Phosphorylation of AZT-resistant human immunodeficiency virus type 1 reverse transcriptase by casein kinase II in vitro: effects on inhibitor sensitivity.
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.
Lazaro
J B
JB
Department BCMP, Harvard Medical School, Boston, Massachusetts, USA.
Boretto
J
J
Selmi
B
B
Capony
J P
JP
Canard
B
B
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D047390:Casein Kinase II; D016384:Consensus Sequence / Q000235:genetics; D004247:DNA / Q000096:biosynthesis; D004260:DNA Repair; D054306:Dideoxynucleotides; D004352:Drug Resistance, Microbial; D004789:Enzyme Activation; D017245:Foscarnet / Q000494:pharmacology; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D009154:Mutation; D010449:Peptide Mapping; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis / Q000378:metabolism; D010769:Phosphothreonine / Q000032:analysis / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000494:pharmacology; D015215:Zidovudine / Q000031:analogs & derivatives / Q000494:pharmacology*
D054306:Dideoxynucleotides; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; D010769:Phosphothreonine; D010768:Phosphoserine; D017245:Foscarnet; D015215:Zidovudine; C052873:zidovudine triphosphate; D004247:DNA; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D054303:HIV Reverse Transcriptase; D010744:Phosphoric Monoester Hydrolases