JNK-interacting Protein Research Articles

배양한 흰쥐 해마신경세포에서 c-Jun N-terminal kinase (JNK)-interacting protein (JIP)의 표현

c-Jun N-terminal kinase (JNK)-interacting protein 1(JIP1)은 비계단백질(scaffold protein)로서 신경 세포와 ??장<TEX>${\beta}$</TEX>세포에서 많이 발현된다. 본 연구에서는 배양한 흰쥐 해마신경세포에서 JIP1, JIP-2 및 JIP-3을 모두 인식하는 항체를 이용하여 이들의 세포내 표현을 조사하였다. 전반적으로 JIP은 세포체와 가지돌기에 반점 모양으로 표현되었다. 이 JIP 반점들을 통계적으로 분석한 결과 흥분성 연접후표지인 PSD95 및 <TEX>${\alpha}CaMKII$</TEX> 반점 과 각각 <TEX>$54.8{\pm}4.0%$</TEX> 및 <TEX>$94.1{\pm}4.5%$</TEX>가 겹쳐졌다. 반면에 억제성 연접후표지인 그리신수용체 및 gephyrin 반점과는 각각 단지 <TEX>$8.6{\pm}0.5%$</TEX> 및 <TEX>$7.3{\pm}0.5%$</TEX>만 겹쳐졌다. 한편 lipid raft의 표지인 flotillin 반점의 상당부분<TEX>$(29.3{\pm}1.0%)$</TEX>이 JIP 반점과 겹쳐졌다. 또한, JIP은 일부 가지돌기의 끝부분에 매우 강하게 발편되었으며 축삭에는 표현이 미미하였다. 이 결과들은 JIP 단백질이 흥분성 연접후구역, 일부 lipid raft, 그리고 일부 가지돌기 끝부분에 주로 위치함을 의미한다. c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP1), also known as Islet-brain 1 (IB1), is a scaffold protein that is highly expressed in neurons and pancreatic <TEX>${\beta}-cells$</TEX>. In this study subcellular localization of JIP was investigated in cultured rat hippocampal neurons using an antibody that recognize all variants of JIP1, JIP-2 and JIP-3. The overall expression profile of JIP is punctate throughout soma and dendrites. Statistic analysis showed that <TEX>$54.8{\pm}4.0%\;and\;94.1{\pm}4.5%$</TEX> of total JIP immunopuncta overlapped with those of excitatory postsynaptic markers SD-95 and <TEX>${\alpha}Camik$</TEX>, respectively. In contrast, only <TEX>$8.6{\pm}0.5%\;and\;7.3{\pm}0.5%$</TEX> of JIP clusters overlapped with those of inhibitory postsynaptic markers glycine receptor (GlyR) and gephyrin, respectively. JIP clusters overlapped or juxtaposed with SV2 but not GAD, markers for general and inhibitory nerve terminals, respectively. A substantial fraction <TEX>$(29.3{\pm}1.0%)$</TEX> of flotillin immunopuncta, a marker for lipid rafts, clusters overlapped with those of JIP. In addition, JIP was highly expressed in some select ends of dendrites but minimal in axons. These data suggest important roles of JIP in excitatory postsynaptic sites, lipid rafts and dendritic ends.

JNK phosphorylates synaptotagmin-4 and enhances Ca2+-evoked release

Ca2+ influx induced by membrane depolarization triggers the exocytosis of secretory vesicles in various cell types such as endocrine cells and neurons. Peptidyl growth factors enhance Ca2+-evoked release, an effect that may underlie important adaptive responses such as the long-term potentiation of synaptic transmission induced by growth factors. Here, we show that activation of the c-Jun N-terminal kinase (JNK) plays an essential role in nerve growth factor (NGF) enhancement of Ca2+-evoked release in PC12 neuroendocrine cells. Moreover, JNK associated with phosphorylated synaptotagmin-4 (Syt 4), a key mediator of NGF enhancement of Ca2+-evoked release in this system. NGF treatment led to phosphorylation of endogenous Syt 4 at Ser135 and translocation of Syt 4 from immature to mature secretory vesicles in a JNK-dependent manner. Furthermore, mutation of Ser135 abrogated enhancement of Ca2+-evoked release by Syt 4. These results provide a molecular basis for the effect of growth factors on Ca2+-mediated secretion.

