蛋白激酶C分子的成熟与活化

蛋白激酶C(Protein kinase C,PKC)是细胞内一类重要的Ser/Thr激酶,调控多种生命活动的信号转导过程,目前已发现了至少11种亚型,其结构有一定的保守性而又有所差别,导致其功能和调控的多样性。新合成的PKC一般需要经历活化茎环(Activation-loop,A-loop)、转角模体(Turn motif,TM)以及疏水模体(hydrophobic motif,HM)的程序性磷酸化过程才能成熟,获得进一步活化的功能。本文综述了近年来PKC的程序性磷酸化成熟以及活化的研究进展情况。     

PKCδ在细胞凋亡中的作用

PKCδ是nPKC家族成员,参与细胞凋亡调控,其激活机制与特异性位点的磷酸化和半胱天冬酶3(caspase-3)的剪切密切联系.PKCδ激活后可通过多种途径介导细胞凋亡:激活多种蛋白激酶级联启动细胞凋亡信号,转位至线粒体诱导细胞色素C等凋亡因子的释放,核转位启动核内凋亡通路诱导细胞凋亡.本文综述了PKCδ的分子结构、激活机制以及调控细胞凋亡的最新研究进展.     

PKCδ与JNK共同调控喜树碱诱导的白血病细胞凋亡

采用流式细胞术、蛋白质免疫印迹法检测了喜树碱诱导白血病细胞凋亡过程中蛋白激酶Cδ(protein kinase Cδ,PKCδ)与c-Jun氨基末端激酶(c-Jun N-termital kinase,JNK)的作用。结果发现,50 nmol/L喜树碱诱导处理U937细胞24、36或48 h后,细胞发生明显凋亡,并且PKCδ和JNK均被激活。用化学抑制剂rottlerin抑制PKCδ的活化可以降低喜树碱诱导细胞凋亡过程中JNK的磷酸化,进而抑制细胞凋亡;而用化学抑制剂SP600125抑制JNK的磷酸化也会降低PKCδ的剪切活化,进而一定程度地阻断细胞凋亡;同时,JNK抑制剂SP600125也可以阻断过表达PKCδ活性片段诱导的细胞凋亡。这些结果提示,PKCδ和JNK介导的信号通路可以相互调控,共同促进细胞凋亡。该研究对理解细胞凋亡的精细调控机制以及肿瘤的治疗都有一定的借鉴意义。     

蛋白激酶C_ζ对小鼠受精卵发育早期基因组转录活化的影响

为研究蛋白激酶Cζ (proteinkinaseCζ ,PKCζ)在小鼠受精卵细胞早期发育过程中对胚胎基因组活化影响 ,采用免疫印迹和细胞免疫荧光的方法 ,观察PKCζ的抑制剂对小鼠受精卵 1 细胞期G1和G2 不同时期小鼠受精卵基因组活化的影响 .小鼠 1 细胞期受精卵蛋白激酶C (PKC)的活性不断增加 ,并在G2 期达到最高 .PKC的抑制剂calphostinC可以明显抑制PKC的活性达 4 7% .同时calphostinC对受精卵 1 细胞期基因组的早期活化具有显著的抑制作用 (P <0 0 1) .在小鼠 1 细胞期受精卵的G2 期 ,具有活性的磷酸化PKCζ的含量明显多于G1期和卵母细胞MⅡ期 ,分别比它们高2 7%和 110 % .PKCζ的特异性抑制剂可以抑制受精卵 1 细胞期基因的转录和活化 (P <0 0 5 ) .实验结果表明 ,PKCζ参与了小鼠受精卵基因组早期转录的调控     

