Myristoyl moiety of HIV Nef is involved in regulation of the interaction with calmodulin in vivo.
Human immunodeficiency virus Nef is a myristoylated protein expressed early in infection by HIV. In addition to the well known down-regulation of the cell surface receptors CD4 and MHCI, Nef is able to alter T-cell signaling pathways. The ability to alter the cellular signaling pathways suggests that Nef can associate with signaling proteins. In the present report, we show that Nef can interact with calmodulin, the major intracellular receptor for calcium. Coimmunoprecipitation analyses with lysates from the NIH3T3 cell line constitutively expressing the native HIV-1 Nef protein revealed the presence of a stable Nef-calmodulin complex. When lysates from NIH3T3 cells were incubated with calmodulin-agarose beads in the presence of CaCl(2) or EGTA, calcium ion drastically enhanced the interaction between Nef and calmodulin, suggesting that the binding is under the influence of Ca(2+) signaling. Glutathione S-transferase-Nef fusion protein bound directly to calmodulin with high affinity. Using synthetic peptides based on the N-terminal sequence of Nef, we determined that within a 20-amino-acid N-terminal basic domain was sufficient for calmodulin binding. Furthermore, the myristoylated peptide bound to calmodulin with higher affinity than nonmyris-toylated form. Thus, the N-terminal myristoylation domain of Nef plays an important role in interacting with calmodulin. This domain is highly conserved in several HIV-1 Nef variants and resembles the N-terminal domain of NAP-22/CAP23, a myristoylated calmodulin-binder. These results for the interaction between HIV Nef and calmodulin in the cells suggested that the Nef might interfere with intracellular Ca(2+) signaling through calmodulin-mediated interactions in infected cells.
Matsubara
Mamoru
M
Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan.
Jing
Tao
T
Kawamura
Kumi
K
Shimojo
Naoshi
N
Titani
Koiti
K
Hashimoto
Keiichiro
K
Hayashi
Nobuhiro
N
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D015704:CD4 Antigens / Q000737:chemistry; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002122:Calcium Chloride / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry*; D002417:Cattle; D004533:Egtazic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015693:Gene Products, nef / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000737:chemistry; D041681:NIH 3T3 Cells; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D013601:T-Lymphocytes / Q000378:metabolism; D013997:Time Factors
D015704:CD4 Antigens; D002147:Calmodulin; D015693:Gene Products, nef; D010455:Peptides; D011993:Recombinant Fusion Proteins; D019814:Myristic Acid; D004533:Egtazic Acid; D005982:Glutathione Transferase; D002122:Calcium Chloride; D002118:Calcium