Protein Phosphatase-1 Activates CDK9 by Dephosphorylating Ser175

Abstract

The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). CDK9 activity is regulated by protein phosphatase-1 (PP1) which was previously shown to dephosphorylate CDK9 Thr186. Here, we analyzed the effect of PP1 on RNAPII phosphorylation and CDK9 activity. The selective inhibition of PP1 by okadaic acid and by NIPP1 inhibited phosphorylation of RNAPII CTD in vitro and in vivo. Expression of the central domain of NIPP1 in cultured cells inhibited the enzymatic activity of CDK9 suggesting its activation by PP1. Comparison of dephosphorylation of CDK9 phosphorylated by (32P) in vivo and dephosphorylation of CDK9's Thr186 analyzed by Thr186 phospho-specific antibodies, indicated that a residue other than Thr186 might be dephosphorylated by PP1. Analysis of dephosphorylation of phosphorylated peptides derived from CDK9's T-loop suggested that PP1 dephosphorylates CDK9 Ser175. In cultured cells, CDK9 was found to be phosphorylated on Ser175 as determined by combination of Hunter 2D peptide mapping and LC-MS analysis. CDK9 S175A mutant was active and S175D – inactive, and dephosphorylation of CDK9's Ser175 upregulated HIV-1 transcription in PP1-dependent manner. Collectively, our results point to CDK9 Ser175 as novel PP1-regulatory site which dephosphorylation upregulates CDK9 activity and contribute to the activation of HIV-1 transcription.

Role of Protein Phosphatase 1 in Dephosphorylation of Ebola Virus VP30 Protein and Its Targeting for the Inhibition of Viral Transcription

“…Analysis of VP30 Phosphorylation-Phosphorylation of EBOV VP30 in cells was analyzed as described previously (17). Briefly, FLAG-tagged VP30 was expressed in 293T cells or Vero-E6 cells.…”

Feedback regulation mediated by Bcl-2 and DARPP-32 regulates inositol 1,4,5-trisphosphate receptor phosphorylation and promotes cell survival

“…Also described are methods of peptide synthesis, immunoblotting and immunoprecipitation (6), T-cell stimulation by anti-CD3 antibodies and Ca 2+ imaging (6), RNA interference (6), and in vitro phosphatase assays (24). Details of these methods and statistical analysis are in SI Materials and Methods.…”

“…Immune-captured InsP 3 R-1 was incubated with purified PP1 or PP2A enzymes in a phosphatase assay buffer (24). Under these conditions, recombinant PP1 and PP2A retain their substrate specificities (24). PP1, but not PP2A, causes a dose-dependent decrease of InsP 3 R-1 P-Ser1755 (Fig.…”

“…WEHI7.2 cells were pretreated with forskolin to increase InsP 3 R-1 P-Ser1755 levels, and then InsP 3 R1s were immunoprecipitated from cell lysates. Immune-captured InsP 3 R-1 was incubated with purified PP1 or PP2A enzymes in a phosphatase assay buffer (24). Under these conditions, recombinant PP1 and PP2A retain their substrate specificities (24).…”

An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb

“…Such contact between Hexim1 and the Cdk9 activation segment accounts for its inhibition. Importantly, phosphorylation of S175 and T186 in the "T-loop" is required for P-TEFb assembly with Hexim1 and 7SK RNA (21,48,49). Like Hexim1, the HIV viral protein Tat interacts mainly with cyclin T1 and is known to compete with Hexim1 for association with P-TEFb (5,22).…”