受体激酶介导的油菜素内酯信号转导途径

油菜素内酯（brassinosteroids,BRs）是一类重要的类固醇激素,参与调控植物生长发育的许多过程。结合应用遗传学、生物化学以及蛋白质组学等研究手段现已基本阐明了BR信号转导的主要过程。BRI1作为受体在细胞表面感知BR,BRI1抑制子BKI1从质膜上解离下来,使BRI1与其共受体BAK1结合。BRI1和BAK1通过顺序磷酸化将BR信号完全激活。活化的BRI1将BSK磷酸化激活,BSK活化BSU1,BSU1将BIN2去磷酸化使其失活,解除BIN2对BES1/BZR1的抑制功能。PP2A可以将BES1/BZR1去磷酸化激活,又可以将受体BRI1去磷酸化促使其降解。BR信号的传递最终使去磷酸化状态的BES1/BZR1在细胞内累积,激活BR信号通路下游的转录调控。     

尼罗罗非鱼PKCθ蛋白和Spartin蛋白的 原核表达、抗体制备及其组织表达

为了研究尼罗罗非鱼（Oreochromis niloticus）蛋白激酶C theta（PKCθ）和Spartin的蛋白表达情况,本实验在大肠杆菌（Escherichia coli）中表达和提纯了尼罗罗非鱼PKCθ和Spartin的重组蛋白,并利用日本大耳兔（Oryctolagus cuniculus）制备了相应的多克隆抗体。用间接ELISA技术检测抗体效价,Western Blot鉴定抗体的特异性,并检测其在罗非鱼肝、脾、肠和肌肉组织中的表达情况。结果表明,实验成功构建了原核表达载体pET-B2m-PKCθ和pET-B2m-Spartin,实现了重组蛋白的原核表达和纯化。PKCθ重组蛋白主要存在于包涵体中,分子量约为56 ku;Spartin重组蛋白分子量约为30 ku,以包涵体蛋白的形式存在。获得的多克隆抗体效价均高达1︰512 000,Western Blot检测结果表明,制备的抗体能特异性识别尼罗罗非鱼PKCθ和Spartin多种异构体;PKCθ蛋白在罗非鱼肝、脾、肠与肌肉组织中均有表达;Spartin蛋白在罗非鱼的肝、脾和肠组织中不表达,在肌肉组织中表达。研究表明,尼罗罗非鱼PKCθ和Spartin重组蛋白在大肠杆菌中成功表达,获得了高效价的多克隆抗体,并明确了尼罗罗非鱼PKCθ和Spartin在不同组织中的表达情况,为进一步研究尼罗罗非鱼PKCθ和Spartin的功能及其作用机制奠定了基础。     

GnRH类似物对培养的大鼠胃平滑肌细胞蛋白激酶C活性的影响

研究GnRH类似物阿拉瑞林（Alarelin)对大鼠胃平滑肌细胞蛋白酶C（PKC）活性的影响,采用放射性同位素法测定PKC的活性,发现：（1）阿拉瑞林可使培养的大鼠胃平滑肌细胞中PKC活性明显升高,3min时达高峰,10min后作用显著降低,且成剂量依赖性。当阿拉瑞林为10^-5mol/L时,PKC活性最高,10^-9mol/L时其对PCK的作用基本消失;（2）当用佛波醇酯（phorbol 12 myristate 13-acetate,PMA)短期作用激活PKC后,再加入阿拉瑞林,仍可使PKC活性略有升高,但无显著性差异;当用PMA长期持续作用耗PKC后,再加入阿拉瑞林作用3min,它不能激活PKC,与单纯的阿拉瑞林作用3min组相比差异显著;（3）在无外Ca^2 时,阿拉瑞林也可使PKC活性升高,但不如单独用阿拉瑞林作用3min组的高。因此PKC活性的增高,并不完全依赖细胞外Ca^2 ,它可以动员胞内Ca^2 的释放激活PKC,说明GnRH类似物可使胃平滑肌细胞中PKC活性增加,PKC参与阿拉瑞林对胃平滑肌细胞调控的信号转导过程。     

