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Myristoyl moiety of HIV Nef is involved in regulation of the interaction with calmodulin in vivo

Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan

(RECEIVED July 8, 2004; FINAL REVISION October 9, 2004; ACCEPTED October 21, 2004)

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 CaCl₂ or EGTA, calcium ion drastically enhanced the interaction between Nef and calmodulin, suggesting that the binding is under the influence of Ca²⁺ 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 nonmyristoylated 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 Ca²⁺ signaling through calmodulin-mediated interactions in infected cells.

Keywords: HIV nef; calmodulin; myristoylation; protein-protein interaction

Abbreviations: HIV, human immunodeficiency virus • SIV, simian immunodeficiency virus • GST, glutathione S-transferase • MARCKS, myristoylated alanine-rich protein kinase C substrate • PKC, protein kinase C

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04969605.

Reprint requests to: Nobuhiro Hayashi, Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan; e-mail: nhayashi{at}fujita-hu.ac.jp; fax: 562-93-8832.

¹ Present address: Carna Biosciences Inc., 5-5-2 Minatojima-Minamima-chi, Kobe, 650-0047, Japan.

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