SOCS3 suppresses AP-1 transcriptional activity in neuroblastoma cells through inhibition of c-Jun N-terminal kinase

Transduction and activation of an inducible form of STAT3 (signal transducer and activator of transcription) sufficed to increase VIP (vasoactive intestinal protein) mRNA concentrations in neuroblastoma cells. Overexpression of SOCS3 (suppressor of cytokine signaling) inhibited and mutant SOCS3 (with an inactivating point mutation in amino acid 25) enhanced the induction of VIP mRNA by CNTF (ciliary neurotrophic factor). Because mutant SOCS3 did not augment the increase in STAT transcriptional activity following CNTF stimulation, the enhancement by mutant SOCS3 of the actions of CNTF cannot be attributed to changes in STAT3 signaling. Mutant SOCS3 increased AP-1 (activator protein) transcriptional activity and JNK (c-Jun N-terminal kinase) activity and SOCS3 bound to the scaffolding protein, JNK-interacting protein-1: these observations provide a plausible explanation for the enhancement by mutant SOCS3 of the actions of CNTF. We conclude that endogenous SOCS3 inhibits AP-1 activity through blocking of JNK phosphorylation.

Neural-specific ablation of the scaffold protein JSAP1 in mice causes neonatal death

We previously identified c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1, also known as JNK-interacting protein 3) as a scaffolding factor for JNK intracellular signaling pathways. Targeted gene-disruption studies have shown that JSAP1-null mice are unable to breathe and die shortly after birth. Although neural defects might be responsible for their death, there has been no convincing evidence for this. Here we first generated genetically engineered mice carrying a loxP-flanked (floxed) jsap1 gene. To evaluate the validity of this deletion as a jsap1 conditional knockout (KO), we created mice in which the same exon was deleted in all cell lineages, and compared their phenotypes with those of the jsap1 conventional KO mice reported previously. The two KO lines showed indistinguishable phenotypes, i.e., neonatal death and morphological defects in the telencephalon, indicating that the conditional deletion was a true null mutation. We then introduced the floxed jsap1 deletion mutant specifically into the neural lineage, and found that the jsap1 conditional KO mice showed essentially the same phenotypes as the JSAP1-null mice. These results strongly suggest that the neonatal death of jsap1-deficient mice is caused by defects in the nervous system.

Splice variant-specific stabilization of JNKs by IB1/JIP1

Islet-Brain 1 (IB1) (also called JNK-interacting protein 1; JIP1) is a scaffold protein that tethers components of the JNK mitogen-activated protein kinase pathway inducing a modulation of the activity and the target specificity of the JNK kinases. Dysfunctions in IB1 have been associated with diseases such as early type II diabetes. To gain more insight in the functions of IB1, its ability to modulate the expression levels of the various JNK proteins was assessed. Each of the three JNK genes gives rise to several splice variants encoding short or long proteins. The expression levels of the short JNK proteins, but not of the long variants, were systematically higher in rat tissues and in transformed cell lines expressing high IB1 levels compared to tissues and cells with no or low IB1 expression. HEK293 cells bearing a tetracycline-inducible IB1 construct showed a specific increase of the short JNK endogenous splice variants in the presence of tetracycline. The augmented expression level of the short JNK splice variants induced by IB1 resulted from an increased stability towards degradation. Modulation of the stability of specific JNK splice variants represents therefore a newly identified mechanism used by IB1 to regulate the JNK MAPK pathway.

