TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2

The I kappa B kinase (IKK), consisting of the IKK1 and IKK2 catalytic subunits and the NEMO (also known as IKK gamma) regulatory subunit, phosphorylates I kappa B proteins, targeting them for degradation and thus inducing activation of NF-kappa B (reviewed in refs 1, 2). IKK2 and NEMO are necessary for NF-kappa B activation through pro-inflammatory signals. IKK1 seems to be dispensable for this function but controls epidermal differentiation independently of NF-kappa B. Previous studies suggested that NF-kappa B has a function in the growth regulation of epidermal keratinocytes. Mice lacking RelB or I kappa B alpha, as well as both mice and humans with heterozygous NEMO mutations, develop skin lesions. However, the function of NF-kappa B in the epidermis remains unclear. Here we used Cre/loxP-mediated gene targeting to investigate the function of IKK2 specifically in epidermal keratinocytes. IKK2 deficiency inhibits NF-kappa B activation, but does not lead to cell-autonomous hyperproliferation or impaired differentiation of keratinocytes. Mice with epidermis-specific deletion of IKK2 develop a severe inflammatory skin disease, which is caused by a tumour necrosis factor-mediated, alpha beta T-cell-independent inflammatory response that develops in the skin shortly after birth. Our results suggest that the critical function of IKK2-mediated NF-kappa B activity in epidermal keratinocytes is to regulate mechanisms that maintain the immune homeostasis of the skin.     

Ia binding ligands and cAMP stimulate nuclear translocation of PKC in B lymphocytes

Altered subcellular distribution and activity of protein kinase C (PKC) is associated with transmembrane signalling in a variety of systems in which receptor occupancy leads to increased hydrolysis of polyphosphoinositides. Here we report evidence that in B lymphocytes, cyclic-cAMP-generating signal transduction pathways can activate translocation of PKC from the cytosol to the nucleus. Elevated cAMP levels and translocation of PKC to the nucleus are induced by antibodies against Ia antigens in normal B lymphocytes. Further, cAMP analogues mediate the translocation of PKC to the nucleus of these cells. These findings suggest that in physiological situations, ligation of B-lymphocyte Ia molecules by helper T cells leads to increased cAMP production which in turn causes PKC translocation to the nucleus. In view of recent observations that antibodies against Ia antigens induce differentiation of B cells, we conclude that nuclear PKC may function in the regulation of gene expression.     

Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase

In vertebrate cells, the nuclear entry of Cdc2-cyclin B1 (MPF) during prophase is thought to be essential for the induction and coordination of M-phase events. Phosphorylation of cyclin B1 is central to its nuclear translocation, but the kinases that are responsible remain unknown. Here we have purified a protein kinase from Xenopus M-phase extracts that phosphorylates a crucial serine residue (S147) in the middle of the nuclear export signal sequence of cyclin B1. We have identified this kinase as Plx1 (ref. 16), a Xenopus homologue of Polo-like kinase (Plk)-1. During cell-cycle progression in HeLa cells, a change in the kinase activity of endogenous Plk1 toward S147 and/or S133 correlates with a kinase activity in the cell extracts. An anti-Plk1 antibody depletes the M-phase extracts of the kinase activity toward S147 and/or S133. An anti-phospho-S147 antibody reacts specifically with cyclin B1 only during G2/M phase. A mutant cyclin B1 in which S133 and S147 are replaced by alanines remains in the cytoplasm, whereas wild-type cyclin B1 accumulates in the nucleus during prophase. Co-expression of constitutively active Plk1 stimulates nuclear entry of cyclin B1. Our results indicate that Plk1 may be involved in targeting MPF to the nucleus during prophase.     

