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The DNA damage checkpoint arrests cell cycle progression to allow time for repair. Once DNA repair is completed, checkpoint signaling is terminated. Currently little is known about the mechanism by which checkpoint signaling is terminated, and the disappearance of DNA lesions is considered to induce the end of checkpoint signaling; however, here we show that the termination of checkpoint signaling is an active process promoted by Src family tyrosine kinases. Inhibition of Src activity delays recovery from the G2 phase DNA damage checkpoint following DNA repair. Src activity is required for the termination of checkpoint signaling, and inhibition of Src activity induces persistent activation of ataxia telangiectasia mutated (ATM)- and Rad3-related (ATR) and Chk1 kinases. Src-dependent nuclear protein tyrosine phosphorylation and v-Src expression suppress the ATR-mediated Chk1 and Rad17 phosphorylation induced by DNA double strand breaks or DNA replication stress. Thus, Src family kinases promote checkpoint recovery through termination of ATR- and Chk1-dependent G2 DNA damage checkpoint. These results suggest a model according to which Src family kinases send a termination signal between the completion of DNA repair and the initiation of checkpoint termination.

Mimosine is an effective cell synchronization reagent used for arresting cells in late G1 phase. However, the mechanism underlying mimosine-induced G1 cell cycle arrest remains unclear. Using highly synchronous cell populations, we show here that mimosine blocks S phase entry through ATM activation. HeLa S3 cells are exposed to thymidine for 15 h, released for 9 h by washing out the thymidine, and subsequently treated with 1 mM mimosine for a further 15 h (thymidine → mimosine). In contrast to thymidine-induced S phase arrest, mimosine treatment synchronizes >90% of cells at the G1-S phase boundary by inhibiting the transition of the prereplication complex to the preinitiation complex. Mimosine treatment activates ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint signaling without inducing DNA damage. Inhibition of ATM activity is found to induce mimosine-arrested cells to enter S phase. In addition, ATM activation by mimosine treatment is mediated by reactive oxygen species (ROS). These results suggest that, upon mimosine treatment, ATM blocks S phase entry in response to ROS, which prevents replication fork stalling-induced DNA damage.

Protein tyrosine phosphorylation regulates a wide range of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine phosphorylation by Src family kinases (SFKs) induces chromatin structural changes. In this study, we identify KRAB-associated protein 1 (KAP1/TIF1β/TRIM28), a component of heterochromatin, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of KAP1 is induced by several tyrosine kinases, such as Src, Lyn, Abl, and Brk. Among SFKs, Src strongly induces tyrosine phosphorylation of KAP1. Nucleus-targeted Lyn potentiates tyrosine phosphorylation of KAP1 compared with intact Lyn, but neither intact Fyn nor nucleus-targeted Fyn phosphorylates KAP1. Substitution of the three tyrosine residues Tyr-449/Tyr-458/Tyr-517, located close to the HP1 binding-motif, into phenylalanine ablates tyrosine phosphorylation of KAP1. Immunostaining and chromatin fractionation show that Src and Lyn decrease the association of KAP1 with heterochromatin in a kinase activity-dependent manner. KAP1 knockdown impairs the association of HP1α with heterochromatin, because HP1α associates with KAP1 in heterochromatin. Intriguingly, tyrosine phosphorylation of KAP1 decreases the association of HP1α with heterochromatin, which is inhibited by replacement of endogenous KAP1 with its phenylalanine mutant (KAP1-Y449F/Y458F/Y517F, KAP1-3YF). In DNA damage, KAP1-3YF repressed transcription of p21. These results suggest that nucleus-localized tyrosine kinases, including SFKs, phosphorylate KAP1 at Tyr-449/Tyr-458/Tyr-517 and inhibit the association of KAP1 and HP1α with heterochromatin.

Rab27, a small GTPase, is generally recognized as an important regulator of secretion that interacts with Rab27-specific effectors to regulate events in a wide variety of cells, including endocrine and exocrine cells. However, the mechanisms governing the spatio-temporal regulation of GTPase activity of Rab27 are not firmly established, and no GTPase-activating protein (GAP) specific for Rab27 has been identified in secretory cells. We previously showed that expression of EPI64, a Tre-2/Bub2/Cdc16 (TBC)-domain-containing protein, in melanocytes inactivates endogenous Rab27A on melanosomes (Itoh, T., and Fukuda, M. (2006) J. Biol. Chem. 281, 31823-31831), but the EPI64 role in secretory cells has never been investigated. In this study, we investigated the effect of EPI64 on Rab27 in isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Subcellular fractionation and immunohistochemical analyses indicated that EPI64 was enriched on the apical plasma membrane of parotid acinar cells. We found that an antibody against the TBC/Rab-GAP domain of EPI64 inhibited the reduction in levels of the endogenous GTP-Rab27 in streptolysin-O-permeabilized parotid acinar cells and suppressed amylase release in a dose-dependent manner. We also found that the levels of EPI64 mRNA and EPI64 protein increased after IPR stimulation, and that treatment with actinomycin D or antisense-EPI64 oligonucleotides suppressed the increase of EPI64 mRNA/EPI64 protein and the amount of amylase released. Our findings indicated that EPI64 acted as a physiological Rab27-GAP that enhanced GTPase activity of Rab27 in response to IPR stimulation, and that this activity is required for IPR-induced amylase release.

