Purification and characterization of a cytosolic 65-kilodalton phosphoprotein in human leukocytes whose phosphorylation is augmented by stimulation with interleukin 1.
We have recently shown that glucocorticoids dramatically increase the number of interleukin 1 (IL 1) receptors on human peripheral blood mononuclear cells (PBMC) and that IL 1 selectively induces the phosphorylation of a cytosolic 65-kilodalton (kDa) protein (pp 65) in glucocorticoid-pretreated PBMC. We describe here the purification and biochemical characteristics of pp 65. 32P-Labeled pp 65 was purified to homogeneity from the cytosol fraction of IL 1 stimulated [32P]orthophosphate-labeled PBMC by sequential chromatography on Sephacryl S-200, high-performance liquid chromatography (HPLC) anion exchange, and hydroxyapatite HPLC. The purified pp 65 was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The unphosphorylated 65-kDa protein (p 65) was also purified to homogeneity in a similar way. About 40 micrograms of purified 65-kDa protein was recovered from 5 x 10(8) PBMC. Analysis of the amino-terminal sequence of the purified pp 65 revealed the amino terminus of pp 65 to be blocked. Amino acid sequence analysis of a cyanogen bromide cleaved peptide showed pp 65 to be a unique protein whose protein sequence has not yet been reported. Studies of the distribution of p(p) 65 based on Western blotting using specific polyclonal rabbit antibody to p(p) 65 showed that p(p) 65 exists in a variety of cells such as neutrophils, monocytes, B lymphocytes, and myeloid cells. It could not be detected in the T cell leukemia cell line (MOLT), melanoma cells, and fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)
Matsushima
K
K
Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick, Maryland 21701-1013.
Shiroo
M
M
Kung
H F
HF
Copeland
T D
TD
D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007375:Interleukin-1 / Q000502:physiology*; D007962:Leukocytes / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010750:Phosphoproteins / Q000097:blood* / Q000302:isolation & purification; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000494:pharmacology*
D007375:Interleukin-1; D010446:Peptide Fragments; D010750:Phosphoproteins; D011994:Recombinant Proteins; D003488:Cyanogen Bromide