Inhibitors of protein kinase C

Protein kinase catalyzes the transfer of the γ-phosphoryl group from ATP to the hydroxyl groups o fprotein side chains, which plays critical roles in signal transduction pathways by transmitting extracellular signals across the plasma membrane and nuclear membrane to the destination sites in the cytoplasm and the nucleus. Protein kinase C (PKC) is a superfamily of phospholipid-dependent Ser/Thr kinase. There are at least 12 isozymes in PKC family.They are distributed in different tissues and play different roles in physiological processes. On account of their concern with a variety of pathophysiologic states, such as cancer,inflammatory conditions, autoimmune disorder, and cardiac diseases, the inhibitors, which can inhibit the activity of PKC and the interaction of cytokine with receptor, and interfere signal transduction pathway, may be candidates of therapeutic drugs. Therefore, intense efforts have been made to develop specific protein kinase inhibitors as biological tools and therapeutic agents. This article reviews the recent development of some of PKC inhibitors based on their interaction with different conserved domains and different inhibition mechanisms.     

Corticosterone activates Erk1/2 mitogen-activated protein kinase in primary hippocampal cells through rapid nongenomic mechanism

Nongenomic effects of glucocorticoids (GC) in various cell types have been well documented, but it still remains unknown whether the mechanism also works in hippocampus which is a crucial target of glucocorticoids in neural system during physiological and/or pathophysiological processes. We present here that corticosterone (B) could rapidly activate Erk1/2 mitogen-activated protein kinase (MAPK) in primarily cultured hippocampal cells within minutes, with a bell-shaped time dependent curve which peaked at 15min and then went down to normal level in 30 min. This activation was blocked by protein kinase C (PKC) inhibitor (Go6976), G protein inhibitor (GDPβs), and MEK(MAPK/extracellular signal-regulated kinase kinase) inhibitor(PD98059), but not by protein kinase A (PKA) inbibitor (H89), tyrosine kinase inhibitor (genistein), and glucocorticoid receptor ( GR ) antagonist (RU38486). Thus, the rapid activation of Erk1/2 MAPK in primary hippocampal cells induced by B was likely mediated by a G protein coupled receptor (GPCR) pathway with involvement of PKC, which belonged to the nongenomic rather than genomic mechanism of GC' s effects.     

Roles of protein kinase C in oocyte meiotic maturation and fertilization

Protein kinase C (PKC) is a superfamily of Ser/Thr protein kinases that is distributed widely in eukaryotes. It plays key regulatory roles at multiple steps of oocyte meiotic maturation and fertilization. During the process of meiotic maturation, the activation of PKC in cumulus cells stimulates meiotic maturation, whereas the activation of PKC in oocytes results in the inhibition of germinal vesicle breakdown. PKC activity increases following the meiotic maturation, and decreases at the transition of metaphase/anaphase in meiosis I, so as to facilitate the release of the first polar body and the entry of meiosis II. In fertilization of mammalian oocytes, PKC may act as one of the downstream targets of Ca2+ to stimulate the cortical granule exocytosis, release the oocytes from MII arrest and to induce pronucleus formation. PKC is also involved in the regulation of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Several PKC isoforms have been identified in mammalian oocytes, and there is evidence showing that classical PKCs may be the principal mediator of oocyte cortical reaction.     

In vitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase.

The protein kinase MAP kinase, also called MAP2 kinase, is a serine/threonine kinase whose activation and phosphorylation are induced by a variety of mitogens, and which is thought to have a critical role in a network of protein kinases in mitogenic signal transduction. A burst in kinase activation and protein phosphorylation may also be important in triggering the dramatic reorganization of the cell during the transition from interphase to mitosis. The interphase-metaphase transition of microtubule arrays is under the control of p34cdc2 kinase, a central control element in the G2-M transition of the cell cycle. Here we show that a Xenopus kinase, closely related to the mitogen-activated mammalian MAP kinase, is phosphorylated and activated during M phase of meiotic and mitotic cell cycles, and that the interphase-metaphase transition of microtubule arrays can be induced by the addition of purified Xenopus M phase-activated MAP kinase or mammalian mitogen-activated MAP kinase to interphase extracts in vitro.     