M1乙酰胆碱受体对AMPA受体GluA1-Ser831 位点的调控作用

目的:从海马神经元谷氨酸离子型受体--AMPA受体亚基GluA1的831位丝氨酸(GluA1Ser831)磷酸化角度,探讨M1乙酰胆碱受体对AMPA受体GluA1亚基的调控作用及作用机制。方法:本研究以成熟的原代海马神经元为实验对象,用不易被降解的卡巴胆碱(Carbachol,CCh)作为胆碱受体激动剂,以免疫印迹法作为蛋白和磷酸化蛋白的主要检测手段,结合不同蛋白抑制剂研究M1受体调控AMPA受体GluA1亚基的关键信号分子及其机制。结果:1与对照组相比,CCh组Ser831的磷酸化水平显著升高。2CCh促进Ser831磷酸化的现象在M1受体选择性拮抗剂哌仑西平(Pirenzepine)+CCh组消失,CCh升高GluA1-Ser831磷酸化水平的作用由M1受体介导。3蛋白激酶C(ProteinkinaseC,PKC)抑制剂白屈菜红碱(Chelerythrinechloride,CHCL)能对抗CCh促进GluA1-Ser831位点磷酸化的作用,而钙/钙调素依赖性蛋白激酶II(Calcium/calmodulin-dependentkinaseII,CaMKII)抑制剂KN62不能对抗CCh的作用。4为检测体内GluA1-Ser831的磷酸化情况,用小鼠海马组织定位注射CCh和CHCL,CCh组小鼠海马组织GluA1-Ser831位点的磷酸化水平升高,CHCL能对抗这种作用,PKC介导了M1受体激活所导致的GluA1-Ser831磷酸化水平的升高。结论:M1受体通过激活PKC促进GluA1-Ser831的磷酸化。     

过表达PKCε和PKCη在HCC1806细胞中的抗凋亡作用

为探讨PKCε、PKCη对乳腺癌细胞中TNFα诱导凋亡的影响,采用PCR技术,从人肌肉cDNA文库中克隆了全长PKCε和PKCη序列,亚克隆至pcDNA3,转化HCC1806细胞,并选择出稳定的转化细胞株.然后用流式细胞仪检测了PKCε、PKCη过表达对DNA断裂的影响,用Western印迹方法检测了PKCε、PKCη过表达对PARP、caspase 3和caspase 8水解的影响,以及对Bcl-2,Bax表达的影响,和对MAPK磷酸化的影响.流式细胞仪分析DNA含量的结果表明：TNFα处理后,HCC1806/PC、HCC1806/ε、HCC1806/η的sub_G1状态的DNA含量分别为：57.7%、23.3%、14.6%.Western印迹结果表明,当用0.01nmol/L TNFα处理,HCC1806/η中PARP、caspase3、caspase8的水解程度最低,HCC1806/ε中次之,HCC1806/PC最严重;与HCC1806/PC比较,无论用不用TNFα处理,HCC1806/ε都表达更高的Bcl_2,而HCC1806/η不影响Bcl_2的表达.过表达PKCη而不是PKCε抑制了由TNFα引起的p38和JNK的磷酸化,并且是JNK的抑制剂SP600125而不是p38的抑制剂SB220025抑制了PARP的裂解.以上结果显示,过表达PKCε、PKCη可能通过不同的信号途径在HCC1806中起抗凋亡作用,PKCε上调了bcl_2的表达,而PKCη抑制了JNK的磷酸化.     

Peptidyl-prolyl isomerase Pin1 controls down-regulation of conventional protein kinase C isozymes

The down-regulation or cellular depletion of protein kinase C (PKC) attendant to prolonged activation by phorbol esters is a widely described property of this key family of signaling enzymes. However, neither the mechanism of down-regulation nor whether this mechanism occurs following stimulation by physiological agonists is known. Here we show that the peptidyl-prolyl isomerase Pin1 provides a timer for the lifetime of conventional PKC isozymes, converting the enzymes into a species that can be dephosphorylated and ubiquitinated following activation induced by either phorbol esters or natural agonists. The regulation by Pin1 requires both the catalytic activity of the isomerase and the presence of a Pro immediately following the phosphorylated Thr of the turn motif phosphorylation site, one of two C-terminal sites that is phosphorylated during the maturation of PKC isozymes. Furthermore, the second C-terminal phosphorylation site, the hydrophobic motif, docks Pin1 to PKC. Our data are consistent with a model in which Pin1 binds the hydrophobic motif of conventional PKC isozymes to catalyze the isomerization of the phospho-Thr-Pro peptide bond at the turn motif, thus converting these PKC isozymes into species that can be efficiently down-regulated following activation.     

