Osamu Higuchi

Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization

Rac is a small GTPase of the Rho family that mediates stimulus-induced actin cytoskeletal reorganization to generate lamellipodia. Little is known about the signalling pathways that link Rac activation to changes in actin filament dynamics. Cofilin is known to be a potent regulator of actin filament dynamics, and its ability to bind and depolymerize actin is abolished by phosphorylation of serine residue at 3 (refs 11, 12); however, the kinases responsible for this phosphorylation have not been identified. Here we show that LIM-kinase 1 (LIMK-1), a serine/threonine kinase containing LIM and PDZ domains, phosphorylates cofilin at Seru20093, both in vitro and in vivo. When expressed in cultured cells, LIMK-1 induces actin reorganization and reverses cofilin-induced actin depolymerization. Expression of an inactive form of LIMK-1 suppresses lamellipodium formation induced by Rac or insulin. Furthermore, insulin and an active form of Rac increase the activity of LIMK-1. Taken together, our results indicate that LIMK-1 participates in Rac-mediated actin cytoskeletal reorganization, probably by phosphorylating cofilin.

Protein-Protein Interaction of Zinc Finger LIM Domains with Protein Kinase C

The LIM domain comprising two zinc-finger motifs is found in a variety of proteins and has been proposed to direct protein-protein interactions. During the identification of protein kinase C (PKC)-interacting proteins by a yeast two-hybrid assay, a novel protein containing three LIM domains, designated ENH, was shown to associate with PKC in an isoform-specific manner. Deletion analysis demonstrated that any single LIM domain of ENH associates with the NH2-terminal region of PKC. ENH associated with PKC in COS-7 cells and was phosphorylated by PKC in vitro. Upon treatment of the cells with phorbol ester, ENH in the membrane fraction was translocated to the cytosol fraction in vivo. Other LIM domain-containing proteins, such as Enigma and LIM-kinase 1, also interacted with PKC through their LIM domains. These results suggest that the LIM domain is one of the targets of PKC and that the LIM-PKC interaction may shed light on undefined roles of LIM domain-containing proteins.

Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2 : studies with PEX5-defective CHO cell mutants

ABSTRACT To investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. PEX5 cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R’s deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of PEX5 complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations inPEX5 were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.

Identification and change in the receptor for hepatocyte growth factor in rat liver after partial hepatectomy or induced hepatitis

Specific binding of 125I-labeled human recombinant HGF to the primary cultured rat hepatocytes or liver plasma membranes was observed to be temperature- and time-dependent. Scatchard analysis indicated the presence of a single class of high affinity receptors with a dissociation constant (Kd) of 24-32 pM, a value in good accord with half maximum dose for HGF activity and a receptor density of about 500-600 sites/cell. Affinity cross-linking of the receptor with 125I-HGF revealed the HGF receptor in rat liver membranes to be a polypeptide of Mr approximately 220,000. After partial hepatectomy, specific binding of 125I-HGF to the membranes of residual livers decreased by 60-70% between 3 and 6 h, and was scanty at 12 h after hepatectomy. After one week, the binding was recovered to the 1.7 fold level in the untreated rat liver. This rapid down-regulation of HGF receptors was also observed in plasma membranes of rat livers in the presence of hepatitis induced by CCl4. We propose that HGF which can be immediately supplied to the liver after hepatic injury will function as a trigger for regeneration of this organ.

Expression of c‐met proto‐oncogene in COS cells induces the signal transducing high‐affinity receptor for hepatocyte growth factor

By transfection of the expression plasmid containing a human c‐met cDNA into COS‐7 cells, high‐affinity binding sites specific for HGF with a K d value of 30 pM were newly detected. Furthermore, only in the c‐met transfected COS‐7 cells, but not in the control COS‐7 cells, DNA synthesis was markedly induced in response to HGF. Thus, transient expression of exogenous c‐met cDNA resulted in the appearance of high‐affinity receptor for HGF and conversion of the normally non‐responsive COS‐7 cells into the HGF‐responsive cells. These results provide evidence for identifying the c‐met product as a signal transducing high‐affinity receptor for HGF.

