A Wild-Type Nanopore Sensor for Protein Kinase Activity

The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type v-fms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolytically processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane.

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Cell surface expression of v-fms-coded glycoproteins is required for transformation

Molecular and Cellular Biology ◽

10.1128/mcb.4.10.1999-2009.1984 ◽

1984 ◽

Vol 4 (10) ◽

pp. 1999-2009

Author(s):

M F Roussel ◽

C W Rettenmier ◽

A T Look ◽

C J Sherr

Keyword(s):

Amino Acids ◽

Protein Kinase ◽

Cell Surface ◽

Gene Product ◽

Kinase Activity ◽

Specific Protein ◽

Protein Kinase Activity ◽

Wild Type ◽

Specific Protein Kinase

The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type v-fms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolytically processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane.

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Protein kinase and ATP-binding activity associated with the 72-kdalton single-stranded DNA-binding protein from early region 2A of human adenovirus type 5

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o85-117 ◽

1985 ◽

Vol 63 (9) ◽

pp. 941-952 ◽

Cited By ~ 3

Author(s):

Philip E. Branton ◽

Mike Evelegh ◽

David T. Rowe ◽

Frank L. Graham ◽

Silvia Bacchetti

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Human Adenovirus ◽

Adenovirus Type ◽

Atp Binding ◽

Protein Kinase Activity ◽

Wild Type ◽

Amino Terminal ◽

Early Region ◽

Adenovirus Type 5

We have developed monoclonal antibodies which react specifically with the human adenovirus type 5 early region 2A 72-kdalton phosphoprotein (72 kDa) and its 48-kdalton proteolytic cleavage product (48 kDa) and have used these antibodies to study a number of properties of these viral polyeptides. Fluorescent antibody staining indicated that the 72 kDa was found almost entirely in the nucleus, generally in discreet patches. Preparations of the 72 kDa, purified by immunoprecipitation or by single-stranded DNA-cellulose column chromatography and incubated with [γ-32P]ATP, were found to contain protein kinase activity. Using photoaffinity labelling with 8-azido-[α-32P]ATP, the 72 kDa was shown to be an ATP-binding protein. The ATP-binding site was probably in the amino terminal region because the 48 kDa which lacks approximately 120 residues at the amino terminus failed to bind ATP. Experiments carried out with immunoprecipitates from wild-type and temperature-sensitive (ts125) infected cells suggested that the ts mutant-induced kinase activity was not more thermolabile than the wild-type protein. Thus although the present results indicated that protein kinase activity is associated with the 72 kDa, it is still unclear whether such activity is intrinsic to the 72 kDa or present in an enzyme associated with it. While it is probably not intrinsic to the carboxy terminal region affected by the ts125 mutation, it is still possible that it resides in an amino terminal domain.

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Involvement of a Protein Kinase Activity Inducer in DNA Double Strand Break Repair and Radioresistance of Deinococcus radiodurans

Journal of Bacteriology ◽

10.1128/jb.00026-08 ◽

2008 ◽

Vol 190 (11) ◽

pp. 3948-3954 ◽

Cited By ~ 32

Author(s):

Yogendra S. Rajpurohit ◽

Roja Gopalakrishnan ◽

Hari S. Misra

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Deinococcus Radiodurans ◽

Strand Break ◽

Pyrroloquinoline Quinone ◽

Protein Kinase Activity ◽

Wild Type ◽

Dna Double Strand Break ◽

Break Repair ◽

Mutant Cells

ABSTRACT Transgenic bacteria producing pyrroloquinoline quinone, a known cofactor for dehydrogenases and an inducer of a periplasmic protein kinase activity, show resistance to both oxidative stress and protection from nonoxidative effects of radiation and DNA-damaging agents. Deinococcus radiodurans R1 encodes an active pyrroloquinoline quinone synthase, and constitutive synthesis of pyrroloquinoline quinone occurred in wild-type bacteria. Disruption of a genomic copy of pqqE resulted in cells that lacked this cofactor. The mutant showed a nearly 3-log decrease in γ radiation resistance and a 2-log decrease in mitomycin C tolerance compared to wild-type cells. The mutant cells did not show sensitivity to UVC radiation. Expression of pyrroloquinoline quinone synthase in trans showed that there was functional complementation of γ resistance and mitomycin C tolerance in the pqqE mutant. The sensitivity to γ radiation was due to impairment or slow kinetics of DNA double strand break repair. Low levels of 32P incorporation were observed in total soluble proteins of mutant cells compared to the wild type. The results suggest that pyrroloquinoline quinone has a regulatory role as a cofactor for dehydrogenases and an inducer of selected protein kinase activity in radiation resistance and DNA strand break repair in a radioresistant bacterium.

