'-terminal region of the c-myb coding sequence. This possibility was investigated with T-cell-specific and B-cell-specific DNA probes in a Northern (RNA) blot analysis of mRNAs from different hematopoietic cell types and from chicken embryo fibroblasts. The hematopoietic tissues analyzed were bone marrow, bursa of Fabricius, and thymus from 1-day-old chicks, 13-day yolk sac, and spleen from 16-day embryos. At least three different c-myb mRNA species were found to have 5'-terminal heterogeneity that was specific for either B cells, T cells, or the other hematopoietic cells and chicken embryo fibroblasts. This lineage-specific heterogeneity in the c-myb transcript was found to be expressed in the bone marrow precursors of B and T cells before they migrated to their definitive differentiation sites. S1 nuclease protection analysis of the UE3 exon, part of which appeared to be coding sequences for thymic c-myb mRNA, revealed that this exon is utilized either in its entirety or partially in a cell-lineage-specific manner by all six tissues analyzed. Also, the 5'-terminal exon(s) present in the thymus cDNA was absent in c-myb mRNAs from the other cell types analyzed.

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