Regulation of apoptosis signal‐regulating kinase 1 degradation by Gα13

Apoptosis signal-regulating kinase (ASK1) is a mitogen-activated protein kinase (MAPK) that transduces apoptotic signals from a variety of stresses. We have shown previously that alpha subunits of heterotrimeric G12 and G13 proteins stimulate ASK1 kinase activity and ASK1-dependent apoptosis. Here, we report a novel mechanism of G-protein-dependent regulation of ASK1. We demonstrated that G alpha13 forms a complex with ASK1 in an activation-independent manner. Both N- and C-terminal regulatory domains of ASK1 were essential for the efficient interaction, while its kinase domain was not required. Formation of the G alpha13-ASK1 complex was enhanced by JNK-interacting leucine zipper protein, JLP. Constitutively activated G alpha13Q226L increased ASK1 expression. Short-term activation of a serotonin 5-HT4 receptor that is coupled to G alpha13 also increased ASK1 expression. Importantly, prolonged activation of 5-HT4 receptor in COS-7 cells or prolonged treatment of human umbilical vein endothelial cells with thrombin concomitantly down-regulated both G alpha13 and ASK1. Data showed that G alpha13Q226L reduced the rate of ASK1 degradation, decreased ASK1 ubiquitination, and reduced association of ASK1 with an E3 ubiquitin ligase CHIP, previously shown to mediate ASK1 degradation. Our findings indicate that ASK1 expression levels can be regulated by G alpha13, at least in part via control of ASK1 ubiquitination and degradation.

T-Lymphokine–Activated Killer Cell–Originated Protein Kinase Functions as a Positive Regulator of c-Jun-NH2-Kinase 1 Signaling and H-Ras–Induced Cell Transformation

T-lymphokine-activated killer cell-originated protein kinase (TOPK) is overexpressed in highly proliferating tumors such as leukemias and myelomas, and seems to play a key role in tumorigenesis or metastasis. However, the precise role and regulatory mechanism explaining the effects of TOPK on tumor cells still remain elusive. Here, we reported that TOPK regulates UVB-induced c-Jun-NH2-kinase 1 (JNK1) activity, and is essential for H-Ras-induced activator protein-1 activity and cell transformation. We showed that TOPK associated with and phosphorylated JNK1 following UVB irradiation in vitro or in vivo. Moreover, UVB-induced JNK1 activity was greatly augmented in mouse epidermal JB6 Cl41 cells that stably expressed TOPK cDNA. On the other hand, JNK1 activity was markedly attenuated by stable expression of small interfering RNA against TOPK in malignant melanoma RPMI 7951 cells. Interestingly, TOPK interacted with JNK-interacting protein 1 and caused an elevation of JNK-interacting protein 1 scaffolding activity, thereby enhancing JNK1 activity. Furthermore, JNK1 was required for TOPK-mediated activator protein-1 transcriptional activity and transformed foci induced by UVB or H-Ras. Taken together, these findings showed that TOPK positively modulated UVB-induced JNK1 activity and played a pivotal role in JNK1-mediated cell transformation induced by H-Ras. These studies might also provide a novel molecular mechanism for the role of TOPK in UVB-mediated skin carcinogenesis.

Regulation of the Pro-apoptotic Scaffolding Protein POSH by Akt

POSH (Plenty of SH3 domains) binds to activated Rac and promotes apoptosis by acting as a scaffold to assemble a signal transduction pathway leading from Rac to JNK activation. Overexpression of POSH induces apoptosis in a variety of cell types, but apoptosis can be prevented by co-expressing the pro-survival protein kinase Akt. We report here that POSH is a direct substrate for phosphorylation by Akt in vivo and in vitro, and we identify a major site of Akt phosphorylation as serine 304 of POSH, which lies within the Rac-binding domain. We further show that phosphorylation of POSH results in a decreased ability to bind activated Rac, as does phosphomimetic S304D and S304E mutation of POSH. S304D mutant POSH also shows a strongly reduced ability to induce apoptosis. These findings identify a novel mechanism by which Akt promotes cell survival.

