Purification and characterization of a cytosolic 65-kilodalton phosphoprotein in human leukocytes whose phosphorylation is augmented by stimulation with interleukin 1

Abstract

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)

Phosphorylation on Ser5 increases the F-actin-binding activity of L-plastin and promotes its targeting to sites of actin assembly in cells

“…In line with previous data (Lin et al, 1998) phosphorylation of L-plastin is triggered by signals activating immune cells (Henning et al, 1994;Jones and Brown, 1996;Matsushima et al, 1988;Paclet et al, 2004), pathways controlling L-plastin phosphorylation may be constitutively activated in adherent fibroblasts and epithelial cells (Lin et al, 1998). Consistently, constitutive phosphorylation was observed in activated, adherent macrophages and leucocytes (Jones and Brown, 1996;Messier et al, 1993).…”

“…Discussion L-plastin, a major F-actin-bundling protein of leucocytes and epithelial-or mesenchymal-derived cancer cells, is phosphorylated upon activation of pathways triggering adhesion and migration, concomitantly to remodelling the actin (Henning et al, 1994;Matsushima et al, 1988;Shibata et al, 1993;Wang and Brown, 1999). Here, we gained consistent evidence that phosphorylation of L-plastin positively regulates its F-actin-binding and cross-linking activities and that it might function as a regulatory switch in the assembly of actin-rich structures involved in cell migration and signalling.…”

“…Initially detected in leucocytes (Matsushima et al, 1988) and cancer cells derived from solid tissues (Goldstein et al, 1985;Lapillonne et al, 2000;Lin et al, 1993a;Lin et al, 1993b;Park et al, 1994) L-plastin was identified as a closely related isoform of intestinal brush-border fimbrin (de Arruda et al, 1990) and is sometimes referred to as L-fimbrin. Several lines of evidence support an important role for L-plastin in the assembly of the actin cytoskeleton.…”

Interleukin 1 and tumour necrosis factor increase phosphorylation of fibroblast proteins

“…ILl has been reported to cause changes in phosphorylation of proteins in some other cell types; a 65 kDa cytosolic protein in glucocorticoidtreated mononuclear leucocytes [18], a 41 kDa membrane protein in K562 cells [19] and 52, 46 and 20 kDa membrane proteins in mesangial cells [20]. The K562 protein and the 46 kDa mesangial cell protein were phosphorylated on tyrosine, the others on serine and threonine.…”

Interaction of a Dictyostelium member of the plastin/fimbrin family with actin filaments and actin-myosin complexes.

“…The first fimbrin was identified as a major component of the microvilli in the intestinal brush border (Bretscher and Weber, 1980;Bretscher, 1981;Glenney et al, 1981). The sequence of chicken fimbrin (de Arruda et al, 1990) shows a high degree of homology to human L-plastin, a protein phosphorylated in response to growth factors interleukin 1 and interleukin 2 and to phorbol esters (Goldstein et al, 1985;Matsushima et al, 1988). In addition to L-plastin, two isoforms, Tplastin and I-plastin, have been identified in man (Lin et al, 1988(Lin et al, , 1994.…”