Interactions among yeast protein-disulfide isomerase proteins and endoplasmic reticulum chaperone proteins influence their activities

We previously reported that the reductive activities of yeast protein-disulfide isomerase (PDI) family proteins did not completely explain their contribution to the viability of Saccharomyces cerevisiae (Kimura, T., Hosoda, Y., Kitamura, Y., Nakamura, H., Horibe, T., and Kikuchi, M. (2004) Biochem. Biophys. Res. Commun. 320, 359-365). In this study, we examined oxidative refolding activities and found that Mpd1p, Mpd2, and Eug1p exhibit activities of 13.8, 16.0, and 2.16%, respectively, compared with Pdi1p and that activity for Eps1p is undetectable. In analyses of interactions between yeast PDI proteins and endoplasmic reticulum molecular chaperones, we found that Mpd1p alone does not have chaperone activity but that it interacts with and inhibits the chaperone activity of Cne1p, a homologue of mammalian calnexin, and that Cne1p increases the reductive activity of Mpd1p. These results suggest that the interface between Mpd1p and Cne1p is near the peptide-binding site of Cne1p. In addition, Eps1p interacts with Pdi1p, Eug1p, Mpd1p, and Kar2p with dissociation constants (KD) in the range of 10(-7) to 10(-6). Interestingly, co-chaperone activities were completely suppressed in Eps1p-Pdi1p and Eps1p-Mpd1p complexes, although only Eps1p and Pdi1p have chaperone activity. The in vivo consequences of these results are discussed.     

RA-RhoGAP, Rap-activated Rho GTPase-activating protein implicated in neurite outgrowth through Rho

Rap1 and Rho small G proteins have been implicated in the neurite outgrowth, but the functional relationship between Rap1 and Rho in the neurite outgrowth remains to be established. Here we identified a potent Rho GTPase-activating protein (GAP), RA-RhoGAP, as a direct downstream target of Rap1 in the neurite outgrowth. RA-RhoGAP has the RA and GAP domains and showed GAP activity specific for Rho, which was enhanced by the binding of the GTP-bound active form of Rap1 to the RA domain. Overexpression of RA-RhoGAP induced inactivation of Rho for promoting the neurite outgrowth in a Rap1-dependent manner. Knockdown of RA-RhoGAP reduced the Rap1-induced neurite outgrowth. These results indicate that RA-RhoGAP transduces a signal from Rap1 to Rho and regulates the neurite outgrowth.     

PKA-catalyzed phosphorylation of tomosyn and its implication in Ca2+-dependent exocytosis of neurotransmitter

Neurotransmitter is released from nerve terminals by Ca2+-dependent exocytosis through many steps. SNARE proteins are key components at the priming and fusion steps, and the priming step is modulated by cAMP-dependent protein kinase (PKA), which causes synaptic plasticity. We show that the SNARE regulatory protein tomosyn is directly phosphorylated by PKA, which reduces its interaction with syntaxin-1 (a component of SNAREs) and enhances the formation of the SNARE complex. Electrophysiological studies using cultured superior cervical ganglion (SCG) neurons revealed that this enhanced formation of the SNARE complex by the PKA-catalyzed phosphorylation of tomosyn increased the fusion-competent readily releasable pool of synaptic vesicles and, thereby, enhanced neurotransmitter release. This mechanism was indeed involved in the facilitation of neurotransmitter release that was induced by a potent biological mediator, the pituitary adenylate cyclase-activating polypeptide, in SCG neurons. We describe the roles and modes of action of PKA and tomosyn in Ca2+-dependent neurotransmitter release.     

Attenuated phosphorylation of heat shock protein 27 correlates with tumor progression in patients with hepatocellular carcinoma

Heat shock protein 27 (HSP27) is expressed at high levels in human hepatocellular carcinoma (HCC). We examined correlations of total HSP27 and serine phosphorylated (Ser-15, Ser-78, and Ser-82) HSP27 levels in HCC tissues with clinical and pathologic characteristics in 48 resected HCC specimens. The levels of total and Ser-phosphorylated HSP27 were evaluated by Western blot analysis. Immunohistochemical analysis of HSP27 expression was also performed on some samples. Phosphorylation of HSP27 was detected in all 48 HCC tissues. Levels of phosphorylated HSP27 were correlated inversely with tumor size, microvascular invasion of HCC, and tumor stage by TNM classification. In contrast, only microvascular invasion showed an inverse correlation with total HSP27 levels. The decrease in phosphorylated HSP27 in progressed HCC was also observed by immunohistochemistry. Levels of phosphorylated HSP27 gradually decreased in parallel with HCC progression. Our findings suggest that phosphorylated HSP27 may have a suppressive role in progression of human HCC.     

Involvement of LMO7 in the association of two cell-cell adhesion molecules, nectin and E-cadherin, through afadin and alpha-actinin in epithelial cells

Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules that are involved in formation of cadherin-based adherens junctions (AJs). The nectin-based cell-cell adhesion induces activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of AJs through reorganization of the actin cytoskeleton. Although evidence has accumulated that nectins recruit cadherins to the nectin-based cell-cell adhesion sites through their cytoplasm-associated proteins, afadin and catenins, it is not fully understood how nectins are physically associated with cadherins. Here we identified a rat counterpart of the human LIM domain only 7 (LMO7) as an afadin- and alpha-actinin-binding protein. Rat LMO7 has two splice variants, LMO7a and LMO7b, consisting of 1,729 and 1,395 amino acids, respectively. LMO7 has calponin homology, PDZ, and LIM domains. Western blotting revealed that LMO7 was expressed ubiquitously in various rat tissues. Immunofluorescence and immunoelectron microscopy revealed that LMO7 localized at cell-cell AJs, where afadin localized, in epithelial cells of rat gallbladder. In addition, LMO7 localized at the cytoplasmic faces of apical membranes in the same epithelial cells. We furthermore revealed that LMO7 bound alpha-actinin, an actin filament-bundling protein, which bound to alpha-catenin. Immunoprecipitation analysis revealed that LMO7 was associated with both the nectin-afadin and E-cadherin-catenin systems. LMO7 was assembled at the cell-cell adhesion sites after both the nectin-afadin and E-cadherin-catenin systems had been assembled. These results indicate that LMO7 is an afadin- and alpha-actinin-binding protein that connects the nectin-afadin and E-cadherin-catenin systems through alpha-actinin.     

