Phosphorylation and regulation of Raf by Akt (protein kinase B)

Activation of the protein kinase Raf can lead to opposing cellular responses such as proliferation, growth arrest, apoptosis, or differentiation. Akt (protein kinase B), a member of a different signaling pathway that also regulates these responses, interacted with Raf and phosphorylated this protein at a highly conserved serine residue in its regulatory domain in vivo. This phosphorylation of Raf by Akt inhibited activation of the Raf-MEK-ERK signaling pathway and shifted the cellular response in a human breast cancer cell line from cell cycle arrest to proliferation. These observations provide a molecular basis for cross talk between two signaling pathways at the level of Raf and Akt.     

Phosphorylation of muscle membranes: identification of a membrane-bound protein kinase

A membrane-bound protein kinase occurs in membranes derived from rat skeletal muscle and appears limited to a surface membrane fraction. The enzyme is magnesium dependent, is only minimally stimulated by cyclic nucleotides, and phosphorylates serine and to a lesser extent threonine residues of three membrane proteins with molecular weights of less than 30,000.     

Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy

Adenosine monophosphate-activated protein kinase (AMPK) is a conserved sensor of intracellular energy activated in response to low nutrient availability and environmental stress. In a screen for conserved substrates of AMPK, we identified ULK1 and ULK2, mammalian orthologs of the yeast protein kinase Atg1, which is required for autophagy. Genetic analysis of AMPK or ULK1 in mammalian liver and Caenorhabditis elegans revealed a requirement for these kinases in autophagy. In mammals, loss of AMPK or ULK1 resulted in aberrant accumulation of the autophagy adaptor p62 and defective mitophagy. Reconstitution of ULK1-deficient cells with a mutant ULK1 that cannot be phosphorylated by AMPK revealed that such phosphorylation is required for mitochondrial homeostasis and cell survival during starvation. These findings uncover a conserved biochemical mechanism coupling nutrient status with autophagy and cell survival.     

Cl- channels in CF: lack of activation by protein kinase C and cAMP-dependent protein kinase

Secretory chloride channels can be activated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase in normal airway epithelial cells but not in cells from individuals with cystic fibrosis (CF). In excised, inside-out patches of apical membrane of normal human airway cells and airway cells from three patients with CF, the chloride channels exhibited a characteristic outwardly rectifying current-voltage relation and depolarization-induced activation. Channels from normal tissues were activated by both cAMP-dependent protein kinase and protein kinase C. However, chloride channels from CF patients could not be activated by either kinase. Thus, gating of normal epithelial chloride channels is regulated by both cAMP-dependent protein kinase and protein kinase C, and regulation by both kinases is defective in CF.     

Targeted protein degradation and synapse remodeling by an inducible protein kinase

Synaptic plasticity involves the reorganization of synapses at the protein and the morphological levels. Here, we report activity-dependent remodeling of synapses by serum-inducible kinase (SNK). SNK was induced in hippocampal neurons by synaptic activity and was targeted to dendritic spines. SNK bound to and phosphorylated spine-associated Rap guanosine triphosphatase activating protein (SPAR), a postsynaptic actin regulatory protein, leading to degradation of SPAR. Induction of SNK in hippocampal neurons eliminated SPAR protein, depleted postsynaptic density-95 and Bassoon clusters, and caused loss of mature dendritic spines. These results implicate SNK as a mediator of activity-dependent change in the molecular composition and morphology of synapses.     

AMP-activated protein kinase regulates neuronal polarization by interfering with PI 3-kinase localization

Axon-dendrite polarization is crucial for neural network wiring and information processing in the brain. Polarization begins with the transformation of a single neurite into an axon and its subsequent rapid extension, which requires coordination of cellular energy status to allow for transport of building materials to support axon growth. We found that activation of the energy-sensing adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway suppressed axon initiation and neuronal polarization. Phosphorylation of the kinesin light chain of the Kif5 motor protein by AMPK disrupted the association of the motor with phosphatidylinositol 3-kinase (PI3K), preventing PI3K targeting to the axonal tip and inhibiting polarization and axon growth.     

