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PMID: 2431288 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Retrovirus shuttle vector for study of kinase activities of pp60c-src synthesized in vitro and overproduced in vivo.

Molecular and cellular biology ·Vol. 6 ·No. 6 ·1986-06-00 ·Pages 2033-40

Piwnica-Worms H, Kaplan DR, Whitman M, Roberts TM

Abstract

We have constructed a recombinant murine retrovirus which efficiently transduces avian pp60c-src into murine cells and which is easily rescued from infected cells in plasmid form. To characterize the virus, several randomly selected NIH 3T3 lines were isolated after infection with recombinant retroviral stocks. All lines overproduced avian pp60c-src and appeared morphologically normal. Immunoprecipitates made from these lines with antisera specific for pp60c-src were tested for their kinase activities in vitro. We find that both autokinase and enolase kinase activities increase proportionately with the level of pp60c-src in the immunoprecipitates. To further test the authenticity of the pp60c-src encoded by the retroviral vector, these analyses were repeated in the presence of polyomavirus middle T antigen. Avian pp60c-src was activated as a protein kinase, indicating that the virally encoded pp60c-src interacts normally with middle T antigen. Interestingly, by increasing the intracellular levels of pp60c-src 15-fold over normal endogenous levels, we were unable to obtain a proportionate increase in the amount of middle-T-antigen-pp60c-src complex. Finally, using the shuttle features designed into the vector, we have isolated the first fully processed cDNA encoding functional avian pp60c-src X pp60c-src synthesized in vitro with this cDNA had intrinsic protein kinase activity and no detectable phosphatidylinositol kinase activity.

MeSH Terms
Animals Cell Line DNA/genetics Genetic Vectors Mice Proto-Oncogene Proteins/genetics Proto-Oncogene Proteins pp60(c-src) Retroviridae/genetics
Chemicals
Proto-Oncogene Proteins DNA Proto-Oncogene Proteins pp60(c-src)
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Piwnica-Worms H
Kaplan D R
Whitman M
Roberts T M
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39 references, click to expand
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.6.6.2033-2040.1986
Published
1986-06-00
Pages
2033-40
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC367743
Subset
IM
Grants
NCI NIH HHS · CA 30002 · United States
NCI NIH HHS · CA07634 · United States
External Links
PubMed source DOI: 10.1128/mcb.6.6.2033-2040.1986 PMC Full Text View journal details
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