Phosphorylation of intracellular precursors of human IL-1.
The human IL-1 molecules (IL-1 alpha and IL-1 beta) are post-translationally cleaved from 31-kDa precursor to 18-kDa biologically active molecules. During the course of studies of post-translational modifications of human IL-1, we have observed that although LPS induced the production of both intracellular IL-1 alpha and IL-1 beta in human monocytes, [32P]orthophosphate labeling of these cells revealed that intracellular precursor of IL-1 alpha (pre-IL-1 alpha) to be phosphorylated at least 10-fold more than intracellular pre-IL-1 beta. However, no 32P-incorporation could be detected in the 18-kDa processed IL-1 alpha and IL-1 beta. Analysis by TLC revealed that the major phosphorylation site occurred at serine residue(s). The 32P was incorporated into multiply cleaved precursors of IL-1 alpha, which appeared in the absence of protease inhibitors. Since the smallest Mr pre-IL-1 alpha that was labeled with 32P was 22 kDa, the phosphorylated serine residue is presumably located adjacent to a sequence of four basic amino acids located in the 4-kDa region at the amino terminus of the 22-kDa precursor of IL-1 alpha. This serine residue might also be a major phosphorylation site for a cAMP-dependent protein kinase. This hypothesis was substantiated by the demonstration that a synthetic peptide analogue of this region (residue 84 to 112) could be similarly phosphorylated in vitro by a cAMP-dependent protein kinase. Furthermore, a truncated pre-IL-1 alpha (residue 64 to 271) and a "fusion" protein containing staphylococcal protein A and an amino-terminal half-portion of pre-IL-1 alpha (residue 1 to 112), but not mature IL-1 alpha (residue 113 to 271), could also be phosphorylated by cAMP-dependent protein kinase. There is no comparable amino acid sequence in IL-1 beta which could be expected to be phosphorylated by a cAMP-dependent protein kinase. The physiologic relevance of phosphorylation of pre-IL-1 alpha was investigated. The data showed that phosphorylation of truncated pre-IL-1 alpha greatly enhanced its susceptibility to digestion by trypsin and promoted the conversion of pre-IL-1 alpha to the more biologically active IL-1. Although the precise role of the rather selective phosphorylation of pre-IL-1 alpha is not known, our findings do suggest that the phosphorylation of serine close to dibasic/tetrabasic amino acid sequence functions to facilitate the processing and/or release of IL-1 alpha.
Kobayashi
Y
Y
Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick, MD 21701.
Appella
E
E
Yamada
M
M
Copeland
T D
TD
Oppenheim
J J
JJ
Matsushima
K
K
D000595:Amino Acid Sequence; D001826:Body Fluids / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D007375:Interleukin-1 / Q000031:analogs & derivatives / Q000378:metabolism* / Q000502:physiology; D007424:Intracellular Fluid / Q000378:metabolism*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism* / Q000502:physiology; D014357:Trypsin
D007375:Interleukin-1; D010455:Peptides; D011498:Protein Precursors; C043830:interleukin 1 precursor; D011494:Protein Kinases; D014357:Trypsin