[Show abstract][Hide abstract] ABSTRACT: The c-Jun NH(2)-terminal protein kinase (JNK) plays important roles in a broad range of physiological processes. JNK is controlled by two upstream regulators, mitogen-activated protein kinase kinase (MKK) 4 and MKK7, which are activated by various MAPKKKs. Studies employing knockout mice have demonstrated that the JNK signaling pathway is involved in diverse phenomena in the brain, regulating brain development and maintenance as well as animal metabolism and behavior. Furthermore, examination of single or combined knockout mice of Jnk1, Jnk2, and Jnk3 has revealed both functional differences and redundancy among JNK1, JNK2, and JNK3. Phenotypic differences between knockouts of MKK4 and MKK7 have also been observed, suggesting that the JNK signaling pathway in the brain has a complex nature and is intricately regulated. This paper summarizes the functional properties of the major JNK signaling components in the developing and adult brain.
[Show abstract][Hide abstract] ABSTRACT: The stress kinase mitogen-activated protein kinase kinase 7 (MKK7) is a specific activator of c-Jun N-terminal kinase (JNK), which controls various physiological processes, such as cell proliferation, apoptosis, differentiation, and migration. Here we show that genetic inactivation of MKK7 resulted in an extended period of oscillation in circadian gene expression in mouse embryonic fibroblasts. Exogenous expression in cultured mammalian cells of an MKK7-JNK fusion protein that functions as a constitutively active form of JNK induced phosphorylation of PER2, an essential circadian component. Furthermore, JNK interacted with PER2 at both the exogenous and endogenous levels, and MKK7-mediated JNK activation increased the half-life of PER2 protein by inhibiting its ubiquitination. Notably, the PER2 protein stabilization induced by MKK7-JNK fusion protein reduced the degradation of PER2 induced by casein kinase 1ε. Taken together, our results support a novel function for the stress kinase MKK7 as a regulator of the circadian clock in mammalian cells at steady state.
Preview · Article · Jan 2012 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The c-Jun NH(2)-terminal protein kinase (JNK), which belongs to the mitogen-activated protein kinase family, plays important roles in a broad range of physiological processes. JNK is controlled by two upstream regulators, mitogen-activated protein kinase kinase (MKK) 7 and MKK4. To elucidate the physiological functions of MKK7, we used Nestin-Cre to generate a novel mouse model in which the mkk7 gene was specifically deleted in the nervous system (Mkk7(flox/flox) Nestin-Cre mice). These mice were indistinguishable from their control littermates in gross appearance during embryogenesis but died immediately after birth without breathing. Histological examination showed that the mutants had severe defects in brain development, including enlarged ventricles, reduced striatum, and minimal axon tracts. Electron microscopy revealed abnormal accumulations of filamentous structures and autophagic vacuoles in Mkk7(flox/flox) Nestin-Cre brain. Further analysis showed that MKK7 deletion decreased numbers of TAG-1-expressing axons and delayed neuronal migration in the cerebrum. Neuronal differentiation was not altered. In utero electroporation studies showed that contralateral projection of axons by layer 2/3 neurons was impaired in the absence of MKK7. Moreover, MKK7 regulated axon elongation in a cell-autonomous manner in vivo, a finding confirmed in vitro. Finally, phosphorylation levels of JNK substrates, including c-Jun, neurofilament heavy chain, microtubule-associated protein 1B, and doublecortin, were reduced in Mkk7(flox/flox) Nestin-Cre brain. Our findings demonstrate that the phenotype of Mkk7(flox/flox) Nestin-Cre mice differs substantially from that of Mkk4(flox/flox) Nestin-Cre mice, and establish that MKK7-mediated regulation of JNK is uniquely critical for both axon elongation and radial migration in the developing brain.
