A mammalian two-hybrid screening system for inhibitors of interaction between HIV Nef and the cellular tyrosine kinase Hck.
In the scope of the search for new anti-HIV agents interacting with a new target, we developed a high-throughput screening system to detect the interactions between Nef and Hck. Nef is an accessory protein of HIV, which is involved in the pathogenicity of the acquired immunodeficiency syndrome (AIDS). Nef is also a signaling molecule because it binds to many host molecules. It has especially high affinity to Hck, a member of src family tyrosine kinase. Using a mammalian two-hybrid system, the interaction between Nef and the SH3 domain of Hck induced luciferase activity with high sensitivity and a Nef-PXXP peptide inhibited this interaction; and so did the anticancer drug adriamycin. We also developed another assay system by coexpression of full-length Hck and Nef, and found that Hck kinase was activated depending on the dose of Nef plasmid. Using the second system, we found that adriamycin interfered with the Nef-Hck interaction by reducing the amount of the Hck protein. The mammalian two-hybrid system may show utility in screening inhibitors of Nef-Hck interaction.
Murakami
Yuko
Y
Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, 162-8640, Tokyo, Japan.
Fukazawa
Hidesuke
H
Kobatake
Takae
T
Yamagoe
Satoshi
S
Takebe
Yutaka
Y
Tobiume
Minoru
M
Matsuda
Michiyuki
M
Uehara
Yoshimasa
Y
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D004317:Doxorubicin / Q000494:pharmacology; D015693:Gene Products, nef / Q000378:metabolism*; D006678:HIV / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051572:Proto-Oncogene Proteins c-hck; D020798:Two-Hybrid System Techniques; D054311:nef Gene Products, Human Immunodeficiency Virus; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism
D015693:Gene Products, nef; D011518:Proto-Oncogene Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D004317:Doxorubicin; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; C496632:Hck protein, mouse; D051572:Proto-Oncogene Proteins c-hck; D019061:src-Family Kinases