mTORC2 phosphorylates protein kinase Cζ to regulate its stability and activity

Protein kinase Cζ (PKCζ) is phosphorylated at the activation loop and the turn motif (TM). However, the TM kinase and functional relevance of TM phosphorylation remain largely unknown. We demonstrate that PKCζ TM is phosphorylated directly by the mTORC2 complex, and this phosphorylation is required for maintaining PKCζ kinase activity and stability. Functionally, mTORC2 regulates the activity of Rho family of GTPases, and therefore the organization of the actin cytoskeleton, through the control of PKCζ activity. Taken together, our findings identify PKCζ as a novel substrate and downstream effector of mTORC2 signaling.     

Activation of focal adhesion kinase in human lung cancer cells involves multiple and potentially parallel signaling events

Integrins are adhesion receptors that transmit signals bidirectionally across the plasma membrane. In our previous report we have shown that the squamous lung cancer cell line, Calu-1, binds to collagen type IV (Coll IV) through beta1-integrin and results in phosphorylation of focal adhesion kinase (FAK) (Ann Thorac Surg 2004; 78:450-457). Considering the critical role of FAK in cell migration, proliferation, and survival, here we investigated potential mechanisms of its activation and regulation in Calu-1 cells. We observed the phosphorylation of Tyr397 of FAK (the autophosphorylation site of FAK) and paxillin, the immediate downstream substrate of FAK following the adhesion of Calu-1 cells to Coll IV. FAK remains phosphorylated during proliferation either on Coll IV or on uncoated plates for 72 h, as determined by peroxivanadate treatment. Exposure of Calu-1 cells with 60 microM genistein, reduces FAK phosphorylation (7.6 fold) and cell proliferation. Extracellular signal regulated kinases (ERKs) were also phosphorylated after Coll IV attachment. Disruption of Calu-1 cell cytoskeleton integrity by 1-5 muM Cytochalasin D resulted in the inhibition of cell adhesion (50% to 75%, p<0.19 - 6.6 x 10(7)) and ERKs phosphorylation (2 fold) without any effect on FAK phosphorylation. Protein Kinase C inhibitor, Calphostin C at 100 and 250 nM concentrations did not block Coll IV induced FAK phosphorylation but activated the ERKs in a dose dependent manner. beta1-integrin is essential for Coll IV induced FAK activation, but it is not physically associated with FAK as determined by immunodetection assay. Collectively, this report defines the existence of multiple and potentially parallel Coll IV/beta1-integrin mediated signaling events in Calu-1 cells, which involve FAK, ERKs, and PKC.     

Diacylglycerol kinase α enhances protein kinase Cζ-dependent phosphorylation at Ser311 of p65/RelA subunit of nuclear factor-κB

We recently reported that diacylglycerol kinase (DGK) α enhanced tumor necrosis factor-α (TNF-α)-induced activation of nuclear factor-κB (NF-κB). However, the signaling pathway between DGKα and NF-κB remains unclear. Here, we found that small interfering RNA-mediated knockdown of DGKα strongly attenuated protein kinase C (PKC) ζ-dependent phosphorylation of a large subunit of NF-κB, p65/RelA, at Ser311 but not PKCζ-independent phosphorylation at Ser468 or Ser536. Moreover, knockdown and overexpression of PKCζ suppressed and synergistically enhanced DGKα-mediated NF-κB activation, respectively. These results strongly suggest that DGKα positively regulates TNF-α-dependent NF-κB activation via the PKCζ-mediated Ser311 phosphorylation of p65/RelA.     

