SH3-mediated Hck tyrosine kinase activation and fibroblast transformation by the Nef protein of HIV-1.
Tyrosine kinases of the Src family are regulated via their Src homology 2 (SH2) and SH3 domains. The Nef protein of human immunodeficiency virus-1 (HIV-1) has previously been shown to bind with high affinity and specificity in vitro to the SH3 domain of Hck, a Src family member expressed primarily in myeloid cells. However, the effect of Nef on Hck activity in living cells is unknown. Here we show that Rat-2 fibroblasts co-expressing Hck and Nef rapidly developed transformed foci, whereas control cells expressing either protein alone did not. Nef formed a stable complex with Hck and stimulated its tyrosine kinase activity in vivo. Mutagenesis of the Nef proline-rich motif essential for SH3 binding completely blocked complex formation, kinase activation, and transformation, indicating that the Nef SH3-binding function is required for its effects on Hck. These results provide direct evidence that SH3 engagement is sufficient to activate a Src family kinase in vivo and suggest that Hck may be activated by Nef in HIV-infected macrophages.
Briggs
S D
SD
Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA.
Sharkey
M
M
Stevenson
M
M
Smithgall
T E
TE
D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D005347:Fibroblasts; D015693:Gene Products, nef / Q000378:metabolism*; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051572:Proto-Oncogene Proteins c-hck; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae; D054311:nef Gene Products, Human Immunodeficiency Virus; D018909:src Homology Domains
D015693:Gene Products, nef; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; C496633:Hck protein, rat; D051572:Proto-Oncogene Proteins c-hck