title: Constitutive turnover of phosphorylation at Thr-412 of human p57/coronin-1 regulates the interaction with actin.
abstract: The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in hematopoietic cells and plays crucial roles in the immune response through reorganization of the actin cytoskeleton. We previously reported that p57/coronin-1 is phosphorylated by protein kinase C, and the phosphorylation down-regulates the association of this protein with actin. In this study we analyzed the phosphorylation sites of p57/coronin-1 derived from HL60 human leukemic cells by MALDI-TOF-MS, two-dimensional gel electrophoresis, and Phos-tag® acrylamide gel electrophoresis in combination with site-directed mutagenesis and identified Ser-2 and Thr-412 as major phosphorylation sites. A major part of p57/coronin-1 was found as an unphosphorylated form in HL60 cells, but phosphorylation at Thr-412 of p57/coronin-1 was detected after the cells were treated with calyculin A, a Ser/Thr phosphatase inhibitor, suggesting that p57/coronin-1 undergoes constitutive turnover of phosphorylation/dephosphorylation at Thr-412. A diphosphorylated form of p57/coronin-1 was detected after the cells were treated with phorbol 12-myristate 13-acetate plus calyculin A. We then assessed the effects of phosphorylation at Thr-412 on the association of p57/coronin-1 with actin. A co-immunoprecipitation experiment with anti-p57/coronin-1 antibodies and HL60 cell lysates revealed that β-actin was co-precipitated with the unphosphorylated form but not with the phosphorylated form at Thr-412 of p57/coronin-1. Furthermore, the phosphorylation mimic (T412D) of p57/coronin-1 expressed in HEK293T cells exhibited lower affinity for actin than the wild-type or the unphosphorylation mimic (T412A) did. These results indicate that the constitutive turnover of phosphorylation at Thr-412 of p57/coronin-1 regulates its interaction with actin.
authors.affiliation: Department of Microbiology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Tokyo 142-8501, Japan.
authors.forename: Teruaki
authors.initials: T
authors.lastname: Oku
authors.forename: Mai
authors.initials: M
authors.lastname: Nakano
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kaneko
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Ando
authors.forename: Hiroki
authors.initials: H
authors.lastname: Kenmotsu
authors.forename: Saotomo
authors.initials: S
authors.lastname: Itoh
authors.forename: Makoto
authors.initials: M
authors.lastname: Tsuiji
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Seyama
authors.forename: Satoshi
authors.initials: S
authors.lastname: Toyoshima
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Tsuji
mesh_terms: D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D053119:Benzophenanthridines / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D057809:HEK293 Cells; D018922:HL-60 Cells; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010587:Phagocytosis / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012694:Serine / Q000378:metabolism
chemical_list: D000199:Actins; D053119:Benzophenanthridines; D008840:Microfilament Proteins; D050505:Mutant Proteins; D047428:Protein Kinase Inhibitors; D010769:Phosphothreonine; C072061:coronin proteins; D012694:Serine; C016299:chelerythrine; D011493:Protein Kinase C