Interaction Analyses of 14-3-3ζ, Dok1, and Phosphorylated Integrin β Cytoplasmic Tails Reveal a Bi-molecular Switch in Integrin Regulation

Integrins are hetero-dimeric (α and β subunits) type I transmembrane proteins that facilitate cell adhesion and migration. The cytoplasmic tails (CTs) of integrins interact with a plethora of intra-cellular proteins that are required for integrin bidirectional signaling. In particular, the β CTs of integrins are known to recruit a variety of cytosolic proteins that often have overlapping recognition sites. However, the chronological sequence of β CTs/cytosolic proteins interactions remains to be fully characterized. Previous studies have shown that the scaffold protein 14-3-3ζ binds to phosphorylated β CTs in activated integrins, whereas interactions of Dok-1 with phosphorylated β CTs maintained integrins in the resting state. In this study, we examined the binding interactions between 14-3-3ζ, Dok1, and phosphorylated integrin β2 and β3 CTs. We show that the scaffold protein 14-3-3ζ interacts with the phosphotyrosine binding (PTB) domain of Dok1 even in the absence of the phosphorylated integrin β CTs. The interactions were mapped onto the β-sheet region of the PTB domain of Dok1. Furthermore, we provide evidence that the 14-3-3ζ/Dok1 binary complex is able to bind to their cognate phosphorylated sequence motifs in the integrin β CTs. We demonstrate that Thr phosphorylated pTTT β2 CT or pTST β3 CT can bind to 14-3-3ζ that is in complex with the Dok1 PTB domain, whereas Ser phosphorylated β2 CT or Tyr phosphorylated β3 CT interacted with Dok1 in 14-3-3ζ/Dok1 complex. Based on these data, we propose that 14-3-3ζ/Dok1 complex could serve as a molecular switch providing novel molecular insights into the regulating integrin activation.     

The selective inhibition of nuclear PKCζ restores the effectiveness of chemotherapeutic agents in chemoresistant cells

The atypical protein kinase C (PKC) isoform zeta (PKCζ) has been implicated in the intracellular transduction of mitogenic and apoptotic signals by acting on different signaling pathways. The key role of these processes in tumorigenesis suggests a possible involvement of PKCζ in this event. PKCζ is activated by cytotoxic treatments, inhibits apoptotic cell death and reduces the sensitivity of cancer cells to chemotherapeutic agents. Here, using pharmacological and DNA recombinant approaches, we show that oxidative stress triggers nuclear translocation of PKCζ and induces resistance to apoptotic agents. Accordingly, chemoresistant cells show accumulation of PKCζ within the nucleus, and a nuclear-targeted PKCζ transfected in tumor cells decreases sensitivity to apoptosis. We thus developed a novel recombinant protein capable of selectively inhibiting the nuclear fraction of PKCζ that restored the susceptibility to apoptosis in cells in which PKCζ was enriched in the nuclear fraction, including chemoresistant cells. These findings establish the importance of PKCζ as a possible target to increase the effectiveness of anticancer therapies and highlight potential sites of intervention.     

Protein kinase C (PKC)ζ-mediated Gαq stimulation of ERK5 protein pathway in cardiomyocytes and cardiac fibroblasts

Gq-coupled G protein-coupled receptors (GPCRs) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced Gα(q)-mediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that Gα(q) acts as an adaptor protein that facilitates PKCζ-mediated activation of ERK5 in epithelial cells. Because the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in cardiovascular Gq-dependent signaling using both cultured cardiac cell types and chronic administration of angiotensin II in mice. We find that PKCζ is required for the activation of the ERK5 pathway by Gq-coupled GPCR in neonatal and adult murine cardiomyocyte cultures and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNA-mediated silencing of PKCζ in primary cultures of cardiac cells and in neonatal cardiomyocytes isolated from PKCζ-deficient mice. Moreover, upon chronic challenge with angiotensin II, these mice fail to promote the changes in the ERK5 pathway, in gene expression patterns, and in hypertrophic markers observed in wild-type animals. Taken together, our results show that PKCζ is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts and indicate a key cardiac physiological role for the Gα(q)/PKCζ/ERK5 signaling axis.     