Regulation of myosin phosphatase by a specific interaction with cGMP- dependent protein kinase Ialpha

Contraction and relaxation of smooth muscle are regulated by myosin light-chain kinase and myosin phosphatase through phosphorylation and dephosphorylation of myosin light chains. Cyclic guanosine monophosphate (cGMP)-dependent protein kinase Ialpha (cGKIalpha) mediates physiologic relaxation of vascular smooth muscle in response to nitric oxide and cGMP. It is shown here that cGKIalpha is targeted to the smooth muscle cell contractile apparatus by a leucine zipper interaction with the myosin-binding subunit (MBS) of myosin phosphatase. Uncoupling of the cGKIalpha-MBS interaction prevents cGMP-dependent dephosphorylation of myosin light chain, demonstrating that this interaction is essential to the regulation of vascular smooth muscle cell tone.     

猪CDK9基因的克隆及其过表达对SUVEC细胞周期的影响

【目的】克隆猪细胞周期蛋白激酶9(CDK9)基因,观察该基因过表达对猪脐静脉血管内皮细胞(SU-VEC)周期的影响。【方法】采用电子克隆方法筛选得到猪CDK9基因全长序列,并对其编码的蛋白进行生物信息学分析。利用RT-PCR方法从猪脾脏中扩增出包含完整开放读码框架(ORF)的CDK9 cDNA片段,将其与真核表达载体pEGFP-C1连接,构建重组真核表达载体pEGFP-CDK9。将pEGFP-CDK9以脂质体介导法转染至SUVEC细胞,采用绿色荧光标记和Western blot检测SUVEC细胞中CDK9基因的表达,用流式细胞仪检测转染CDK9基因后SUVEC细胞周期的变化。【结果】得到猪CDK9基因全长1820bp的cDNA序列,其ORF为1119bp,编码372个氨基酸;CDK9分子质量为42.76ku,等电点为9.04;CDK9基因定位于猪的1号染色体上。酶切和测序结果证明,重组真核表达载体pEGFP-CDK9构建成功。pEGFP-CDK9转染SUVEC细胞48h后,荧光显微镜和Western blot检测到目的基因序列在SUVEC细胞中成功表达;试验组各期SUVEC细胞比例与空载体组相比,差异无统计学意义(P0.05),细胞周期未发生显著变化。【结论】克隆了猪CDK9基因,并在SUVEC细胞中成功进行了表达。CDK9基因的过表达未引起细胞周期发生显著变化,其可能并不参与细胞周期的调控。     

基于乌金猪深度发掘的优质高效新品种选育

目的揭示宣和猪PRKAG3基因外显子5多态性及其与肉质性状的关联。方法采用PCR产物直接测序法检测了120头宣和猪PRKAG3基因外显子5区域的SNP位点,借助最小二乘模型分析了各位点不同基因型及合并基因型对6个肉质性状的影响。结果宣和猪PRKAG3基因外显子5区域检出3个SNP位点,包括2个同义突变位点(T579C、T580C)和1个错义突变位点(G595A),分别以TT、TT、GA基因型和T、T、G等位基因的频率最高,且都处于Hardy-Weinberg平衡状态。T579C、T580C位点的TT和TC基因型可显著提高大理石纹(P<0.05)和降低失水率(P<0.01),分别呈显著的加性效应及加性—显性效应(P<0.05或P<0.01);G595A位点的AA基因型失水率最低(P<0.05)、熟肉率最高(P<0.05),呈显著的加性效应(P<0.05);3个位点的合并基因型TTTTGG、TCTCGG和TTTTAA分别具有最高的大理石纹、pH₁和最低的失水率(P<0.05或P<0.01),CCCCGG基因型的大理石纹和pH₁最低、失水率最高(P<0.05或P<0.01)。结论研究结果进一步确认宣和猪PRKAG3基因外显子5多态性与肌肉大理石纹、pH值和失水率显著关联,T579C、T580C位点的CC基因型和G595A位点的GG基因型具有协同降低猪肉品质的效应。     

Phosphorylation of proteins by inositol pyrophosphates

The inositol pyrophosphates IP7 and IP8 contain highly energetic pyrophosphate bonds. Although implicated in various biologic functions, their molecular sites of action have not been clarified. Using radiolabeled IP7, we detected phosphorylation of multiple eukaryotic proteins. We also observed phosphorylation of endogenous proteins by endogenous IP7 in yeast. Phosphorylation by IP7 is nonenzymatic and may represent a novel intracellular signaling mechanism.     

