1 AbstractThe specific and efficient activation of mitogen-activated protein kinase (MAPK) signaling modules is mediated, at least in part, by scaffold proteins. c-Jun NH 2 -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 G α13 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.

The ADP-ribosylation factor 6 (ARF6) GTPase is important in cytokinesis and localizes to the midbody. However, the mechanism and regulation of ARF6's recruitment to the midbody are largely unknown. Here, we investigated the functions of two binding partners of active ARF6, c-Jun NH 2 -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's localization to the midbody and delayed cytokinesis. These defects were almost completely rescued by wildtype 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.

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 NH 2 -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 fulllength shRNA-resistant mouse JLP rescued the impaired cell migration of the JLPdepleted HeLa cells; whereas, a C-terminal deletion mutant of mouse JLP, which failed to bind the G protein G a13 , showed little or no effect on the cell migration defect. Furthermore, although a constitutively active G a13 enhanced the migration of control HeLa cells, the G a13 -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 G a13 .Key words: cell migration, MAP kinase, p38, RNA interference, scaffold protein.Abbreviations: DMEM, Dulbecco's modified Eagle's medium; EDTA, ethylenediaminetetraacetic acid; GST, glutathione S-transferase; HEK, human embryonic kidney; JLP, JNK-associated leucine zipper protein; JNK, c-Jun NH2-terminal kinase; JSAP1, JNK/stress-activated protein kinase-associated protein 1; KD, knockdown; MAPK, MAP kinase; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; shRNA, short hairpin RNA.MAP kinase (MAPK) cascades transmit signals through the sequential phosphorylation and activation of threetier kinase modules, consisting of MAPK kinase kinase, MAPK kinase and MAPK. Mammalian MAPK signalling pathways play a key role in multiple cellular functions, such as proliferation, migration and apoptosis (1, 2). The specificity of MAPK signalling is mediated, at least in part, by scaffold proteins. Scaffold proteins for MAPK cascades organize the MAPK signalling components into functional modules, thereby enabling the efficient activation of specific MAPK pathways (3-5). The scaffolding complexes also protect the signalling components within the relevant MAPK modules from undesired activation by other signalling molecules in cells.c-Jun NH 2 -terminal kinase (JNK)-associated leucine zipper protein (JLP) was originally identified as a binding protein for the transcription factor Max, and further biochemical study indicated that JLP functions as a scaffold protein for the JNK and p38 MAPK signalling modules (6). JLP is broadly expressed, and is structurally related to JNK/stress-activated protein kinase-associated protein 1 (JSAP1 or JNK-interacting protein 3), a scaffold protein for JNK cascades (7-9).Studies of JLP have mainly focused on identifying its interacting proteins, which include kinesin light chain 1 and G a13 , the a-subunit of the heteromeric G 13 protein (10,11). JLP has also been reported to play an important role in myogenesis by interacting with the cell-surface p...

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 NH 2 -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 UVBinduced 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.