Full-text · Article · Nov 2011 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: After partial hepatectomy (PH), regenerating liver accumulates unknown lipid species. Here, we analyzed lipids in murine liver and adipose tissues following PH by thin-layer chromatography (TLC), imaging mass spectrometry (IMS), and real-time RT-PCR. In liver, IMS revealed that a single TLC band comprised major 19 TG species. Similarly, IMS showed a single phospholipid TLC band to be major 13 species. In adipose tissues, PH induced changes to expression of genes regulating lipid metabolism. Finally, IMS of phosphatidylcholine species demonstrated distribution gradients in lobules that resembled hepatic zonation. IMS is thus a novel and power tool for analyzing lipid species with high resolution.
No preview · Article · Apr 2011 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Stress-induced Sapk/Jnk signaling is involved in cell survival and apoptosis. Recent studies have increased our understanding of the physiological roles of Jnk signaling in embryonic development. However, still unclear is the precise function of Jnk signaling during gastrulation, a critical step in the establishment of the vertebrate body plan. Here we use morpholino-mediated knockdown of the zebrafish orthologs of the Jnk activators Mkk4 and Mkk7 to examine the effect of Jnk signaling abrogation on early vertebrate embryogenesis. Depletion of zebrafish Mkk4b led to abnormal convergent extension (CE) during gastrulation, whereas Mkk7 morphants exhibited defective somitogenesis. Surprisingly, Mkk4b morphants displayed marked upregulation of wnt11, which is the triggering ligand of CE and stimulates Jnk activation via the non-canonical Wnt pathway. Conversely, ectopic activation of Jnk signaling by overexpression of an active form of Mkk4b led to wnt11 downregulation. Mosaic lineage tracing studies revealed that Mkk4b-Jnk signaling suppressed wnt11 expression in a non-cell-autonomous manner. These findings provide the first evidence that wnt11 itself is a downstream target of the Jnk cascade in the non-canonical Wnt pathway. Our work demonstrates that Jnk activation is indispensable for multiple steps during vertebrate body plan formation. Furthermore, non-canonical Wnt signaling may coordinate vertebrate CE movements by triggering Jnk activation that represses the expression of the CE-triggering ligand wnt11.
No preview · Article · Jul 2010 · Journal of Cellular Biochemistry
[Show abstract][Hide abstract] ABSTRACT: SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase) belongs to the MAPK (mitogen-activated protein kinase) family and is important in many biological contexts. JNK activation is regulated by phosphorylation of specific tyrosine and threonine residues sequentially catalysed by MKK4 and MKK7, which are both dual-specificity MAPKKs (MAPK kinases). Previously, we reported that tyrosine-phosphorylation of JNK by MKK4 precedes threonine-phosphorylation by MKK7, and that both are required for synergistic JNK activation. In the present study, we identify the actin-binding protein-280 (Filamin A) as a presumed 'binder' protein that can bind to MKK7, as well as to MKK4, connecting them in close proximity. We show that Filamin family members A, B and C interact with MKK4 and MKK7, but not with JNK. Filamin A binds to an N-terminal region (residues 31-60) present in the MKK7gamma and MKK7beta splice isoforms, but cannot bind to MKK7alpha which lacks these amino acids. This same N-terminal region is crucial for the intracellular co-localization of MKK7gamma with actin stress fibres and Filamin A. Experiments using Filamin-A-deletion mutants revealed that the MKK7-binding region of Filamin A differs from its MKK4-binding region, and that MKK7gamma (but not MKK7alpha) can form a complex with Filamin A and MKK4. Finally, we used Filamin-A-deficient cells to show that Filamin A enhances MKK7 activation and is important for synergistic stress-induced JNK activation in vivo. Thus Filamin A is a novel member of the group of scaffold proteins whose function is to link two MAPKKs together and promote JNK activation.