Regulation of a heart potassium channel by protein kinase A and C

The enzymes adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (protein kinase A) and protein kinase C regulate the activity of a diverse group of cellular proteins including membrane ion channel proteins. When protein kinase A was stimulated in cardiac ventricular myocytes with the membrane-soluble cAMP analog 8-chlorphenylthio cAMP (8-CPT cAMP), the amplitude of the delayed-rectifier potassium current (IK) doubled when recorded at 32 degrees C but was not affected at 22 degrees C. In contrast, modulation of the calcium current (ICa) by 8-CPT cAMP was independent of temperature with similar increases in ICa occurring at both temperatures. Stimulation of protein kinase C by phorbol 12,13-dibutyrate also enhanced IK in a temperature-dependent manner but failed to increase ICa at either temperature. Thus, cardiac delayed-rectifier potassium but not calcium channels are regulated by two distinct protein kinases in a similar temperature-dependent fashion.     

Cell cycle regulation of myosin-V by calcium/calmodulin-dependent protein kinase II

Organelle transport by myosin-V is down-regulated during mitosis, presumably by myosin-V phosphorylation. We used mass spectrometry phosphopeptide mapping to show that the tail of myosin-V was phosphorylated in mitotic Xenopus egg extract on a single serine residue localized in the carboxyl-terminal organelle-binding domain. Phosphorylation resulted in the release of the motor from the organelle. The phosphorylation site matched the consensus sequence of calcium/calmodulin-dependent protein kinase II (CaMKII), and inhibitors of CaMKII prevented myosin-V release. The modulation of cargo binding by phosphorylation is likely to represent a general mechanism regulating organelle transport by myosin-V.     

MicroRNA-370-5p inhibits pigmentation and cell proliferation by downregulating mitogen-activated protein kinase kinase kinase 8 expression in sheep melanocytes

In mammals, microRNAs (miRNAs) play key roles in multiple biological processes by regulating the expression of target genes.  Studies have found that the levels of miR-370-5p expression differ significantly in the skins of sheep with different hair colors; however, its function remains unclear.  In this study, we investigated the roles of miR-370-5p in sheep melanocytes and found that the overexpression of miR-370-5p significantly inhibited cell proliferation (P<0.01), tyrosinase activity (P=0.001) and significantly reduced (P<0.001) melanin production.  Functional prediction revealed that the 3´-untranslated region (UTR) of MAP3K8 has a putative miR-370-5p binding site, and the interaction between these two molecules was confirmed using luciferase reporter assays.  In situ hybridization assays revealed that MAP3K8 is expressed in the cytoplasm of melanocytes.  The results of quantitative RT-PCR and Western blotting analyses revealed that overexpression of miR-370-5p in melanocytes significantly inhibits (P<0.01) MAP3K8 expression via direct targeting of its 3´ UTR.  Inhibition of MAP3K8 expression by siRNA-MAP3K8 transfection induced a significant inhibition (P<0.01) of melanocyte proliferation and significant reduction (P<0.001) in melanin production, which is consistent with our observations for miR-370-5p.  Target gene rescue experiments indicated that the expression of MAP3K8 in melanocytes co-transfected with miR-370-5p and MAP3K8-cDNA (containing sites for the targeted binding to miR-370-5p) was significantly rescued (P≤0.001), which subsequently promoted significant increases in cell proliferation (P<0.001) and melanin production (P<0.01).  Collectively, these findings indicate that miR-370-5p plays a functional role in inhibiting sheep melanocyte proliferation and melanogenesis by downregulating the expression of MAP3K8.       