The role of C1QBP in CSF-1-dependent PKCζ activation and macrophage migration

Macrophages view as double agents in tumor progression. Trafficking of macrophages to the proximity of tumors is mediated by colony-stimulating factor-1 (CSF-1), a growth factor. In this study, we investigated the role of complement1q-binding protein (C1QBP)/ atypical protein kinase C ζ (PKCζ) in CSF-1-induced macrophage migration. Disruption of C1QBP expression impaired chemotaxis and adhesion of macrophage. Phosphorylation of PKCζ is an essential component in macrophage chemotaxis signaling pathway. C1QBP could interact with PKCζ in macrophage. C1QBP knockdown inhibited CSF-1 induced phosphorylation of PKCζ and integrin-β1. However, C1QBP knockdown didn’t affect the phosphorylation of PKCζ induced by MCP-1. Furthermore, CSF-1 from RCC cell condition medium promoted macrophage chemotaxis and adhesion. Taken together, our results demonstrated that C1QBP plays an essential role in CSF-1 induced migration of macrophages.     

Generation of point‐mutant FAK knockin mice

Focal adhesion kinase is a non‐receptor protein tyrosine kinase with signaling functions downstream of integrins and growth factor receptors. In addition to its role in adhesion, migration, and proliferation it also has non‐kinase scaffolding functions in the nucleus. Focal adhesion kinase (FAK) activation involves the following: (1) ligand bound growth factors or clustered integrins activate FAK kinase domain; (2) FAK autophosphorylates tyrosine (Y) 397; (3) Src binds pY397 and phosphorylates FAK at various other sites including Y861; (4) downstream signaling of activated FAK elicits changes in cellular behavior. Although many studies have demonstrated roles for the kinase domain, Y397 and Y861 sites, in vitro much less is known about their functions in vivo. Here, we report the generation of a series of FAK‐mutant knockin mice where mutant FAK, either kinase dead, non‐phosphorylatable mutants Y397F and Y861F, or mutant Y397E—containing a phosphomimetic site that results in a constitutive active Y397, can be expressed in a Cre inducible fashion driven by the ROSA26 promoter. In future studies, intercrossing these mice with FAKflox/flox mice and inducible cre‐expressing mice will enable the in vivo study of mutant FAK function in the absence of endogenous FAK in a spatially and temporally regulated fashion within the whole organism. genesis 52:907–915, 2014. © 2014 Wiley Periodicals, Inc.     

(+)-Nootkatone inhibits tumor necrosis factor α/interferon γ-induced production of chemokines in HaCaT cells

Chemokines are important mediators of cell migration, and thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) are well-known typical inflammatory chemokines involved in atopic dermatitis (AD). (+)-Nootkatone is the major component of Cyperus rotundus. (+)-Nootkatone has antiallergic, anti-inflammatory, and antiplatelet activities. The purpose of this study was to investigate the effect of (+)-nootkatone on tumor necrosis factor α (TNF-α)/interferon γ (IFN-γ)-induced expression of Th2 chemokines in HaCaT cells. We found that (+)-nootkatone inhibited the TNF-α/IFN-γ-induced expression of TARC/CCL17 and MDC/CCL22 mRNA in HaCaT cells. It also significantly inhibited TNF-α/IFN-γ-induced activation of nuclear factor kappa B (NF-κB), p38 mitogen-activated protein kinase (MAPK), and protein kinase Cζ (PKCζ). Furthermore, we showed that PKCζ and p38 MAPK contributed to the inhibition of TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression by blocking IκBα degradation in HaCaT cells. Taken together, these results suggest that (+)-nootkatone may suppress TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression in HaCaT cells by inhibiting of PKCζ and p38 MAPK signaling pathways that lead to activation of NF-κB. We propose that (+)-nootkatone may be a useful therapeutic candidate for inflammatory skin diseases such as AD.     

