JSAP1 and JLP are required for ARF6 localization to the midbody in cytokinesis

13301甲第4104号博士（医学）金沢大学博士論文本文Full 以下に掲載：Genes to Cells 19(9) pp.692-703 2014. MBSJ and Wiley. 共著者：Baljinnyam Tuvshintugs,　Tokiharu Sato, Radnaa, Enkhtuya, Katsumi Yamashita, Katsuji Yoshiok

JSAP1 and JLP are required for ARF6 localization to the midbody in cytokinesis

The ADP-ribosylation factor 6 (ARF6) GTPase is important in cytokinesis and localizes to the midbody. However, the mechanism and regulation of ARF6\u27s recruitment to the midbody are largely unknown. Here, we investigated the functions of two binding partners of active ARF6, c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) and JNK-associated leucine zipper protein (JLP), by gene knockout and rescue experiments in mouse embryonic fibroblasts. Depleting both JSAP1 and JLP impaired ARF6\u27s localization to the midbody and delayed cytokinesis. These defects were almost completely rescued by wild-type JSAP1 or JLP, but not by JSAP1 or JLP mutants that were unable to interact with active ARF6 or with the kinesin heavy chain (KHC) of kinesin-1. In transfected cells, a constitutively active form of ARF6 associated with KHC only when co-expressed with wild-type JSAP1 or JLP and not with a JSAP1 or JLP mutant. These findings suggest that JSAP1 and JLP, which might be paralogous to each other, are critical and functionally redundant in cytokinesis and control ARF6 localization to the midbody by forming a tripartite complex of JSAP1/JLP, active ARF6, and kinesin-1. © 2014 The Authors. Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

The Scaffold Protein c-Jun NH2-Terminal Kinase-associated Leucine Zipper Protein Regulates Cell Migration through Interaction with the G Protein G13

金沢大学がん研究所がん分子細胞制御Scaffold proteins for MAP kinase (MAPK) signalling modules play an important role in the specific and efficient signal transduction of the relevant MAPK cascades. Here, we investigated the function of the scaffolding protein c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP) by depleting it in cultured cells using a short hairpin RNA (shRNA) against human JLP. HeLa and DLD-1 cells stably expressing the shRNA showed a defect in cell migration. The re-expression of full-length shRNA-resistant mouse JLP rescued the impaired cell migration of the JLP-depleted HeLa cells; whereas, a C-terminal deletion mutant of mouse JLP, which failed to bind the G protein Gα13, showed little or no effect on the cell migration defect. Furthermore, although a constitutively active Gα13 enhanced the migration of control HeLa cells, the Gα13-induced cell migration was significantly suppressed in the JLP-depleted HeLa cells. Taken together, these results suggest that JLP regulates cell migration through an interaction with Gα13. © The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

The scaffold protein c-Jun NH2-terminal kinase-associated leucine zipper protein regulates cell migration through interaction wity the G protein Gα13

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The scaffold protein JLP plays a key role in regulating ultraviolet B-induced apoptosis in mice

The ultraviolet B (UVB) component of sunlight can cause severe damage to skin cells and even induce skin cancer. Growing evidence indicates that the UVB-induced signaling network is complex and involves diverse cellular processes. In this study, we investigated the role of c-Jun NH2-terminal kinase-associated leucine zipper protein (JLP), a scaffold protein for mitogen-activated protein kinase (MAPK) signaling cascades, in UVB-induced apoptosis. We found that UVB-induced skin epidermal apoptosis was prevented in Jlp knockout (KO) as well as in keratinocyte-specific Jlp KO mice. Analysis of the repair of UVB-induced DNA damage over time showed no evidence for the involvement of JLP in this process. In contrast, UVB-stimulated p38 MAPK activation in the skin was impaired in both Jlp KO and keratinocyte-specific Jlp KO mice. Moreover, topical treatment of UVB-irradiated mouse skin with a p38 inhibitor significantly suppressed the epidermal apoptosis in wild-type mice, but not in Jlp KO mice. Our findings suggest that JLP in skin basal keratinocytes plays an important role in UVB-induced apoptosis by modulating p38 MAPK signaling pathways. This is the first study to show a critical role for JLP in an in vivo response to environmental stimulation. © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

Ablation of the scaffold protein JLP causes reduced fertility in male mice

金沢大学がん研究所がん分子細胞制御The specific and efficient activation of mitogen-activated protein kinase (MAPK) signaling modules is mediated, at least in part, by scaffold proteins. c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP) was identified as a scaffold protein for JNK and p38 MAPK signaling modules. JLP is expressed nearly ubiquitously and is involved in intracellular signaling pathways, such as the Gα13 and Cdo-mediated pathway, in vitro. To date, however, JLP expression has not been analyzed in detail, nor are its physiological functions well understood. Here we investigated the expression of JLP in the mouse testis during development. Of the tissues examined, JLP was strongest in the testis, with the most intense staining in the elongated spermatids. Since the anti-JLP antibody used in this study can recognize both JLP and sperm-associated antigen 9 (SPAG9), a splice variant of JLP that has been studied extensively in primates, we also examined its expression in macaque testis samples. Our results indicated that in mouse and primate testis, the isoform expressed at the highest level was JLP, not SPAG9. We also investigated the function of JLP by disrupting the Jlp gene in mice, and found that the male homozygotes were subfertile. Taken together, these observations may suggest that JLP plays an important role in testis during development, especially in the production of functionally normal spermatozoa. © 2008 Springer Science+Business Media B.V

JSAP1 and JLP are required for ARF6 localization to the midbody in cytokinesis

Cytokinesis is the final stage of cell division, in which a single cell is physically separated into two daughter cells. The failure of cytokinesis can lead to aneuploidy, which is often associated with cancer. There is growing evidence that membrane trafficking plays an important role during cytokinesis in animal cells (Albertson et al. 2005; Montagnac et al. 2008). Although several small GTPases, including ADP-ribosylation factor 6 (ARF6), are known to regulate membrane trafficking during cytokinesis (D'Souza-Schorey & Chavrier 2006; Schweitzer et al. 2011; Schiel & Prekeris 2013), the mechanisms that control these GTPases spatiotemporally are not well understood. ARF6, like other small GTPases, cycles between an active GTP-bound and an inactive GDP-bound state. Recent studies have shown that ARF6 is crucial for proper cytokinesis, and that it localizes t