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Molecular events in cells transformed by Rous Sarcoma virus.

The Journal of Cell Biology ◽

10.1083/jcb.87.2.319 ◽

1980 ◽

Vol 87 (2) ◽

pp. 319-325 ◽

Cited By ~ 52

Author(s):

R L Erikson ◽

A F Purchio ◽

E Erikson ◽

M S Collett ◽

J S Brugge

Keyword(s):

Protein Kinase ◽

Rous Sarcoma Virus ◽

Kinase Activity ◽

Protein Kinase Activity ◽

Wild Type ◽

Dependent Protein Kinase ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Dependent Protein ◽

Transforming Gene

The Rous sarcoma virus (RSV) transforming gene product has been identified and characterized as a phosphoprotein with a molecular weight of 60,000, denoted pp60src. Partially purified pp60src displays a closely associated phosphotransferase activity with the unusual specificity of phosphorylating tyrosine residues in a variety of proteins. That the enzymatic activity observed is actually encoded by the RSV-transforming gene is indicated by the comparison of the pp60src-protein kinase isolated from cells tranformed by a wild-type RSV or by a RSV temperature-sensitive transformation mutant; these experiments revealed that the latter enzyme had a half-life of 3 min at 41 degrees C, whereas that of the wild-type enzyme was 20 min. Evidence is now beginning to accumulate showing that viral pp60src expresses its protein kinase activity in transformed cells as well as in vitro because at least one cellular protein has been identified as a substrate for this activity of pp60src. Although the protein kinase activity associated with pp60src is itself cyclic AMP (cAMP) independent, the molecule contains at least one serine residue that is directly phosphorylated by the cellular cAMP-dependent protein kinase, thus suggesting that the viral transforming gene product may be regulated indirectly by the level of cAMP. The significance of this latter observation must be regarded from the point of view that the RSV src gene is apparently derived from a normal cellular gene that seemingly expresses in normal uninfected cells a phosphoprotein structurally and functionally closely related to pp60src. This celluar protein, found in all vertebrate species tested, also is a substrate for a cAMP-dependent protein kinase of normal cells, and, therefore, may be evolved to function in a regulatory circuit involving cAMP.

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Effect of Thrombin on Phosphorylation of Platelet Membrane Proteins

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647962 ◽

1976 ◽

Vol 35 (03) ◽

pp. 635-642 ◽

Cited By ~ 4

Author(s):

M Steiner

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Qualitative Change ◽

Protein Kinase Activity ◽

Protein Substrates ◽

Phosphoprotein Phosphatase ◽

Progressive Loss ◽

Platelet Membranes ◽

Endogenous Protein ◽

Considerable Loss

SummaryThe effect of thrombin on the phosphorylating activity of platelet membranes was compared to that of trypsin. Preincubation of non-32P phosphorylated platelet membranes with or without either of these two enzymes resulted in a considerable loss of membrane protein kinase activity which was most severe when trypsin was used. Protein kinase activity and endogenous protein acceptors decreased in parallel. 32P-phosphorylated membranes showed a slow but progressive loss of label which was accelerated by trypsin. Thrombin under these conditions prevented the loss of 32P-phosphate. These results are interpreted to indicate a thrombin-induced destruction of a phosphoprotein phosphatase. The protein kinase activity of phosphorylated platelet membranes using endogenous or exogenous protein substrates showed a significant reduction compared to non-phosphorylated membranes suggesting a deactivation of protein kinase by phosphorylation of platelet membranes. Neither thrombin nor trypsin caused a qualitative change in the membrane polypeptides accepting 32P-phosphate but resulted in quantitative alterations of their ability to become phosphorylated.

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