GSK3 beta mediates acentromeric spindle stabilization by activated PKC zeta

Upon fertilization, the mammalian egg undergoes a precise series of signaling events that orchestrate its conversion into a zygote. Mouse eggs contain acentrosomal spindle poles when arrested at meiotic metaphase II. The meiotic spindle is thought to provide a scaffold that mediates spatial and temporal regulation of the signaling pathways orchestrating post-fertilization events. Many kinases have been found to be enriched at the MII meiotic spindle, such as Protein Kinase C (PKC), and are thought to have an important role in regulating signaling events initiated through fertilization. In this study phosphorylated PKCζ (p-PKCζ) and Glycogen Synthase Kinase 3β (GSK3β) were found to be enriched at both acentrosomal spindle poles and the kinetochore region. Phosphorylated PKCζ (p-PKCζ) was immunopurified from MII eggs and was found to co-localize with known microtubule stabilizing components found in somatic cells, including GSK3β and Partition deficit protein 6 (Par6). Both fluorescence resonance energy transfer (FRET) and immunofluorescence confirmed the existence and close association of these proteins with p-PKCζ at the meiotic spindle. When GSK3β is phosphorylated on ser9 its activity is inhibited and the spindle is stabilized. However, when GSK3β is dephosphorylated (on ser9) it becomes active and the spindle is destabilized. The mechanism by which p-PKCζ maintains spindle organization appears to be through GSK3β and suggests that p-PKCζ phosphorylates GSK3β on the ser9 position inactivating GSK3β and consequently maintaining spindle stability during meiotic metaphase arrest.     

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     

Bronchial inflammation induced PKCζ over-expression: involvement in mechanical properties of airway smooth muscle

Protein kinase C variants (PKCs) have been involved in the control of airway smooth muscle (ASM) tone, and abnormalities in PKC-dependent signaling have been associated with respiratory diseases such as asthma. In this study, the role of atypical PKCζ in airway hyperresponsiveness was investigated, using an in-vitro model of TNFα-treated human bronchi and an in vivo guinea pig model of chronic asthma. Our results demonstrated that PKCζ-specific inhibition produced a significant increase in isoproterenol sensitivity in TNFα-treated bronchi and ovalbumin (OVA)-sensitized guinea pig bronchi. The role of epoxy-eicosanoids, known to exert anti-inflammatory effects in lung, on PKCζ expression and activity in these models was evaluated. An enhanced PKCζ protein expression was delineated in TNFα-treated bronchi when compared with control (untreated) and epoxy-eicosanoid-treated bronchi. Measurements of Ca(2+) sensitivity, performed in TNFα-treated bronchi, demonstrated that treatment with myristoylated (Myr) PKCζ peptide inhibitor resulted in significant reductions of pCa-induced tension. Epoxy-eicosanoid treatments had similar effects on Ca(2+) sensitivity in TNFα-treated bronchi. In control and epoxy-eicosanoid-treated bronchi, the phosphorylated forms of p38MAPK and CPI-17 were significantly decreased compared with the TNFα-treated bronchi. An enhanced expression of PKCζ was ascertained in our in-vivo model of allergic asthma. Hence an increased Ca(2+) sensitivity could be explained by the phosphorylation of p38-MAPK, which in turn leads to phosphorylation and activation of the CPI-17 regulatory protein. This process was reversed upon treatment with the Myr-PKCζ-peptide inhibitor. The present data provide relevant evidence regarding the role of PKCζ in human and rodent models of airways inflammation.     

Src介导骨桥蛋白诱导的血管平滑肌细胞黏附和迁移过程

为阐明酪氨酸激酶Src在整合素被骨桥蛋白(OPN)激活所触发的细胞黏附和迁移信号途径中所起的作用,应用Src特异性抑制剂PP2阻断Src,观察OPN诱导的血管平滑肌细胞(VSMC)黏附和迁移活性的改变,并利用免疫沉淀检查PP2对整合素下游信号分子黏着斑激酶(FAK)和整合素偶联激酶(ILK)磷酸化及其相互作用的影响。结果显示,PP2可明显抑制OPN诱导的VSMC黏附和伤口愈合(黏附和迁移活性分别为对照组的76.6%和33.8%);OPN可显著诱导FAK磷酸化(磷酸化水平达对照组的1.9倍),促进ILK去磷酸化,并使FAK与ILK的结合减少(降至对照组的46.4%)。10μmol/LPP2可明显抑制OPN诱导的FAK磷酸化、拮抗OPN诱导对ILK的去磷酸化作用、促进FAK与ILK之间的结合。研究结果表明,Src作为OPN-整合素-FAK信号途径中的信号分子,通过影响FAK和ILK的磷酸化以及两者之间的相互作用来调节VSMC的黏附和迁移活性。     