The phosphatase PHLPP controls the cellular levels of protein kinase C

The life cycle of protein kinase C (PKC) is controlled by multiple phosphorylation and dephosphorylation steps. The maturation of PKC requires three ordered phosphorylations, one at the activation loop and two at COOH-terminal sites, the turn motif and the hydrophobic motif, to yield a stable and signaling-competent enzyme. Dephosphorylation of the enzyme leads to protein degradation. We have recently discovered a novel family of protein phosphatases named PH domain leucine-rich repeat protein phosphatase (PHLPP) whose members terminate Akt signaling by dephosphorylating the hydrophobic motif on Akt. Here we show that the two PHLPP isoforms, PHLPP1 and PHLPP2, also dephosphorylate the hydrophobic motif on PKC betaII, an event that shunts PKC to the detergent-insoluble fraction, effectively terminating its life cycle. Deletion mutagenesis reveals that the PH domain is necessary for the effective dephosphorylation of PKC betaII by PHLPP in cells, whereas the PDZ-binding motif, required for Akt regulation, is dispensable. The phorbol ester-mediated dephosphorylation of the hydrophobic site, but not the turn motif or activation loop, is insensitive to okadaic acid, consistent with PHLPP, a PP2C family member, controlling the hydrophobic site. In addition, knockdown of PHLPP expression reduces the rate of phorbol ester-triggered dephosphorylation of the hydrophobic motif, but not turn motif, of PKC alpha. Last, we show that depletion of PHLPP in colon cancer and normal breast epithelial cells results in an increase in conventional and novel PKC levels. These data reveal that PHLPP controls the cellular levels of PKC by specifically dephosphorylating the hydrophobic motif, thus destabilizing the enzyme and promoting its degradation.     

DNA damage targets PKCη to the nuclear membrane via its C1b domain

Translocation to cellular membranes is one of the hallmarks of PKC activation, occurring as a result of the generation of lipid secondary messengers in target membrane compartments. The activation-induced translocation of PKCs and binding to membranes is largely directed by their regulatory domains. We have previously reported that PKCη, a member of the novel subfamily and an epithelial specific isoform, is localized at the cytoplasm and ER/Golgi and is translocated to the plasma membrane and the nuclear envelope upon short-term activation by PMA. Here we show that PKCη is shuttling between the cytoplasm and the nucleus and that upon etoposide induced DNA damage is tethered at the nuclear envelope. Although PKCη expression and its phosphorylation on the hydrophobic motif (Ser675) are increased by etoposide, this phosphorylation is not required for its accumulation at the nuclear envelope. Moreover, we demonstrate that the C1b domain is sufficient for translocation to the nuclear envelope. We further show that, similar to full-length PKCη, the C1b domain could also confer protection against etoposide-induced cell death. Our studies demonstrate translocation of PKCη to the nuclear envelope, and suggest that its spatial regulation could be important for its cellular functions including effects on cell death.     

The hydrophobic phosphorylation motif of conventional protein kinase C is regulated by autophosphorylation.

