The human immunodeficiency virus type 1 Nef protein binds the Src-related tyrosine kinase Lck SH2 domain through a novel phosphotyrosine independent mechanism.
Primate lentiviruses encode for an unique nef gene with an essential function in both viral replication and pathogenicity in the host. The molecular basis for this function remains however poorly defined. Several Nef-binding cellular proteins are thought to be instrumental in its function. Indeed, Nef contains a proline-rich motif implicated in the binding to the Src-like tyrosine kinase Hck and also to a Ser/Thr kinase of molecular weight 62 kDa. The disruption of this motif affects the binding to both these kinases as well as viral replication. Whereas Hck is expressed in the myeloid lineage and hence may account for the nef function in infected monocytes, we and others have reported previously that Nef also interacts with the T-lymphocyte Src-kinase Lck, leading to specific cell signaling impairment. This interaction occurs through the binding of Nef to both Lck SH2 and SH3 domains. Both the proline motif and phosphorylation of Nef on tyrosine residue were proposed to account for these interactions. Here, we investigate the mechanism of Lck SH2 binding by HIV-1 Nef. Using recombinant fusion proteins to precipitate lysates, we show that although SH2 binding is dependent on phosphorylation events, it occurs in a tyrosine independent manner because it requires neither tyrosine residues in Nef nor the phosphotyrosine binding pocket from the Lck SH2 domain, hence suggesting a role for a phosphoserine or a phosphothreonine residue. Further, we show that Hck SH2 does not interact with Nef, indicating that Hck SH3 binding is sufficient for Nef binding, whereas Lck SH2 cooperate together with SH3 to allow Nef binding to a level similar to Hck SH3. Together, our results establish different mechanisms for Hck and Lck binding by HIV-1 Nef protein, and identify a novel mechanism for Src-like tyrosine kinase targeting by a viral protein.
Unité 119 Instituto National de la Santé et de la Recherche Médicale, Marseille, France.
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Dutartre
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Harris
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Olive
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Collette
D015693:Gene Products, nef / Q000378:metabolism*; D016391:Genes, src; D015497:HIV-1 / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D019169:Jurkat Cells; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism*; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051572:Proto-Oncogene Proteins c-hck; D014779:Virus Replication; D054311:nef Gene Products, Human Immunodeficiency Virus; D018909:src Homology Domains
D015693:Gene Products, nef; D011518:Proto-Oncogene Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051572:Proto-Oncogene Proteins c-hck