Specific involvement of atypical PKCζ/PKMζ in spinal persistent nociceptive processing following peripheral inflammation in rat

Background

Central sensitization requires the activation of various intracellular signalling pathways within spinal dorsal horn neurons, leading to a lowering of activation threshold and enhanced responsiveness of these cells. Such plasticity contributes to the manifestation of chronic pain states and displays a number of features of long-term potentiation (LTP), a ubiquitous neuronal mechanism of increased synaptic strength. Here we describe the role of a novel pathway involving atypical PKCζ/PKMζ in persistent spinal nociceptive processing, previously implicated in the maintenance of late-phase LTP.

Results

Using both behavioral tests and in vivo electrophysiology in rats, we show that inhibition of this pathway, via spinal delivery of a myristoylated protein kinase C-ζ pseudo-substrate inhibitor, reduces both pain-related behaviors and the activity of deep dorsal horn wide dynamic range neurons (WDRs) following formalin administration. In addition, Complete Freund's Adjuvant (CFA)-induced mechanical and thermal hypersensitivity was also reduced by inhibition of PKCζ/PKMζ activity. Importantly, this inhibition did not affect acute pain or locomotor behavior in normal rats and interestingly, did not inhibited mechanical allodynia and hyperalgesia in neuropathic rats. Pain-related behaviors in both inflammatory models coincided with increased phosphorylation of PKCζ/PKMζ in dorsal horn neurons, specifically PKMζ phosphorylation in formalin rats. Finally, inhibition of PKCζ/PKMζ activity decreased the expression of Fos in response to formalin and CFA in both superficial and deep laminae of the dorsal horn.

Conclusions

These results suggest that PKCζ, especially PKMζ isoform, is a significant factor involved in spinal persistent nociceptive processing, specifically, the manifestation of chronic pain states following peripheral inflammation.

     

Role-Shifting PKCζ Fosters Its Own Proapoptotic Destruction by Complexing with Bcl10 at the Nuclear Envelope of Human Cervical Carcinoma Cells: A Proteomic and Biochemical Study

Many features of deadly human cervical cancers (HCCs) still require elucidation. Among HCC-derived cell lines, here we used the C4-I one since its quantitative gene expression pattern most closely mimics invasive HCCs, including protein kinase-Cζ (PKCζ) overexpression. Via proteomic, bioinformatic, and biochemical approaches we identified 31 and 33 proteins co-immunoprecipitating with PKCζ from nuclear membranes (NMs) of, respectively, untreated or VP-16-exposed C4-I cells. Such proteins belonged to eight functional groups, whose compositions and relative sizes changed with either context. Of the 56 proteins identified, only eight were shared between the two subproteomes, including Bcl10. Surprisingly, proteins known to associate with Bcl10, like Carma1/3 and Malt1, in so-called CBM signalosomes were absent. Notably, in VP-16-treated C4-I cells, PKCζ?Bcl10 complexes increasingly accrued at NMs, where PKCζ phosphorylated Bcl10, as PKCζ also did in vitro and in cell-free systems, both processes being thwarted by interfering RNA (iRNA) PKCζ depletion. Caspase-3 was associated with PKCζ?Bcl10 complexes and proteolyzed PKCζ leading to its inactivation/destruction; both events were prevented by Bcl10 iRNA suppression. Thus, PKCζ's molecular interactions and functional roles changed strikingly according to the untreated or apoptogen-treated cells context, and by complexing with Bcl10, PKCζ surprisingly favored its own demise, which suggests both proteins as HCCs therapeutic targets.     