Evidence that tubulobulbar complexes in the seminiferous epithelium are involved with internalization of adhesion junctions

Tubulobulbar complexes may be part of the mechanism by which intercellular adhesion junctions are internalized by Sertoli cells during sperm release. These complexes develop in regions where Sertoli cells are attached to adjacent cells by intercellular adhesion junctions termed ectoplasmic specializations. At sites where Sertoli cells are attached to spermatid heads, tubulobulbar complexes consist of fingerlike processes of the spermatid plasma membrane, corresponding invaginations of the Sertoli cell plasma membrane, and a surrounding cuff of modified Sertoli cell cytoplasm. At the terminal ends of the complexes occur clusters of vesicles. Here we show that tubulobulbar complexes develop in regions previously occupied by ectoplasmic specializations and that the structures share similar molecular components. In addition, the adhesion molecules nectin 2 and nectin 3, found in the Sertoli cell and spermatid plasma membranes, respectively, are concentrated at the distal ends of tubulobulbar complexes. We also demonstrate that double membrane bounded vesicles are associated with the ends of tubulobulbar complexes and nectin 3 is present on spermatids, but is absent from spermatozoa released from the epithelium. These results are consistent with the conclusion that Sertoli cell and spermatid membrane adhesion domains are internalized together by tubulobulbar complexes. PKCalpha, a kinase associated with endocytosis of adhesion domains in other systems, is concentrated at tubulobulbar complexes, and antibodies to endosomal and lysosomal (LAMP1, SGP1) markers label the cluster of vesicles associated with the ends of tubulobulbar complexes. Our results are consistent with the conclusion that tubulobulbar complexes are involved with the disassembly of ectoplasmic specializations and with the internalization of intercellular membrane adhesion domains during sperm release.     

Novel prolyl tri/tetra-peptidyl aminopeptidase from Streptomyces mobaraensis: substrate specificity and enzyme gene cloning

The prolyl peptidase that removes the tetra-peptide of pro-transglutaminase was purified from Streptomyces mobaraensis mycelia. The substrate specificity of the enzyme using synthetic peptide substrates showed proline-specific activity with not only tripeptidyl peptidase activity, but also tetrapeptidyl peptidase activity. However, the enzyme had no other exo- and endo-activities. This substrate specificity is different from proline specific peptidases so far reported. The enzyme gene was cloned, based on the direct N-terminal amino acid sequence of the purified enzyme, and the entire nucleotide sequence of the coding region was determined. The deduced amino acid sequence revealed an N-terminal signal peptide sequence (33 amino acids) followed by the mature protein comprising 444 amino acid residues. This enzyme shows no remarkable homology with enzymes belonging to the prolyl oligopeptidase family, but has about 65% identity with three tripeptidyl peptidases from Streptomyces lividans, Streptomyces coelicolor, and Streptomyces avermitilis. Based on its substrate specificity, a new name, "prolyl tri/tetra-peptidyl aminopeptidase," is proposed for the enzyme.     

NIRF induces G1 arrest and associates with Cdk2

NIRF is a RING finger protein with a ubiquitin-like domain, a PHD finger, a YDG/SRA domain, and a RING finger domain. Previous study showed that NIRF is a nuclear protein expressed in association with cell proliferation. In this study, we further characterized NIRF functions in cell cycle regulation. Flow cytometric analysis showed that overexpression of NIRF induced an increase in G1 phase cells. Immunoprecipitation and immunoblotting experiments showed that NIRF bound to the inactive Cdk2-cyclin E complex. There existed phosphorylated NIRF in cells, and dephosphorylated NIRF interacted with Cdk2. NIRF was phosphorylated by Cdk2 in vitro. These results suggest that NIRF may participate in the G1/S transition regulation.     

Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release

We have recently isolated a novel cytomatrix at the active zone (CAZ)-associated protein, CAST, and found it directly binds another CAZ protein RIM1 and indirectly binds Munc13-1 through RIM1; RIM1 and Munc13-1 directly bind to each other and are implicated in priming of synaptic vesicles. Here, we show that all the CAZ proteins thus far known form a large molecular complex in the brain, including CAST, RIM1, Munc13-1, Bassoon, and Piccolo. RIM1 and Bassoon directly bind to the COOH terminus and central region of CAST, respectively, forming a ternary complex. Piccolo, which is structurally related to Bassoon, also binds to the Bassoon-binding region of CAST. Moreover, the microinjected RIM1- or Bassoon-binding region of CAST impairs synaptic transmission in cultured superior cervical ganglion neurons. Furthermore, the CAST-binding domain of RIM1 or Bassoon also impairs synaptic transmission in the cultured neurons. These results indicate that CAST serves as a key component of the CAZ structure and is involved in neurotransmitter release by binding these CAZ proteins.