Because Varp (VPS9-ankyrin-repeat protein)/Ankrd27 specifically binds two small GTPases, Rab32 and Rab38, which redundantly regulate the trafficking of melanogenic enzymes in mammalian epidermal melanocytes, it has recently been implicated in the regulation of trafficking of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes. However, the functional interaction between Rab32/38 and Varp and the involvement of the VPS9 domain (i.e. Rab21-GEF domain) in Tyrp1 trafficking have never been elucidated. In this study, we succeeded in identifying critical residues of Rab32/38 and Varp that are critical for the formation of the Rab32/38·Varp complex by performing Ala-based site-directed mutagenesis, and we discovered that a conserved Val residue in the switch II region of Rab32(Val-92) and Rab38(Val-78) is required for Varp binding activity and that its point mutant, Rab38(V78A), does not support Tyrp1 trafficking in Rab32/38-deficient melanocytes. We also identified two critical residues for Rab32/38 binding in the Varp ANKR1 domain and demonstrated that their point mutants, Varp(Q509A) and Varp(Y550A), do not support peripheral melanosomal distribution of Tyrp1 in Varp-deficient cells. Interestingly, the VPS9 domain point mutants, Varp(D310A) and Varp(Y350A), did support Tyrp1 trafficking in Varp-deficient cells, and knockdown of Rab21 had no effect on Tyrp1 distribution. We also found evidence for the functional interaction between a vesicle SNARE VAMP7/TI-VAMP and Varp in Tyrp1 trafficking. These results collectively indicated that both the Rab32/38 binding activity and VAMP7 binding activity of Varp are essential for trafficking of Tyrp1 in melanocytes but that activation of Rab21 by the VPS9 domain is not necessary for Tyrp1 trafficking.

The recently identified molecule aquaporin-11 (AQP11) has a unique amino acid sequence pattern that includes an Asn-Pro-Cys (NPC) motif, corresponding to the N-terminal Asn-Pro-Ala (NPA) signature motif of conventional AQPs. In this study, we examined the effect of the mutation of the NPC motif on the subcellular localization, oligomerization, and water permeability of AQP11 in transfected mammalian cells. Furthermore, the effect was also assessed using zebrafish. Site-directed mutation at the NPC motif did not affect the subcellular localization of AQP11 but reduced its oligomerization. A cell swelling assay revealed that cells expressing AQP11 with a mutated NPC motif had significantly lower osmotic water permeability than cells expressing wild-type AQP11. Zebrafish deficient in endogenous AQP11 showed a deformity in the tail region at an early stage of development. This phenotype was dramatically rescued by injection of human wild-type AQP11 mRNA, whereas the effect of mRNA for AQP11 with a mutated NPC motif was less marked. Although the NPA motif is known to be important for formation of water-permeable pores by conventional AQPs, our observations suggest that the corresponding NPC motif of AQP11 is essential for full expression of molecular function.

To investigate a cDNA encoding cation current, we isolated an alternatively spliced form of a rat Trp3, designated Trp3sv. Trp3sv encodes 736 amino acids with a unique N terminus and six transmembrane segments. Expression of the cRNA in Xenopus oocytes was successfully performed. The cation selective current appeared after the addition of ionomycin or induced by prolonged depolarization but not by hyperpolarization. This induction was not observed by a treatment with thapsigargin, phorbol ester, or ATP. Na(+), K(+), tetraethylammonium, and divalent cations were permeable, while N-methylglucamine and chloride were nominally impermeable ions. The currents were not inhibited by flufenamate ruthenium red but nonspecifically by 2 mm Gd(3+). Northern as well as Western blot suggested lower levels of the expression observed in some organs, while reverse transcriptase-polymerase chain reaction suggested that it widely spread among various organs. Therefore, we may conclude that N-terminal spliced valiant of Trp3, Trp3sv, encodes a calcium-activated cation channel in various organs.