The role of atypical protein kinase C in CSF-1-dependent Erk activation and proliferation in myeloid progenitors and macrophages

Colony stimulating factor-1 (CSF-1 or M-CSF) is the major physiological regulator of the proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. CSF-1 binds to a receptor tyrosine kinase, the CSF-1 receptor (CSF-1R). Multiple pathways are activated downstream of the CSF-1R; however, it is not clear which pathways regulate proliferation and survival. Here, we investigated the role of atypical protein kinase Cs (PKCζ) in a myeloid progenitor cell line that expressed CSF-1R (32D.R) and in primary murine bone marrow derived macrophages (BMMs). In 32D.R cells, CSF-1 induced the phosphorylation of PKCζ and increased its kinase activity. PKC inhibitors and transfections with mutant PKCs showed that optimal CSF-1-dependent Erk activation and proliferation depended on the activity of PKCζ. We previously reported that CSF-1 activated the Erk pathway through an A-Raf-dependent and an A-Raf independent pathway (Lee and States, Mol. Cell. Biol.18, 6779). PKC inhibitors did not affect CSF-1 induced Ras and A-Raf activity but markedly reduced MEK and Erk activity, implying that PKCζ regulated the CSF-1-Erk pathway at the level of MEK. PKCζ has been implicated in activating the NF-κB pathway. However, CSF-1 promoted proliferation in an NF-κB independent manner. We established stable 32D.R cell lines that overexpressed PKCζ. Overexpression of PKCζ increased the intensity and duration of CSF-1 induced Erk activity and rendered cells more responsive to CSF-1 mediated proliferation. In contrast to 32D.R cells, PKCζ inhibition in BMMs had only a modest effect on proliferation. Moreover, PKCζ -specific and pan-PKC inhibitors induced a paradoxical increase in MEK-Erk phosphorylation suggesting that PKCs targeted a common negative regulatory step upstream of MEK. Our results demonstrated that CSF-1 dependent Erk activation and proliferation are regulated differentially in progenitors and differentiated cells.     

Protein kinase Cζ phosphorylates occludin and promotes assembly of epithelial tight junctions

Protein kinases play an important role in the regulation of epithelial tight junctions. In the present study, we investigated the role of PKCζ (protein kinase Cζ) in tight junction regulation in Caco-2 and MDCK (Madin-Darby canine kidney) cell monolayers. Inhibition of PKCζ by a specific PKCζ pseudosubstrate peptide results in redistribution of occludin and ZO-1 (zona occludens 1) from the intercellular junctions and disruption of barrier function without affecting cell viability. Reduced expression of PKCζ by antisense oligonucleotide or shRNA (short hairpin RNA) also results in compromised tight junction integrity. Inhibition or knockdown of PKCζ delays calcium-induced assembly of tight junctions. Tight junction disruption by PKCζ pseudosubstrate is associated with the dephosphorylation of occludin and ZO-1 on serine and threonine residues. PKCζ directly binds to the C-terminal domain of occludin and phosphorylates it on threonine residues. Thr403, Thr404, Thr424 and Thr438 in the occludin C-terminal domain are the predominant sites of PKCζ-dependent phosphorylation. A T424A or T438A mutation in full-length occludin delays its assembly into the tight junctions. Inhibition of PKCζ also induces redistribution of occludin and ZO-1 from the tight junctions and dissociates these proteins from the detergent-insoluble fractions in mouse ileum. The present study demonstrates that PKCζ phosphorylates occludin on specific threonine residues and promotes assembly of epithelial tight junctions.     