Mixed lineage kinases (MLKs): a role in dendritic cells, inflammation and immunity?

This review summarizes current knowledge about the mixed lineage kinases (MLKs) and explores their potential role in inflammation and immunity. MLKs were identified initially as signalling molecules in the nervous system. They were also shown to play a role in the cell cycle. Further studies documented three groups of MLKs, and showed that they may be activated via the c-Jun NH(2) terminal kinase (JNK) pathway, and by Rho GTPases. The biochemistry of the MLKs has been investigated in considerable detail. Homodimerization and heterodimerization can occur, and both autophosphorylation and autoinhibition are seen. The interaction between MLKs and JNK interacting protein (JIP) scaffolds, and the resultant effects on mitogen activated protein kinases, have been identified. Clearly, there is some redundancy within the MLK pathway(s), since mice which lack the MLK3 molecule are not abnormal. However, using a combination of biochemical analysis and pharmacological inhibitors, several recent studies in vitro have suggested that MLKs are not only expressed in cells of the immune system (as well as in the nervous system), but also may be implicated selectively in the signalling pathway that follows on toll-like receptor ligation in innate sentinel cells, such as the dendritic cell.

Kinetic Characterization of Human JNK2α2 Reaction Mechanism Using Substrate Competitive Inhibitors

Jun N-terminal kinase (JNK) is a stress activated serine/threonine protein kinase that phosphorylates numerous cellular protein substrates including the transcription factors c-Jun and ATF2. In this study, we defined the kinetic mechanism for the active form of JNK2alpha2. Double reciprocal plots of initial rates versus concentrations of substrate revealed the sequential nature of the JNK2alpha2 catalyzed ATF2 phosphorylation. Dead-end JNK inhibitors were then used to differentiate ordered and random kinetic mechanisms for the reaction. A peptide inhibitor containing the homology JNK docking sequence for substrate recognition, derived from amino acid residues 153-163 of JNK-interacting protein 1 (JIP-1), inhibited JNK activity via competition with ATF2. This peptide functioned as a noncompetitive inhibitor against ATP. In contrast, the anthrapyrazolone compound, SP600125, exhibited competitive inhibition for ATP and noncompetitive inhibition against ATF2. Furthermore, binding of one substrate had no significant effect on the affinity for the other substrate. The data in this study are consistent with a kinetic mechanism for activated JNK2alpha2 in which (1) substrate binding is primarily due to the distal contacts in the JNK2alpha2 docking groove that allow the delivery of the substrate phosphorylation sequence into the catalytic center, (2) there is minimal allosteric communication between the protein-substrate docking site and the ATP binding site in the catalytic center for activated JNK2alpha2, and (3) the reaction proceeds via a random sequential mechanism.

The novel cargo Alcadein induces vesicle association of kinesin-1 motor components and activates axonal transport

Alcadeinalpha (Alcalpha) is an evolutionarily conserved type I membrane protein expressed in neurons. We show here that Alcalpha strongly associates with kinesin light chain (K(D) approximately 4-8x10(-9) M) through a novel tryptophan- and aspartic acid-containing sequence. Alcalpha can induce kinesin-1 association with vesicles and functions as a novel cargo in axonal anterograde transport. JNK-interacting protein 1 (JIP1), an adaptor protein for kinesin-1, perturbs the transport of Alcalpha, and the kinesin-1 motor complex dissociates from Alcalpha-containing vesicles in a JIP1 concentration-dependent manner. Alcalpha-containing vesicles were transported with a velocity different from that of amyloid beta-protein precursor (APP)-containing vesicles, which are transported by the same kinesin-1 motor. Alcalpha- and APP-containing vesicles comprised mostly separate populations in axons in vivo. Interactions of Alcalpha with kinesin-1 blocked transport of APP-containing vesicles and increased beta-amyloid generation. Inappropriate interactions of Alc- and APP-containing vesicles with kinesin-1 may promote aberrant APP metabolism in Alzheimer's disease.