Endothelin-1 (ET-1) can stimulate insulin-responsive glucose transporter (GLUT4) translocation in 3T3-L1 adipocytes (Wu-Wong, J. R., Berg, C. E., Wang, J., Chiou, W. J., and Fissel, B. (1999) J. Biol. Chem. 274, 8103-8110), and in the current study, we have evaluated the signaling pathway leading to this response. First, we inhibited endogenous Galpha(q/11) function by single-cell microinjection using anti-Galpha(q/11) antibody or RGS2 protein (a GTPase activating protein for Galpha(q)) followed by immunostaining to quantitate GLUT4 translocation in 3T3-L1 adipocytes. ET-1-stimulated GLUT4 translocation was markedly decreased by 70 or 75% by microinjection of Galpha(q/11) antibody or RGS2 protein, respectively. Pretreatment of cells with the Galpha(i) inhibitor (pertussis toxin) or microinjection of a Gbetagamma inhibitor (glutathione S-transferase-beta-adrenergic receptor kinase (GST-BARK)) did not inhibit ET-1-induced GLUT4 translocation, indicating that Galpha(q/11 )mediates ET-1 signaling to GLUT4 translocation. Next, we found that ET-1-induced GLUT4 translocation was inhibited by the phosphatidylinositol (PI) 3-kinase inhibitors wortmannin or LY294002, but not by the phospholipase C inhibitor U-73122. ET-1 stimulated the PI 3-kinase activity of the p110alpha subunit (5.5-fold), and microinjection of anti-p110alpha or PKC-lambda antibodies inhibited ET-stimulated GLUT4 translocation. Finally, we found that Galpha(q/11) formed immunocomplexes with the type-A endothelin receptor and the 110alpha subunit of PI 3-kinase and that ET-1 stimulation enhances tyrosine phosphorylation of Galpha(q/11). These results indicate that: 1) ET-1 signaling to GLUT4 translocation is dependent upon Galpha(q/11) and PI 3-kinase; and 2) Galpha(q/11) can transmit signals from the ET(A) receptor to the p110alpha subunit of PI 3-kinase, as does insulin, subsequently leading to GLUT4 translocation.

A new member of the aquaporin (AQP) family has been identified from rat testis. This gene, referred as aquaporin 7 (AQP7), encodes a 269-amino acid protein that contained the conserved NPA motifs of MIP family proteins. AQP7 has the amino acid sequence homology with other aquaporins ( approximately 30%), and it is highest with AQP3 (48%), suggesting that both AQP3 and AQP7 belong to a subfamily in the MIP family. Injection of AQP7-cRNA into Xenopus oocytes expressed a 26-kDa protein detected by immunoblotting. The expression of AQP7 in oocytes stimulated the osmotic water permeability by 10-fold which was not inhibited by 0.3 mM mercury chloride. The Arrhenius activation energy for the stimulated water permeability was low (2.1 kcal/mol). AQP7 also facilitated glycerol and urea transport by 5- and 9-fold, respectively. The activation energy for glycerol was also low (5.3 kcal/mol after the correction of the endogenous glycerol permeability of oocytes). Northern blot analysis revealed a 1.5-kilobase pair transcript expressed abundantly in testis. In situ hybridization of testis revealed the expression of AQP7 at late spermatids in seminiferous tubules. The immunohistochemistry of testis localized the AQP7 expression at late spermatids and at maturing sperms. AQP7 may play an important role in sperm function.

Human aquaporin 3 (AQP3) gene was isolated, and its structural organization was characterized. The gene appeared to exist as a single copy in the human genome and to comprise six exons distributing over 7 kilobases. The sizes of the exons are 171, 127, 138, 119, 218, and 1035 base pairs, and those of introns are approximately 3530, 300, 350, 330, and 90 base pairs, respectively. The initiation site of transcription was identified to locate 64 base pairs upstream of the first ATG codon by primer extension analysis and ribonuclease protection assay. The 5'-flanking region has a TATA box, two Sp1 sequences, and some consensus sequences including AP2 sites. With luciferase assay, the 5'-flanking region was demonstrated to have a promoter activity, which is up-regulated 4-fold by phorbol ester. These findings about the genomic clone of human AQP3 will contribute to elucidate the molecular mechanism of transcriptional regulation of AQP3.

Two species of L-histidine decarboxylase (HDC) mRNA were found in the KU-812-F basophilic cell line, but only the 2.4-kilobase (kb) one encodes the functional HDC (Mamune-Sato, R., Yamauchi, K., Tanno, Y., Ohkawara, Y., Ohtsu, H., Katayose, D., Maeyama, K., Watanabe, T., Shibahara, S., and Takishima, T. (1992) Eur. J. Biochem. 209, 533-539). The 3.4-kb one encodes a truncated HDC protein and is also found in human leukemia-derived cell lines HEL and KCL-22. To clarify the mechanisms that regulate transcription of the HDC gene and generate the two species of mRNA, we have isolated genomic DNA clones coding for the HDC from human genomic libraries. Structural analysis of the isolated clones revealed that the human HDC gene is composed of 12 exons spanning approximately 24 kb. Genomic DNA blot analysis suggested that HDC is encoded by a single copy gene. The structural analysis also demonstrated that the heterogeneity of the HDC mRNA is caused by an insertion of the seventh intron sequence and alternative use of the splicing acceptor site at the 12th exon. The transcription start site of the HDC gene and the nucleotide sequences of the promoter and first exon regions were determined. We found a TATA-like sequence, a GC box, four CACC boxes, four GATA consensus sequences, and six leader-binding protein-1 binding motifs in the promoter region of the HDC gene.