Serum 1α,25-Dihydroxyvitamin D3 Accumulates into the Fracture Callus during Rat Femoral Fracture Healing

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is thought to be an important systemic factor in the fracture repair process, but the mechanism of action of 1,25(OH)2D3 has not been clearly defined. In this study, the role of 1,25(OH)2D3 in the fracture repair process was analyzed in a rat closed femoral fracture model. The plasma concentration of 1,25(OH)2D3 rapidly decreased on day 3 and continued to decrease to 10 days after fracture. We assessed whether this decrease was based on the accelerated degradation or retardation of the synthesis rate of 1,25(OH)2D3, from 25(OH)D3. After radiolabeled 3H-1,25(OH)2D3 or 3H-25(OH)D3 was injected i.v. into fractured or control (unfractured) rats, the concentrations of 25(OH)D3 and 1,25(OH)2D3 metabolites were measured by HPLC. The plasma concentrations of these radiolabeled metabolites in fractured group were similar to those in control rats early after operation. However, radioactivity in the femurs of fractured rats was higher than that of the control group. Furthermore, the radioactivity was concentrated in the callus of the fractured group analyzed by autoradiography. 1,25(OH)2D3 receptor gene expression was detected early after fracture and, additionally, both in the soft and hard callus on days 7 and 13 after fracture. These results showed that the rapid disappearance of 1,25(OH)2D3 in the early stages after fracture was not due to either increased degradation or decreased synthesis of 1,25(OH)2D3, but rather to increased consumption. Further, these results suggest the possibility that plasma 1,25(OH)2D3 becomes localized in the callus and may regulate cellular events in the process of fracture healing.

Self‐association of LIM‐kinase 1 mediated by the interaction between an N‐terminal LIM domain and a C‐terminal kinase domain

LIM‐kinase 1(LIMK1) and 2 (LIMK2) are members of a novel class of protein kinases containing two LIM motifs at the N‐terminus. The LIM motif is thought to be involved in protein‐protein interactions. We report here evidence that LIMK1 self‐associates and also associates with LIMK2. In vivo and in vitro binding analyses using variously deleted mutants of LIMK1 revealed that the self‐association of LIMK1 was caused by interaction between the N‐terminal LIM domain and the C‐terminal kinase domain. The association of LIMK1 with itself and with LIMK2 is important for understanding how activities and functions of LIMK family kinases are regulated.

Inhibition of activated Ras-induced neuronal differentiation of PC12 cells by the LIM domain of LIM-kinase 1

LIM-kinase 1 and 2 (LIMK1 and LIMK2) are members of a novel class of protein kinases with structures composed of two LIM motifs at the N-terminus and an unusual protein kinase domain at the C-terminus. The cellular functions of the LIMK family proteins have remained unknown. In the present study, we examined effects of LIMKs on neuronal differentiation of PC12 pheochromocytoma cells. Transient expression analyses revealed that LIMK1, in itself, had no apparent effect on PC12 cells, but the oncogenic Ras-induced differentiation of PC12 cells was notably inhibited by co-expression with LIMK1 or LIMK2. A mutant of LIMK1 lacking a protein kinase domain (ΔK) similarly inhibited Ras-induced differentiation of PC12 cells, but a mutant lacking a LIM domain (ΔLIM) failed to do so, indicating that a LIM domain but not a protein kinase domain is required for the inhibitory activity. This notion was further supported by the finding that mutation, changing conserved cysteines involved in zinc coordination to glycines in both of two LIM motifs, abolished the inhibitory activity of ΔK. Additionally, we also found that the constitutively activated MAP kinase kinase (MAPKK)-induced differentiation of PC12 cells was inhibited by co-expression with ΔK. Furthermore, ΔK did not inhibit the kinase activity of MAP kinase (MAPK) stimulated by MAPKK, when co-expressed in COS7 cells. These findings suggest that LIMK1 inhibits neuronal differentiation of PC12 cells, through its LIM domain and by interfering with events downstream of MAPK activation.

Suppression of fibroblast cell growth by overexpression of LIM‐kinase 1

LIM‐kinase 1 (LIMK1) is a serine/threonine kinase containing two LIM motifs at the N‐terminus. The functional role of LIMKI has remained unknown. In this study, we examined the role of LIMK1 in cell growth of fibroblasts. Induced expression of LIMK1 in NIH3T3 cells led to growth retardation. Transfection of LIMK1 sense cDNA into NIH3T3 and H‐ras‐transformed FYJ10 fibroblasts significantly suppressed colony formation of these cells. In contrast, transfection with LIMK1 antisense cDNA strongly stimulated colony formation of the NIH3T3 cells. These findings suggest that LIMK1 functions as a negative regulator of fibroblast cell growth, and may play a role in tumor suppression.