坚果改善慢性应激致抑郁症大鼠行为学表现及其机制研究

为探讨坚果(核桃与甜杏仁)在抑郁症中的预防作用和机制.采用慢性温和不可预测应激(CMS)大鼠模型模拟抑郁症,对大鼠进行强迫游泳(FST)、糖水消耗的抑郁症行为学测试,并运用ELISA 试剂盒测定大鼠血浆皮质酮(CORT)含量,Western blot 法检测大鼠海马部脑源性营养因子(BDNF)、蛋白激酶A(PKA)和蛋白激酶C(PKC)的蛋白表达.实验结果表明,模型组FST、糖水消耗的行为学成绩明显低于正常对照组(P＜0.05),预先给予坚果各剂量(60 g·kg－1、120 g·kg－1、240 g·kg－1),可改善抑郁症大鼠的行为学成绩,提示,食用坚果可延缓抑郁症症状.血浆CORT水平测定结果显示,模型组血浆CORT含量显著高于正常对照组(P＜0.05),但坚果各剂量组对血浆CORT含量无影响(P＞0.05),提示,坚果产生延缓抑郁发生的机制可能与作用CORT途径无关联.对大鼠海马部的BDNF、PKA和PKC蛋白表达测定显示,模型组BDNF、PKA和PKC的蛋白表达均低于正常对照组(P ＜ 0.05),而经预先给与坚果的各剂量坚果组,则拮抗了模型组BDNF、PKA和PKC蛋白表达水平的下降(P＜0.05).提示,坚果在预防抑郁症作用机制可能与激活PKA、PKC激酶和增强BDNF蛋白水平的信号通路有关.因此,本实验结果初步明确,预先食用坚果可有助于延缓抑郁症的产生.这将为后续食用坚果类食物在预防抑郁症中的研究提供了一定的理论和实验依据.     

Increased proliferation of B cells and auto-immunity in mice lacking protein kinase Cdelta

Protein kinase C (PKC), which comprises 11 closely related isoforms, has been implicated in a wide variety of cellular processes, such as growth, differentiation, secretion, apoptosis and tumour development. Among the PKC isotypes, PKC-delta is unique in that its overexpression results in inhibition of cell growth. Here we show that mice that lack PKC-delta exhibit expansion of the B-lymphocyte population with the formation of numerous germinal centres in the absence of stimulation. The rate of proliferation in response to stimulation was greater for B cells from PKC-delta-deficient mice than for those from wild-type mice. Adoptive transfer experiments suggested that the hyperproliferation phenotype is B-cell autonomous. Production of interleukin-6 was markedly increased in B cells of PKC-delta-null mice as a result of an increase in the DNA-binding activity of NF-IL6. Furthermore, the PKC-delta-deficient mice contain circulating autoreactive antibodies and display immune-complex-type glomerulonephritis, as well as lymphocyte infiltration in many organs. These results suggest that PKC-delta has an indispensable function in negative regulation of B-cell proliferation, and is particularly important for the establishment of B-cell tolerance.     

Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signalling in neurons

The physiological state of the cell is controlled by signal transduction mechanisms which regulate the balance between protein kinase and protein phosphatase activities. Here we report that a single protein can, depending on which particular amino-acid residue is phosphorylated, function either as a kinase or phosphatase inhibitor. DARPP-32 (dopamine and cyclic AMP-regulated phospho-protein, relative molecular mass 32,000) is converted into an inhibitor of protein phosphatase 1 when it is phosphorylated by protein kinase A (PKA) at threonine 34. We find that DARPP-32 is converted into an inhibitor of PKA when phosphorylated at threonine 75 by cyclin-dependent kinase 5 (Cdk5). Cdk5 phosphorylates DARPP-32 in vitro and in intact brain cells. Phospho-Thr 75 DARPP-32 inhibits PKA in vitro by a competitive mechanism. Decreasing phospho-Thr 75 DARPP-32 in striatal slices, either by a Cdk5-specific inhibitor or by using genetically altered mice, results in increased dopamine-induced phosphorylation of PKA substrates and augmented peak voltage-gated calcium currents. Thus DARPP-32 is a bifunctional signal transduction molecule which, by distinct mechanisms, controls a serine/threonine kinase and a serine/threonine phosphatase.