Full-text · Article · Feb 2010 · Biochemical Journal
[Show abstract][Hide abstract] ABSTRACT: Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) belongs to the mitogen-activated protein kinase (MAPK) family and plays an important role in many biological contexts. JNK is deeply involved in several serious human disorders, including inflammation, obesity, diabetes, neuronal disease and cancer. Accordingly, JNK has been recognized as an appropriate target for the treatment of these diseases, and much effort has been expended over the past 15 years to isolate JNK inhibitors that can inactivate this kinase. In 2001, the compound SP600125 was reported as the first JNK-specific inhibitor. Many researchers have subsequently employed SP600125 in in vitro and in vivo models to evaluate whether certain disease-associated events are JNK-dependent. Indeed, more than 1300 studies citing the use of SP600125 as a JNK inhibitor in cell cultures and animal models have been reported. However, although SP600125 has been employed successfully to inhibit JNK in several situations, there have been questions about its specificity for JNK. SP600125 can bind to a broad range of protein kinases and inhibit some of them with similar or greater potency than JNK, confirming that many additional kinases may be targets of SP600125. In this article, we review both the usefulness of SP60125 as a JNK inhibitor and the limitations to its specificity.
No preview · Article · Feb 2010 · Current Enzyme Inhibition
[Show abstract][Hide abstract] ABSTRACT: Embryonic stem (ES) cells maintain pluripotency by self-renewal. Several homeoproteins, including Oct3/4 and Nanog, are known to be key factors in maintaining the self-renewal capacity of ES cells. However, other genes required for the mechanisms underlying this process are still unclear. Here we report the identification by in silico analysis of a homeobox-containing gene, CrxOS, that is specifically expressed in murine ES cells and is essential for their self-renewal. ES cells mainly express the short isoform of endogenous CrxOS. Using a polyoma-based episomal expression system, we demonstrate that overexpression of the CrxOS short isoform is sufficient for maintaining the undifferentiated morphology of ES cells and stimulating their proliferation. Finally, using RNA interference, we show that CrxOS is essential for the self-renewal of ES cells, and provisionally identify foxD3 as a downstream target gene of CrxOS. To our knowledge, ours is the first delineation of the physiological role of CrxOS in ES cells.
No preview · Article · Oct 2009 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: The fetal liver serves as the predominant hematopoietic organ until birth. However, the mechanisms underlying this link between hematopoiesis and hepatogenesis are unclear. Previously, we reported the isolation of a monoclonal antibody (anti-Liv8) that specifically recognizes an antigen (Liv8) present in murine fetal livers at embryonic day 11.5 (E11.5). Liv8 is a cell surface molecule expressed by hematopoietic cells in both fetal liver and adult mouse bone marrow. Here, we report that Liv8 is also transiently expressed by hepatoblasts at E11.5. Using protein purification and mass spectrometry, we have identified Liv8 as the CD44 protein. Interestingly, the expression of Liv8/CD44 in fetal liver was completely lost in AML1(-/-) murine embryos, which lack definitive hematopoiesis. These results show that hepatoblasts change from Liv8/CD44-negative to Liv8/CD44-positive status in a hematopoiesis-dependent manner by E11.5, and indicate that Liv8/CD44 expression is an important link between hematopoiesis and hepatogenesis during fetal liver development.
No preview · Article · Feb 2009 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: SP600125 is used as a specific inhibitor of c-Jun N-terminal kinase (JNK). We initially aimed to examine physiological roles of JNK in mast cells that play a central role in
inflammatory and immediate allergic responses. We found that Fc receptor for IgE (FcεRI)-induced degranulation (serotonin
release) and cytokine gene expression [interleukin (IL)-6, tumour necrosis factor-α and IL-13] in bone marrow-derived mast
cells, were almost completely inhibited by SP600125. However, the time course of FcεRI-induced JNK activation did not correlate
with that of serotonin release. Furthermore, FcεRI-induced degranulation and cytokine gene expression were not impaired in
a JNK activator, MKK7-deficient mast cells, in which JNK activation was lost. These results indicate that the inhibitory effects
by SP600125 are not due to impaired JNK activation. Instead, we found that SP600125 markedly inhibited the FcεRI-induced activation
of phosphatidylinositol 3-kinase (PI3K) and Akt, the same as a PI3K inhibitor, wortmannin. Finally, we found that SP600125
specifically inhibits delta form of p110 catalytic subunit (p110δ) of PI3K. Thus, SP600125 exerts its influence on mast cell
functions by inhibiting the kinase activity of PI3K, but not JNK.
No preview · Article · Jan 2009 · Journal of Biochemistry