Role of histone kinases in Tat transactivation.

<p>(A) Chromatin immunoprecipitation analysis of Jurkat T cells containing an integrated HIV promoter in the absence or presence of Tat. Immunoprecipitations were performed with α-phospho-histone H3 antibodies (serine 10) followed by radioactive PCR with primers specific for the HIV LTR, the c-fos, or the β-globin genes. (B) Jurkat 1G5 cells containing an integrated HIV LTR luciferase construct were transiently transfected with Tat/FLAG (25 ng) and kinase-deficient (KD) kinase expression vectors (200 ng). (C) Western blot analysis of cellular lysates from 293 cells cotransfected with the indicated expression plasmids. (D) Transfection of CMV luciferase (25 ng) with the KD RSK2 expression plasmid (200 ng) in Jurkat cells. (E) Transfection of 5xUAS luciferase and Gal4-CDK9 (20 ng) with the KD RSK2 expression plasmid (200 ng) in Jurkat cells. Values are means±SEM of three experiments.</p

Superinduction of Tat activity in CLS fibroblasts.

<p>(A) Western blot analysis of cellular extracts of fibroblasts from a patient with CLS and control human fibroblasts. (B) Nuclear microinjection of CLS fibroblasts with synthetic Tat (amino acids 1–72), the HIV LTR luciferase reporter, a CMV-GFP expression plasmid, and either the empty vector, an RSK2 expression construct, or a plasmid expressing kinase-deficient RSK2. Values are means±SEM of five experiments. (C) Coinjection of the 5xUAS luciferase reporter, a plasmid expressing the Gal4-VP16 transactivator and CMV-GFP with either the RSK2-expressing plasmid or the vector alone. Values are means±SEM of three experiments.</p

Activation of RSK2 by Tat.

<p>(A) Autoradiography of radioactive <i>in vitro</i> synthesized RSK2 proteins before (Input) and after binding to biotinylated synthetic Tat (amino acids 1–72) or to beads alone. Increasing amounts of <i>in vitro</i> translated RSK2 were included in the binding reaction. (B) Kinase assay of endogenous RSK2 immunoprecipitated from Cos7 cells transfected with wild type Tat/FLAG, TatF38A/FLAG, or empty vector. Values are means±SEM of four experiments. (C) Western blotting of nuclear extracts isolated from Cos7 cells cotransfected with RSK2/HA and Tat/FLAG or with RSK2/HA and Tat F38A/FLAG constructs. Densitometric quantification of the phospho-S227-specific bands was performed using the NIH Image software. (D) Chromatin immunoprecipitation analysis of the Jurkat T cell line A2, latently infected with an HIV-based lentiviral vector expressing Tat/FLAG from the HIV LTR after treatment with TNF-α. At indicated time points, cells were harvested and immunoprecipitations were performed in duplicate with α-phospho-S227 antibodies followed by PCR with primers specific for the HIV LTR or the c-fos gene.</p