Sensitization of vascular smooth muscle cell to TNF-α-mediated death in the presence of palmitate

Saturated free fatty acids (FFAs), including palmitate, can activate the intrinsic death pathway in cells. However, the relationship between FFAs and receptor-mediated death pathway is still unknown. In this study, we have investigated whether FFAs are able to trigger receptor-mediated death. In addition, to clarify the mechanisms responsible for the activation, we examined the biochemical changes in dying vascular smooth muscle cell (VSMC) and the effects of various molecules to the receptor-mediated VSMC death. Tumor necrosis factor (TNF)-α-mediated VSMC death occurred in the presence of sub-cytotoxic concentration of palmitate as determined by assessing viability and DNA degradation, while the cytokine did not influence VSMC viability in the presence of oleate. The VSMC death was inhibited by the gene transfer of a dominant-negative Fas-associated death domain-containing protein and the baculovirus p35, but not by the bcl-xL or the c-Jun N-terminal kinase (JNK) binding domain of JNK-interacting protein-1, in tests utilizing recombinant adenoviruses. The VSMC death was also inhibited by a neutralizing anti-TNF receptor 1 antibody, the caspase inhibitor z-VAD, and the cathepsin B inhibitor CA074, a finding indicative of the role of both caspases and cathepsin B in this process. Consistent with this finding, caspase-3 activation and an increase in cytosolic cathepsin B activity were detected in the dying VSMC. Palmitate inhibited an increase of TNF-α-mediated nuclear factor kappa B (NF-κB) activity, the survival pathway activated by the cytokine, by hindering the translocation of the NF-κB subunit of p65 from the cytosol into the nucleus. The gene transfer of inhibitor of NF-κB predisposed VSMC to palmitate-induced cell death. To the best of our knowledge, this study is the first report to demonstrate the activation of TNF-α-mediated cell death in the presence of palmitate. The current study proposes that FFAs would take part in deleterious vascular consequences of such patients with elevated levels of FFAs as diabetics and obese individuals via the triggering of receptor-mediated death pathways of VSMC.

The CARMA1-Bcl10 Signaling Complex Selectively Regulates JNK2 Kinase in the T Cell Receptor-Signaling Pathway

Members of the c-Jun NH(2)-terminal kinase (JNK) family play crucial roles in cell activation, differentiation, and apoptosis. Although many studies have indicated that JNK1 and JNK2 have functional differences and redundancy, the upstream signaling pathway that selectively activates JNK1 or JNK2 remains unknown. In this study, we have revealed a selective mechanism of JNK activation, in which JNK2, but not JNK1, was regulated by CARMA1, a scaffold molecule, after stimulation of the T cell receptor (TCR). This CARMA1-dependent regulation of JNK2 worked through the scaffold molecule Bcl10, which was inducibly associated with JNK2 and served as a JNK-interacting protein (JIP)-like scaffold to assemble the kinases JNK2, MKK7, and TAK1. Finally, we showed that CARMA1- and Bcl10-mediated JNK2 activation had a critical role in regulating the amount of c-Jun protein. Together, our studies provide genetic evidence that JNK1 and JNK2 are differentially regulated in the TCR-signaling pathway and play different functions.

Microtubule Acetylation Promotes Kinesin-1 Binding and Transport

Long-distance intracellular delivery is driven by kinesin and dynein motor proteins that ferry cargoes along microtubule tracks . Current models postulate that directional trafficking is governed by known biophysical properties of these motors-kinesins generally move to the plus ends of microtubules in the cell periphery, whereas cytoplasmic dynein moves to the minus ends in the cell center. However, these models are insufficient to explain how polarized protein trafficking to subcellular domains is accomplished. We show that the kinesin-1 cargo protein JNK-interacting protein 1 (JIP1) is localized to only a subset of neurites in cultured neuronal cells. The mechanism of polarized trafficking appears to involve the preferential recognition of microtubules containing specific posttranslational modifications (PTMs) by the kinesin-1 motor domain. Using a genetic approach to eliminate specific PTMs, we show that the loss of a single modification, alpha-tubulin acetylation at Lys-40, influences the binding and motility of kinesin-1 in vitro. In addition, pharmacological treatments that increase microtubule acetylation cause a redirection of kinesin-1 transport of JIP1 to nearly all neurite tips in vivo. These results suggest that microtubule PTMs are important markers of distinct microtubule populations and that they act to control motor-protein trafficking.

