Novel (n)PKC kinases phosphorylate Nef for increased HIV transcription, replication and perinuclear targeting.
The N-terminus of the human immunodeficiency virus (HIV) pathogenicity factor Nef associates with a protein complex (NAKC for Nef-associated kinase complex) that contains at least two kinases: the tyrosine kinase Lck and a serine kinase activity which was found to phosphorylate Lck and the Nef N-terminus. Here we show that this serine kinase activity is mediated by members of the novel Protein Kinase C (nPKC) subfamily, PKCdelta and theta. Association with the Nef N-terminus was sufficient to activate PKC leading to phosphorylation of Nef in vitro on a conserved serine residue at position 6. Mutation of serine 6 or coexpression of a transdominant negative PKC mutant significantly reduced Nef-stimulated HIV transcription and replication in resting PBMC. When analyzing the molecular mechanisms, we found that mutating serine 6 moderately affected myristoylation of Nef and its association with Pak2 activity, whereas CD4 downmodulation was not inhibited. More interestingly, this mutation abolished the typical perinuclear localization of Nef in T cells. We conclude that the activation of nPKCs by Nef is required to increase viral replication/infectivity and direct the subcellular localization of Nef.
Wolf
Dietlinde
D
University of Miami, Miller School of Medicine, Department of Microbiology and Immunology, BCRI 739, Miami, FL 33136, USA.
Giese
Simone I
SI
Witte
Vanessa
V
Krautkrämer
Ellen
E
Trapp
Susanna
S
Sass
Gabriele
G
Haller
Claudia
C
Blume
Katja
K
Fackler
Oliver T
OT
Baur
Andreas S
AS
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D015754:Genes, nef; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism*; D014158:Transcription, Genetic; D014779:Virus Replication; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D009097:Multienzyme Complexes; D054311:nef Gene Products, Human Immunodeficiency Virus; D011493:Protein Kinase C; D051745:Protein Kinase C-delta