Functional interaction between the HIV transactivator Tat and the transcriptional coactivator PC4 in T cells.
The human immunodeficiency virus (HIV) transactivator Tat is a potent activator of transcription from the HIV long terminal repeat and is essential for efficient viral gene expression and replication. Tat has been shown to interact with components of the basal transcription machinery and transcriptional activators. Here we identify the cellular coactivator PC4 as a Tat-interacting protein using the yeast two-hybrid system and confirmed this interaction both in vitro and in vivo by coimmunoprecipitation. We found that this interaction has a functional outcome in that PC4 overexpression enhanced activation of the HIV long terminal repeat in transient transfection studies in a Tat-dependent manner. The domains of PC4 and Tat required for the interaction were mapped. In vitro binding studies showed that the basic transactivation-responsive binding domain of Tat is required for the interaction with PC4. The minimum region of PC4 required for Tat binding was amino acids 22-91, whereas mutation of the lysine-rich domain between amino acids 22 and 43 prevented interaction with Tat. Tat-PC4 interactions may be controlled by phosphorylation, because phosphorylation of PC4 by casein kinase II inhibited interactions with Tat both in vivo and in vitro. We propose that PC4 may be involved in linking Tat to the basal transcription machinery.
Holloway
A F
AF
Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australia.
Occhiodoro
F
F
Mittler
G
G
Meisterernst
M
M
Shannon
M F
MF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D004926:Escherichia coli; D015723:Gene Library; D015696:Gene Products, tat / Q000378:metabolism*; D017930:Genes, Reporter; D006678:HIV / Q000235:genetics*; D016325:HIV Long Terminal Repeat; D006801:Humans; D017874:Immediate-Early Proteins; D019169:Jurkat Cells; D008156:Luciferases / Q000235:genetics; D008239:Lysine; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000502:physiology* / Q000821:virology; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; C490043:IFRD2 protein, human; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D054322:tat Gene Products, Human Immunodeficiency Virus; D008156:Luciferases; D008239:Lysine