Role of the JNK pathway in NMDA-mediated excitotoxicity of cortical neurons

Excitotoxic insults induce c-Jun N-terminal kinase (JNK) activation, which leads to neuronal death and contributes to many neurological conditions such as cerebral ischemia and neurodegenerative disorders. The action of JNK can be inhibited by the D-retro-inverso form of JNK inhibitor peptide (D-JNKI1), which totally prevents death induced by N-methyl-D-aspartate (NMDA) in vitro and strongly protects against different in vivo paradigms of excitotoxicity. To obtain optimal neuroprotection, it is imperative to elucidate the prosurvival action of D-JNKI1 and the death pathways that it inhibits. In cortical neuronal cultures, we first investigate the pathways by which NMDA induces JNK activation and show a rapid and selective phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7), whereas the only other known JNK activator, mitogen-activated protein kinase kinase 4 (MKK4), was unaffected. We then analyze the action of D-JNKI1 on four JNK targets containing a JNK-binding domain: MAPK-activating death domain-containing protein/differentially expressed in normal and neoplastic cells (MADD/DENN), MKK7, MKK4 and JNK-interacting protein-1 (IB1/JIP-1).

C-Jun N-Terminal Kinase Mediates Tumor Necrosis Factor-α Suppression of Differentiation in Myoblasts

The stress kinase c-jun N-terminal kinase (JNK) was recently shown to be involved in the pathophysiology of major inflammatory conditions, including Alzheimer's disease, stroke, obesity, and type II diabetes. However, the role of JNK in regulating inflammatory events in skeletal muscle is only beginning to be explored. IGF-I is the major hormone that promotes muscle growth and development. Here we used a novel, JNK interacting protein (JIP)-derived JNK peptide inhibitor to establish that JNK suppresses the biological activity of IGF-I in skeletal muscle progenitor cells. In these myoblasts, TNFalpha and its downstream receptor substrates, neutral-sphingomyelinase (N-SMase) and N-acetyl-d-sphingosine (C2-ceramide), induce JNK kinase activity in a time-dependent manner. Consistent with these results, TNFalpha induces JNK binding to insulin receptor substrate 1 (IRS-1) but is unable to inhibit IGF-I-induced IRS-1 tyrosine phosphorylation in myoblasts that are treated with the JNK peptide inhibitor. More importantly, JNK activation induced by TNFalpha, C2-ceramide, and N-SMase is associated with reduced expression of the critical muscle transcription factor myogenin as well as the differentiation marker myosin heavy chain (MHC). The JNK peptide inhibitor, but not the control peptide, completely reverses this inhibition of both myogenin and MHC. In the absence of IGF-I, TNFalpha, C2-ceramide, N-SMase and the JNK inhibitor are inactive, as shown by their inability to affect IRS tyrosine phosphorylation and protein expression of myogenin and MHC. These results establish that the resistance of muscle progenitor cells to IGF-I, which is caused by inflammatory stimuli, is mediated by the JNK stress kinase pathway.