BACKGROUND: A growing number of kinases are now known to be controlled by two phosphorylation switches, one on a loop near the entrance to the active site and a second on the carboxyl terminus. For the protein kinase C (PKC) family of enzymes, phosphorylation at the activation loop is mediated by another kinase but the mechanism for carboxy-terminal phosphorylation is still unclear. The latter switch contains two phosphorylation sites - one on a 'turn' motif and the second on a conserved hydrophobic phosphorylation motif - that are found separately or together in a number of other kinases. RESULTS: Here, we investigated whether the carboxy-terminal phosphorylation sites of a conventional PKC are controlled by autophosphorylation or by another kinase. First, kinetic analyses revealed that a purified construct of the kinase domain of PKC betaII autophosphorylated on the Ser660 residue of the hydrophobic phosphorylation motif in an apparently concentration-independent manner. Second, kinase-inactive mutants of PKC did not incorporate phosphate at either of the carboxy-terminal sites, Thr641 or Ser660, when expressed in COS-7 cells. The inability to incorporate phosphate on the hydrophobic site was unrelated to the phosphorylation state of the other key phosphorylation sites: kinase-inactive mutants with negative charge at Thr641 and/or the activation-loop position were also not phosphorylated in vivo. CONCLUSIONS: PKC betaII autophosphorylates at its conserved carboxy-terminal hydrophobic phosphorylation site by an apparently intramolecular mechanism. Expression studies with kinase-inactive mutants revealed that this mechanism is the only one responsible for phosphorylating this motif in vivo. Thus, conventional PKC autoregulates the carboxy-terminal phosphorylation switch following phosphorylation by another kinase at the activation loop switch.     

Lack of constitutive activity of the free kinase domain of protein kinase C zeta. Dependence on transphosphorylation of the activation loop

Following the induction of apoptosis in mammalian cells, protein kinase C zeta (PKC zeta) is processed between the regulatory and catalytic domains by caspases, which increases its kinase activity. The catalytic domain fragments of PKC isoforms are considered to be constitutively active, because they lack the autoinhibitory amino-terminal regulatory domain, which includes a pseudosubstrate segment that plugs the active site. Phosphorylation of the activation loop at Thr(410) is known to be sufficient to activate the kinase function of full-length PKC zeta, apparently by inducing a conformational change, which displaces the amino-terminal pseudosubstrate segment from the active site. Amino acid substitutions for Thr(410) of the catalytic domain of PKC zeta (CAT zeta) essentially abolished the kinase function of ectopically expressed CAT zeta in mammalian cells. Similarly, substitution of Ala for a Phe of the docking motif for phosphoinositide-dependent kinase-1 prevented activation loop phosphorylation and abolished the kinase activity of CAT zeta. Treatment of purified CAT zeta with the catalytic subunit of protein phosphatase 1 decreased activation loop phosphorylation and kinase activity. Recombinant CAT zeta from bacteria lacked detectable kinase activity. Phosphoinositide-dependent kinase-1 phosphorylated the activation loop and activated recombinant CAT zeta from bacteria. Treatment of HeLa cells with fetal bovine serum markedly increased the phosphothreonine 410 content of CAT zeta and stimulated its kinase activity. These findings indicate that the catalytic domain of PKC zeta is intrinsically inactive and dependent on the transphosphorylation of the activation loop.     

Catalytic domain crystal structure of protein kinase C-theta (PKCtheta)

A member of the novel protein kinase C (PKC) subfamily, PKC, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production. Selective involvement of PKC in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes. In this report we describe the crystal structure of the catalytic domain of PKC at 2.0-A resolution. Human recombinant PKC kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine. The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state. Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite. The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKC autocatalytic phosphorylation and activation during bacterial expression. The PKC-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKC selective leads.     

PC phosphorylation increases the ability of AFAP-110 to cross-link actin filaments

