Induction of Sp1 Phosphorylation and NF-κB-Independent HIV Promoter Domain Activity in T Lymphocytes Stimulated by Okadaic Acid

Abstract

In contrast to the purely enhancer-dependent effect of cytokines such as TNF on the activity of the HIV regulatory region (LTR), we observed that okadaic acid (OKA) activates HIV transcription through both the enhancer, responding to the factor NF-kappa B, and the promoter domain of the LTR. The inducibility of HIV LTR-driven luciferase expression constructs in lymphoblastoid cells stimulated by OKA depended on both functional Sp1 binding elements and the ability of the TATA box to bind the protein TBP. In both transformed and normal lymphocytes, OKA stimulation induced intense phosphorylation of the constitutively expressed Sp1 protein in the nucleus, a property of OKA not shared by TNF, phorbol ester, or PHA and interleukin 2. Responsiveness of LTR constructs deleted of kappa B elements to HIV Tat expression was increased upon OKA but not TNF stimulation. Our results suggest that SP1 phosphorylation induced by OKA, a selective inhibitor of the serine-threonine phosphatase PP2A, facilitates the formation of a transcription complex involving general transcription factors, HIV Tat, and Sp1 proteins. The formation of this complex would increase, independently of an in synergy with NF-kappa B, the low basal activity of the HIV LTR observed in normal T lymphocytes.

Protein Phosphatase 2A Activates the HIV-2 Promoter through Enhancer Elements That Include the pets Site

“…This is most likely because of activation of NF-B and Sp1 by OKA as has been reported previously (23,(25)(26)(27). These data support and extend the observations that PP2A phosphatase activity appears to be important in the signaling cascade culminating at the HIV-2 LTR.…”

“…Several published reports implicate PP2A as potentially regulating HIV-1 replication, generally suggesting that this cellular enzyme suppresses HIV-1 transcription or replication (23,(25)(26)(27). Okadaic acid inhibition of PP2A has been shown to activate the HIV-1 LTR, (25)(26)(27).…”

“…PP2A has been shown previously to regulate expression through Sp1 and B sites (23,(25)(26)(27). To test whether these sites play a role in the response of the HIV-2 promoter to PP2A, we transfected U937 cells with plasmids in which the reporter gene (luciferase) was under control of the intact HIV-2 promoter or promoters in which the pets sequence, B site, all upstream enhancer elements (Ϫ107), or the 3Ј Sp1 site were altered or deleted.…”

“…OKA has been reported to activate the HIV-1 LTR by inducing NF-B nuclear translocation and binding to its cognate sites (23)(24)(25)(26) or via an NF-B-independent mechanism involving the Sp1 response elements (26). OKAinduced Sp1-mediated transcription is further enhanced by the presence of Tat (26,27). These studies with OKA suggest the involvement of one or more phosphatases (PP2A or PP1) in regulating transcriptional activation of the HIV-1 LTR.…”

Cooperation of Sp1 and p300 in the induction of the CDK inhibitor p21WAF1/CIP1 during NGF-mediated neuronal differentiation

“…Sp1 has been shown to be the substrate for a double-stranded DNA-dependent protein kinase (Jackson et al, 1990). In addition, Sp1 is phosphorylated in T lymphocytes stimulated with okadaic acid (Vlach et al, 1995) and in vitro by the cAMP-dependent protein kinase (PKA) (Rohl et al, 1997). Finally, phosphorylation of Sp1 occurs during terminal di erentiation in the rat liver and may involve casein kinase II (Legget et al, 1995;Armstrong et al, 1997).…”