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. 1998 Aug 4;95(16):9331-6.
doi: 10.1073/pnas.95.16.9331.

A role for the actin-bundling protein L-plastin in the regulation of leukocyte integrin function

S L Jones  1 , J Wang, C W Turck, E J Brown
Affiliations

A role for the actin-bundling protein L-plastin in the regulation of leukocyte integrin function

S L Jones et al. Proc Natl Acad Sci U S A. .

Abstract

Regulation of leukocyte integrin avidity is a crucial aspect of inflammation and immunity. The actin cytoskeleton has an important role in the regulation of integrin function, but the cytoskeletal proteins involved are largely unknown. Because inflammatory stimuli that activate integrin-mediated adhesion in human polymorphonuclear neutrophils (PMN) and monocytes cause phosphorylation of the actin-bundling protein L-plastin, we tested whether L-plastin phosphorylation was involved in integrin activation. L-plastin-derived peptides that included the phosphorylation site (Ser-5) rapidly induced leukocyte integrin-mediated adhesion when introduced into the cytosol of freshly isolated primary human PMN and monocytes. Substitution of Ala for Ser-5 abolished the ability of the peptide to induce adhesion. Peptide-induced adhesion was sensitive to pharmacologic inhibition of phosphoinositol 3-kinase and protein kinase C, but adhesion induced by a peptide containing a phosphoserine at position 5 was insensitive to inhibition. These data establish a novel role for L-plastin in the regulation of leukocyte adhesion and suggest that many signaling events implicated in integrin regulation act via induction of L-plastin phosphorylation.

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Figures

Figure 1
Figure 1
The domain structure of the fimbrin family and the N-terminal amino acid sequence of LPL and TPL. The fimbrin family is characterized by two EF hand calcium-binding domains in the N-terminal headpiece region and two α-actinin-type actin-binding domains. The sequence of amino acids 1–21 of LPL and TPL is in brackets. The sequence used to derive the LPLtat peptide is underlined. Ser-5, which is critical for LPL phosphorylation, is in bold. Note that TPL, which has not been shown to be phosphorylated, has a Gln at position 5.
Figure 2
Figure 2
The same proximal pathways that activate αMβ2-mediated adhesion induce phosphorylation of serine 5 of LPL. (A) PMN loaded with the fluorophore calcein were treated with control buffer, wortmannin (100 nM), or LY294002 (25 μM) before measurement of adhesion to IC (30 min) or fMLP- or PMA-induced adhesion to FCS (3 min). The data are the mean ± SE of triplicate wells, reported as attachment index (AI), which is the percentage of cells that remain adherent after washing. Wortmannin and LY294002 significantly inhibited adhesion to IC, but did not affect fMLP- or PMA-stimulated adhesion to FCS (<0.05). The data are representative of three separate experiments. (B) PMN loaded with [32P]phosphoric acid were treated with control DMSO, wortmannin (100 nM), or LY294002 (25 μM) and allowed to adhere to plates coated with IC or FCS or stimulated in suspension with fMLP (100 nM) or PMA (50 ng/ml) for 20 min at 37°C. LPL was immunoprecipitated and phosphorylation was quantitated by densitometry of autoradiograms. Data are normalized to maximal LPL phosphorylation induced by PMA. Each point represents the average of two separate experiments. (C) LPL or S5A, S7A, or S5A/S7A mutants of LPL were expressed in HeLa cells. After 6 hr of infection, the cells were loaded with [32P]phosphoric acid and treated with buffer control or the serine phosphatase inhibitor okadaic acid (1 μM) for 30 min at 37°C. LPL phosphorylation (C Upper) was assayed as in B. Loading of LPL was assessed by Coomassie blue stain (C Lower). Results are representative of three separate experiments.
Figure 3
Figure 3
Cell-permeant peptides from the amino terminus of LPL activate leukocyte integrin-mediated adhesion. To measure peptide effects on adhesion, purified PMN (A, B, D, and E) or monocytes (C) loaded with calcein were added to microtiter-plate wells coated with FCS followed by the addition of peptides with or without PMA (50 ng/ml) and incubated for 15 min at 37°C. (A) Peptide dose response. (B and C) One hundred micromoler of various peptides. The data are the mean ± SE of triplicate wells. Results are representative of at least three separate experiments. (D) Effects of cytochlasin D (10 μg/ml), anti-β2 F(ab′)2 mAb IB4, CBRM1/5, or the control anti-β1, anti-β5, or anti-HLA mAbs (all 20 μg/ml) on LPLtat-induced adhesion. (E) Adhesion of purified murine bone marrow PMN from wt (+/+) or CD18 (β2 integrin)-deficient (−/−) mice. (F) PMN were incubated with FITC-conjugated peptides and washed, and total fluorescence was quantitated by flow cytometry after quenching the extracellular fluorescence with the addition of 0.1% trypan blue dye. Data are presented as the mean ± SE fluorescence for three separate experiments.
Figure 4
Figure 4
PI 3-kinase and PKC activity is required for LPLtat, but not S5PO4tat-induced adhesion. (A) Adhesion of PMN treated with DMSO control, wortmannin (100 nM), Gö6976 (50 nM), or LY294002 (25 μM) in response to cell permeant peptides. PMN were allowed to adhere to IC-coated surfaces or the indicated peptides (100 μM) or PMA (50 ng/ml) were added to PMN after allowing the cells to settle on a FCS-coated surface. Each point represents the mean ± SE for triplicate wells. Results are representative of at least three separate experiments. (B) Phosphorylation of endogenous LPL in response to cell-permeant peptides. PMN were treated with DMSO control, wortmannin (100 nM), Gö6976 (100 nM), or LY294002 (25 μM) and then were allowed to adhere to IC or were allowed to settle on an FCS-coated surface before the addition of peptides (100 μM) or PMA (50 ng/ml). LPL phosphorylation was assessed as in Fig. 2. Data are normalized to maximal phosphorylation induced by PMA (50 ng/ml) and represent the average of two separate experiments.
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