The actin filament-associated protein and Src-binding partner, AFAP-110, is an adaptor protein that links signaling molecules to actin filaments. AFAP-110 binds actin filaments directly and multimerizes through a leucine zipper motif. Cellular signals downstream of Src(527F) can regulate multimerization. Here, we determined recombinant AFAP-110 (rAFAP-110)-bound actin filaments cooperatively, through a lateral association. We demonstrate rAFAP-110 has the capability to cross-link actin filaments, and this ability is dependent on the integrity of the carboxy terminal actin binding domain. Deletion of the leucine zipper motif or PKC phosphorylation affected AFAP-110's conformation, which correlated with changes in multimerization and increased the capability of rAFAP-110 to cross-link actin filaments. AFAP-110 is both a substrate and binding partner of PKC. On PKC activation, stress filament organization is lost, motility structures form, and AFAP-110 colocalizes strongly with motility structures. Expression of a deletion mutant of AFAP-110 that is unable to bind PKC blocked the effect of PMA on actin filaments. We hypothesize that upon PKC activation, AFAP-110 can be cooperatively recruited to newly forming actin filaments, like those that exist in cell motility structures, and that PKC phosphorylation effects a conformational change that may enable AFAP-110 to promote actin filament cross-linking at the cell membrane.     

Protein kinase C modulates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by targeting the apical events of death receptor signaling

We have further examined the mechanism by which phorbol ester-mediated protein kinase C (PKC) activation protects against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. We now report that activation of PKC targets death receptor signaling complex formation. Pre-treatment with 12-O-tetradecanoylphorbol-13-acetate (PMA) led to inhibition of TRAIL-induced apoptosis in HeLa cells, which was characterized by a reduction in phosphatidylserine (PS) externalization, decreased caspase-8 processing, and incomplete maturation and activation of caspase-3. These effects of PMA were completely abrogated by the PKC inhibitor, bisindolylmaleimide I (Bis I), clearly implicating PKC in the protective effect of PMA. TRAIL-induced mitochondrial release of the apoptosis mediators cytochrome c and Smac was blocked by PMA. This, together with the observed decrease in Bid cleavage, suggested that PKC activation modulates apical events in TRAIL signaling upstream of mitochondria. This was confirmed by analysis of TRAIL death-inducing signaling complex formation, which was disrupted in PMA-treated cells as evidenced by a marked reduction in Fas-associated death domain protein (FADD) recruitment, an effect that could not be explained by any change in FADD phosphorylation state. In an in vitro binding assay, the intracellular domains of both TRAIL-R1 and TRAIL-R2 bound FADD: activation of PKC significantly inhibited this interaction suggesting that PKC may be targeting key apical components of death receptor signaling. Significantly, this effect was not confined to TRAIL, because isolation of the native TNF receptor signaling complex revealed that PKC activation also inhibited TNF receptor-associated death domain protein recruitment to TNF-R1 and TNF-induced phosphorylation of IkappaB-alpha. Taken together, these results show that PKC activation specifically inhibits the recruitment of key obligatory death domain-containing adaptor proteins to their respective membrane-associated signaling complexes, thereby modulating TRAIL-induced apoptosis and TNF-induced NF-kappaB activation, respectively.     

PKC-mediated stimulation of amphibian CFTR depends on a single phosphorylation consensus site. insertion of this site confers PKC sensitivity to human CFTR

Mutations of the CFTR, a phosphorylation-regulated Cl(-) channel, cause cystic fibrosis. Activation of CFTR by PKA stimulation appears to be mediated by a complex interaction between several consensus phosphorylation sites in the regulatory domain (R domain). None of these sites has a critical role in this process. Here, we show that although endogenous phosphorylation by PKC is required for the effect of PKA on CFTR, stimulation of PKC by itself has only a minor effect on human CFTR. In contrast, CFTR from the amphibians Necturus maculosus and Xenopus laevis (XCFTR) can be activated to similar degrees by stimulation of either PKA or PKC. Furthermore, the activation of XCFTR by PKC is independent of the net charge of the R domain, and mutagenesis experiments indicate that a single site (Thr665) is required for the activation of XCFTR. Human CFTR lacks the PKC phosphorylation consensus site that includes Thr665, but insertion of an equivalent site results in a large activation upon PKC stimulation. These observations establish the presence of a novel mechanism of activation of CFTR by phosphorylation of the R domain, i.e., activation by PKC requires a single consensus phosphorylation site and is unrelated to the net charge of the R domain.