Correction: JNK1 phosphorylation of SCG10 determines microtubule dynamics and axodendritic length

Vol. 173 No. 2, April 24, 2006. Pages [265–277][1]. The following errors occur in this paper. All instances of “JIP” alone should read “JNK-interacting protein”, whereas “JIP1” refers specifically to the JIP family protein also known as MAPK8IP1. The legend for Fig. 9 B should read

Blockade of the translocation and activation of mitogen‐activated protein kinase kinase 4 (MKK4) signaling attenuates neuronal damage during later ischemia–reperfusion

Mitogen-activated protein kinase kinase 4 (MKK4), as an upstream activator of c-Jun NH(2)-terminal kinase (JNK), plays a critical role in response to cellular stresses and pro-inflammatory cytokines. In this study, we investigated the subcellular localization and activation of MKK4 in response to global cerebral ischemia. Our results indicated that MKK4 had two activation peaks in both the cytosol and the nucleus, and translocated from the cytosol to the nucleus at 30 min and 6 h of reperfusion. We also detected the interaction of JNK-interacting protein 3 (JIP3) and MKK4, which reached a maximum at 6 h of reperfusion. To elucidate the mechanism of translocation and activation, we administered N-acetylcysteine, an antioxidant reagent, and a glutamate receptor 6 C-terminus-containing peptide (Tat-GluR6-9c) to rats. The data showed that N-acetylcysteine limited the translocation and activation at 30 min of reperfusion; however, the peptide perturbed the subcellular localization and activation at 6 h of reperfusion, and subsequently provided a protective role against delayed neuronal cell death. Taken together, these results demonstrate that the translocation and activation of MKK4 during early reperfusion are closely associated with reactive oxygen species, whereas, at late reperfusion, MKK4 activation may be involved in brain ischemic injury.

Endodermal differentiation of murine embryonic carcinoma cells by retinoic acid requires JLP, a JNK‐scaffolding protein

Retinoic acid (RA) is a morphogen that induces endodermal differentiation of murine P19 embryonic carcinoma cells. RA-induced differentiation of P19 cells has been used as a model system to define the differentiation programs of pluripotent stem cells. Using this system it has been shown that G alpha13--the alpha-subunit of the heterotrimeric G protein G13--and its activation of JNK-module are critically required for the endodermal differentiation of P19 cells. However, the mechanism through which G alpha13 is linked to JNK-module is unknown. Here, we report that RA stimulates the expression of JNK-interacting leucine zipper protein (JLP), a newly identified JNK-scaffolding protein and its critical role in RA-mediated endodermal differentiation. Our results indicate that there is a physical association between JLP and G alpha13 in RA-stimulated P19 cells. More interestingly, silencing JLP abrogates RA-mediated endodermal differentiation of P19 cells analogous to the effects seen with the silencing of G alpha13 or JNK. Therefore, our studies presented here identify for the first time, a novel role for a newly identified scaffolding protein in RA-mediated endodermal differentiation, providing a new signaling conduit to transmit signals from RA to JNK module.

Selective expression of the scaffold protein JSAP1 in spermatogonia and spermatocytes

Scaffold proteins of mitogen-activated protein kinase (MAPK) intracellular signal transduction pathways mediate the efficient and specific activation of the relevant MAPK signaling modules. Previously, our group and others have identified c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1, also known as JNK-interacting protein 3) as a scaffold protein for JNK MAPK pathways. Although JSAP1 is expressed in the testis in adults, its expression during development has not been investigated. In addition, it is unknown which types of cells in the testis express the scaffold protein. Here, we examined the expression of JSAP1 in the testis of mice aged 14 days, 20 days, 6 weeks, and 12 weeks by immunohistochemistry and Western blotting. The specificity of the anti-JSAP1 antibody was evaluated from its reactivity to exogenously expressed JSAP1 and a structurally related protein, and by antigen-absorption experiments. The immunohistochemical analyses with the specific antibody showed that the JSAP1 protein was selectively expressed in the spermatogonia and spermatocytes, but not in other cell types, including spermatids and somatic cells, during development. However, not all spermatogonia and spermatocytes were immunopositive either, especially in the 12-week-old mouse testis. Furthermore, we found by Western blotting that the expression levels of JSAP1 protein vary during development; there is high expression until 6 weeks after birth, which approximately corresponds to the end of the first wave of spermatogenesis. Collectively, these results suggest that JSAP1 function may be important in spermatogenic cells during early postnatal development.