Infection of Macrophage-Like THP-1 Cells with Mycobacterium avium Results in a Decrease in Their Ability to Phosphorylate Nucleolin

Abstract

ABSTRACT This study of the phosphorylation ability of macrophage-like cells upon infection with Mycobacterium avium was undertaken to establish potential targets of the interference with host response mechanisms. Cytosolic and membrane fractions from noninfected and infected cells were incubated with [γ- 32 P]ATP, in the presence of Mg 2+ and the absence of Ca 2+ , and the patterns of phosphoproteins synthesized were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lower levels of a 110-kDa phosphoprotein were observed in association with cytosolic fractions from mycobacterium-infected cells compared to noninfected cells or cells treated with lipopolysaccharide or having ingested Escherichia coli or killed M. avium . The 110-kDa phosphoprotein was present in the soluble fraction (230,000 × g supernatant) after the kinase incubation, from where it was partially purified and identified as phosphonucleolin by amino acid sequencing. The decrease in nucleolin phosphorylation observed was not related to changes in the cytosolic or membrane levels of this protein, and was detected also in the cytosolic fraction of 32 P-labeled intact cells.