Background: Biological functions of actin-binding protein p57/coronin-1 may be regulated by phosphorylation. Results: Ser-2 and Thr-412 were identified as major phosphorylation sites of p57/coronin-1, and the phosphorylation at Thr-412 reduced the binding affinity for actin. Conclusion: Physical interaction between p57/coronin-1 and actin is regulated by phosphorylation at Thr-412. Significance: The results provide mechanistic insight into the actin-related immunological processes.
The p57/coronin-1 protein is a member of the coronin family of actin-binding proteins, which are characterized by the presence of WD (tryptophan/aspartic acid) repeats and a coiled-coil motif in the molecule. It is selectively expressed in immune cells and has been suggested to play crucial roles in leukocyte functions, including cell migration and phagocytosis. In this study we examined the effects of p57/coronin-1 phosphorylation on the association of the protein with actin. Treatment of HL60 human leukemic cells or p57/coronin-1-transfected HEK293 cells with phorbol 12-myristate 13-acetate (PMA) reduced the association of p57/coronin-1 with the actin cytoskeleton, as indicated by cell fractionation experiments and by fluorescence microscopic observation. Two-dimensional gel electrophoresis of HL60 cell lysate revealed that p57/coronin-1 was phosphorylated upon PMA stimulation of the cells, giving two major and two minor spots of phosphorylated forms, each with distinct isoelectric points. The p57/coronin-1 molecules associated with the cytoskeleton in PMA-treated HL60 cells were phosphorylated at lower levels than those recovered in the cytosolic fraction. In addition, p57/coronin-1 co-sedimented with F-actin polymerized in vitro had lower phosphorylation levels than the molecules remaining in the supernatant. By affinity chromatographic analysis using anti-p57/coronin-1 antibody-conjugated Sepharose, p57/coronin-1 derived from PMA-treated HL60 cells showed lower affinity for actin than that from untreated cells. Finally, recovery of p57/coronin-1 in the actin cytoskeleton-rich fraction from neutrophil-like differentiated HL60 cells decreased during phagocytosis, concomitant with enhanced phosphorylation of p57/coronin-1. These results strongly suggest that the phosphorylation of p57/coronin-1 down-regulates its association with actin and modulates the reorganization of actin-containing cytoskeleton.Actin-binding proteins participate in a variety of leukocyte functions, including chemotaxis, phagocytosis, degranulation, and cellular signaling via reorganization of the actin cytoskeleton. Coronin is an actin-binding protein originally found in Dictyostelium discoideum (1) and has been reported to play important roles in migration, phagocytosis, and cell division of myxomycetes (2-7). Many homologous proteins to coronin were successively identified in various eukaryotes from yeasts to mammals (8). In humans, seven homologues have been identified, and collectively they constitute the coronin protein family. This family is characterized by common structural features, such as five WD (tryptophan-aspartic acid) repeats located at the center of the molecule and a coiled-coil domain at the C terminus. Each coronin member exhibits distinct tissue distribution, suggesting each has a specific function in various actinrelated cellular processes in respective tissues (9). We previously identified p57/coronin-1 as the first mammalian coronin and found its selective expression in immune cells (10). We have also reported tha...
Coronin-1, a hematopoietic cell-specific actin-binding protein, is thought to be involved in the phagocytic process through its interaction with actin filaments. The dissociation of coronin-1 from phagosomes after its transient accumulation on the phagosome surface is associated with lysosomal fusion. We previously reported that 1) coronin-1 is phosphorylated by protein kinase C (PKC), 2) coronin-1 has two phosphorylation sites, Ser-2 and Thr-412, and 3) Thr-412 of coronin-1 is phosphorylated during phagocytosis. In this study, we examined which PKC isoform is responsible for the phosphorylation of coronin-1 at Thr-412 by using isotype-specific PKC inhibitors and small interfering RNAs (siRNAs). Thr-412 phosphorylation of coronin-1 was suppressed by Gö6976, an inhibitor of PKCα and PKCβI. This phosphorylation was attenuated by siRNA for PKCα, but not by siRNA for PKCβ. Furthermore, Thr-412 of coronin-1 was phosphorylated by recombinant PKCα in vitro , but not by recombinant PKCβ. We next examined the effects of Gö6976 on the intracellular distribution of coronin-1 in HL60 cells during phagocytosis. The confocal fluorescence microscopic observation showed that coronin-1 was not dissociated from phagosomes in Gö6976-treated cells. These results indicate that phosphorylation of coronin-1 at Thr-412 by PKCα regulates intracellular distribution during phagocytosis.
Coronin‐1, a hematopoietic cell‐specific actin‐binding protein, is thought to be involved in phagocytic process through its interaction with actin filaments. The dissociation of coronin‐1 from phagosomes after its transient accumulation on the phagosome surface is associated with lysosomal fusion. We previously reported that 1) coronin‐1 is phosphorylated by protein kinase C (PKC), 2) coronin‐1 has two phosphorylation sites, Ser‐2 and Thr‐412, 3) Thr‐412 of coronin‐1 is phosphorylated during phagocytosis. In this study, we examined which PKC isoforms have influence on phosphorylation of coronin‐1 at Thr‐412 using isotype‐specific PKC inhibitors and short interfering RNAs (siRNAs). The phosphorylation of coronin‐1 at Thr‐412 was blocked by Gö6976, an inhibitor of PKCα/β. This phosphorylation was attenuated by PKCα siRNA, but not by PKCβ siRNA. We next analyzed the intracellular distribution of coronin‐1 in Gö6976‐treated HL60 cells during phagocytosis. The confocal fluorescence microscopic observation showed that coronin‐1 was not dissociated from phagosomes. These results indicate that phosphorylation of coronin‐1 at Thr‐412 by PKCα regulates its interaction with actin filaments and intracellular distribution during phagocytosis.
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