Biochemical comparisons of the normal and oncogenic forms of insect cell-expressed neu tyrosine kinases

The rat neu oncogene product is a member of the epidermal growth factor (EGF) receptor subgroup of the superfamily of growth factor receptor tyrosine kinases. The oncogenic activation of the neu protein occurs by a point mutation within its transmembrane region which results in an increase in its ty...

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Bibliographic Details
Published in:The Journal of biological chemistry 1992-07, Vol.267 (20), p.13851-13856
Main Authors: P M Guy, K L Carraway, 3rd, R A Cerione
Format: Article
Language:English
Subjects:
Animals
Baculoviridae - genetics
Base Sequence
Biochemistry & Molecular Biology
Biological and medical sciences
Cell Line
Cell physiology
Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes
DNA - genetics
ErbB Receptors - genetics
Fundamental and applied biological sciences. Psychology
Insecta
Kinetics
Life Sciences & Biomedicine
Molecular and cellular biology
Molecular Sequence Data
Mutagenesis, Site-Directed
Oligodeoxyribonucleotides
Phosphorylation
Plasmids
Protein-Tyrosine Kinases - genetics
Protein-Tyrosine Kinases - metabolism
Proto-Oncogene Proteins - genetics
Proto-Oncogene Proteins - metabolism
Proto-Oncogenes
Rats
Receptor, ErbB-2
Science & Technology
Substrate Specificity
Transfection
tyrosine kinase
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Summary:The rat neu oncogene product is a member of the epidermal growth factor (EGF) receptor subgroup of the superfamily of growth factor receptor tyrosine kinases. The oncogenic activation of the neu protein occurs by a point mutation within its transmembrane region which results in an increase in its tyrosine kinase activity. Using three different forms of neu expressed in insect cells via baculovirus infection, we have examined the biochemical differences between the normal and transforming forms of neu and investigated the role of the transmembrane domain in its tyrosine kinase activity. One form of neu which was expressed in insect cells consisted of the complete tyrosine kinase domain but lacked the extracellular and transmembrane regions (designated NTK). The other two forms consisted of the tyrosine kinase domain, the transmembrane domain, and 40 amino acids of the extracellular domain. One of these transmembrane forms of neu contained the normal valine residue at position 664 within the transmembrane region (MS-N), while the other contained the oncogenic glutamic acid residue at this position (MS-T). Direct comparisons of NTK, MS-N, and MS-T have shown that the NTK protein is capable of the highest extents of both autophosphorylation activity and the tyrosine phosphorylation of exogenous substrate, suggesting that the presence of the transmembrane region of neu suppresses the tyrosine kinase activity of this receptor. In addition, we have found that the oncogenic point mutation within the transmembrane region stimulates the tyrosine kinase activity of the neu protein by allowing it to more effectively utilize Mg2+. Overall, the results of these studies suggest that the valine to glutamic acid substitution at position 664 may at least partially relieve a negative constraint imparted by the membrane-spanning domain on the tyrosine kinase activity of neu and enables a more effective use of Mg2+ in the catalysis of tyrosine phosphorylation of exogenous substrates.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)49646-4