phosphothreonine and zidovudine-triphosphate

Casein kinase II (CKII) phosphorylates wild-type (WT) recombinant reverse transcriptase (RT) mainly in the p66 subunit in vitro. Phosphorylation of T215F RT and D67N/K70R/T215F/K219Q RT (AZT-resistant RT) in vitro increases discrimination against AZTTP 2. 5- and 3.6-fold, respectively. This in vitro resistance can be reversed by treatment of phosphorylated AZT-resistant RT with phosphatase. Phosphorylation has no effect on WT RT. Terminal transferase activity of RT is selectively suppressed on phosphorylated AZT-resistant RT. Resistance to phosphonoformic acid (PFA, foscarnet) increases 3-fold upon phosphorylation of AZT-resistant RT. Although T215, the most important residue for AZT-resistance, is part of a CKII consensus target site, serines are primarily phosphorylated relative to threonines. Mutational analysis shows that phosphorylation can be reduced to 10% that of WT when amino-acid changes are introduced both in the "fingers" subdomain and motif D. These results suggest that phosphorylation of RT might be one factor involved in drug resistance in vivo.

Topics: Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Casein Kinase II; Consensus Sequence; Dideoxynucleotides; DNA; DNA Repair; Drug Resistance, Microbial; Enzyme Activation; Foscarnet; HIV Reverse Transcriptase; HIV-1; Humans; Mutation; Peptide Mapping; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Serine-Threonine Kinases; Recombinant Proteins; Reverse Transcriptase Inhibitors; Substrate Specificity; Thymine Nucleotides; Zidovudine