TRP-PLIK, a bifunctional protein with kinase and ion channel activities

We cloned and characterized a protein kinase and ion channel, TRP-PLIK. As part of the long transient receptor potential channel subfamily implicated in control of cell division, it is a protein that is both an ion channel and a protein kinase. TRP-PLIK phosphorylated itself, displayed a wide tissue distribution, and, when expressed in CHO-K1 cells, constituted a nonselective, calcium-permeant, 105-picosiemen, steeply outwardly rectifying conductance. The zinc finger containing alpha-kinase domain was functional. Inactivation of the kinase activity by site-directed mutagenesis and the channel's dependence on intracellular adenosine triphosphate (ATP) demonstrated that the channel's kinase activity is essential for channel function.     

Phorbol ester action is independent of viral and cellular src kinase levels

Treatment of normal chick embryo fibroblasts with phorbol myristate acetate causes those cells to express many of the phenotypic properties of virally transformed cells and also enhances the expression of transformed properties in Rous sarcoma virus-transformed chick embryo fibroblasts. We report here that phorbol myristate acetate has little or no effect on the level of protein kinases encoded by the viral src or endogenous sarc genes.     

Photoreceptor deactivation and retinal degeneration mediated by a photoreceptor-specific protein kinase C

The protein kinase C (PKC) family of serine-threonine kinases has been implicated in the regulation of a variety of signaling cascades. One member of this family, eye-PKC, is expressed exclusively in the Drosophila visual system. The inaC (inactivation-no-afterpotential C) locus was shown to be the structural gene for eye-PKC. Analysis of the light response from inaC mutants showed that this kinase is required for the deactivation and rapid desensitization of the visual cascade. Light adaptation was also defective in inaC mutant flies. In flies carrying the retinal degeneration mutation rdgB, absence of eye-PKC suppressed photoreceptor cell degeneration. These results indicate that eye-PKC functions in the light-dependent regulation of the phototransduction cascade in Drosophila.     

Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1

Malfolded proteins in the endoplasmic reticulum (ER) induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs). Mammalian homologs of yeast IRE1, which activate chaperone genes in response to ER stress, also activated JNK, and IRE1alpha-/- fibroblasts were impaired in JNK activation by ER stress. The cytoplasmic part of IRE1 bound TRAF2, an adaptor protein that couples plasma membrane receptors to JNK activation. Dominant-negative TRAF2 inhibited activation of JNK by IRE1. Activation of JNK by endogenous signals initiated in the ER proceeds by a pathway similar to that initiated by cell surface receptors in response to extracellular signals.     

Proliferation of functional hair cells in vivo in the absence of the retinoblastoma protein

In mammals, hair cell loss causes irreversible hearing and balance impairment because hair cells are terminally differentiated and do not regenerate spontaneously. By profiling gene expression in developing mouse vestibular organs, we identified the retinoblastoma protein (pRb) as a candidate regulator of cell cycle exit in hair cells. Differentiated and functional mouse hair cells with a targeted deletion of Rb1 undergo mitosis, divide, and cycle, yet continue to become highly differentiated and functional. Moreover, acute loss of Rb1 in postnatal hair cells caused cell cycle reentry. Manipulation of the pRb pathway may ultimately lead to mammalian hair cell regeneration.     

Studies and perspectives of protein kinase C

Protein kinase C, an enzyme that is activated by the receptor-mediated hydrolysis of inositol phospholipids, relays information in the form of a variety of extracellular signals across the membrane to regulate many Ca2+-dependent processes. At an early phase of cellular responses, the enzyme appears to have a dual effect, providing positive forward as well as negative feedback controls over various steps of its own and other signaling pathways, such as the receptors that are coupled to inositol phospholipid hydrolysis and those of some growth factors. In biological systems, a positive signal is frequently followed by immediate negative feedback regulation. Such a novel role of this protein kinase system seems to give a logical basis for clarifying the biochemical mechanism of signal transduction, and to add a new dimension essential to our understanding of cell-to-cell communication.