Lipopolysaccharide-induced cytokine expression in alveolar epithelial cells: role of PKCζ-mediated p47phox phosphorylation

Chronic inflammation incited by bacteria in the saccular lung of premature infants contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). LPS-mediated type II alveolar epithelial cell (AEC) injury induces the expression of pro-inflammatory cytokines that trigger pulmonary neutrophil influx, alveolar matrix degradation and lung remodeling. We hypothesized that NADPH oxidase (Nox)-dependent mechanisms mediate LPS-induced cytokine expression in AEC. We examined the role of p47phox in mediating LPS-dependent inflammatory cytokine expression in A549 cells (which exhibit phenotypic features characteristic of type II AEC) and elucidated the proximal signaling events by which Nox is activated by LPS. LPS-induced ICAM-1 and IL-8 expression was associated with increased superoxide formation in AEC. LPS-mediated oxidative stress and cytokine expression was inhibited by apocynin and augmented by PMA demonstrating that Nox-dependent redox signaling regulates LPS-dependent pro-inflammatory signaling in AEC. In LPS-treated cells, p47phox translocated from the cytoplasm to the perinuclear region and co-localized with gp91phox. LPS also induced a temporal increase in p47phox serine304 phosphorylation in AEC. While inhibition of classical PKC and novel PKC with calphostin and rottlerin did not inhibit ICAM-1 or IL-8 expression, the myristolyated PKCζ pseudosubstrate peptide (a specific inhibitor of PKCζ) inhibited LPS-induced cytokine expression in AEC. Inhibition of PKCζ also attenuated LPS-mediated p47phox phosphorylation and perinuclear translocation in AEC. Consistent with these data, LPS activated PKCζ in AEC as evidenced by increased threonine410 phophorylation. We conclude that PKCζ-mediated p47phox activation regulates LPS-dependent cytokine expression in AEC. Selective inhibition of PKCζ or p47phox might attenuate LPS-mediated inflammation and alveolar remodeling in BPD.     

Structural and biochemical insights into the homotypic PB1-PB1 complex between PKCζ and p62

The atypical PKC isoforms(ζandι)play essential roles in regulating various cellular processes.Both the hetero-interaction between PKCζand p62 through their N-terminal PB1 domains and the homo-oligomerization of p62 via its PB1 domain are critical for the activation of NF-B signaling;however,the molecular mechanisms concerning the formation and regulation of these homotypic complexes remain unclear.Here we determined the crystal structure of PKCζ-PB1 in complex with a monomeric p62-PB1 mutant,where the massive electrostatic interactions between the acidic OPCA motif of PKCζ-PB1 and the basic surface of p62-PB1,as well as additional hydrogen bonds,ensure the formation of a stable and specific complex.The PKCζ-p62 interaction is interfered with the modification of a specific Cys of PKCζby the antiarthritis drug aurothiomalate,though all four cysteine residues in the PKCζ-PB1 domain can be modified in in vitro assay.In addition,detailed structural and biochemical analyses demonstrate that the PB1 domains of aPKCs belong to the type I group,which can depolymerize the high-molecular-weight p62 aggregates into homo-oligomers of lower order.These data together unravel the molecular mechanisms of the homo-or hetero-interactions between p62 and PKCζand provide the basis for designing inhibitors of NF-B signaling.     

Glutamate induces focal adhesion kinase tyrosine phosphorylation and actin rearrangement in heterologous mGluR1-expressing CHO cells via calcium/calmodulin signaling

Group 1 metabotropic glutamate receptors (mGluR1 and mGluR5) stimulate phospholipase C (PLC) and lead to mobilization of intracellular Ca(2+) and activation of protein kinase C (PKC). In this investigation, using heterologous receptor-expressing Chinese hamster ovary (CHO) cells, we showed that stimulation of mGluR1 or mGluR5 with glutamate rapidly increases tyrosine phosphorylation of focal adhesion kinase (FAK) (maximum at 1-3 min) in a dose-dependent manner (half-maximal responses at approximately 2 microM). In mGluR1-expressing cells, the glutamate-induced increase of FAK tyrosine phosphorylation was blocked by not only the PLC inhibitor, U73122, but also depletion of intracellular Ca(2+) and effectively abrogated by calmodulin (CaM) inhibitors, calmidazolium and fluphenazine. However, neither the PKC inhibitor, GF109203X, nor the CaM kinase II inhibitor, KN-62, inhibited glutamate-stimulated FAK tyrosine phosphorylation. Stimulation of mGluR1 caused a marked increase in actin stress fiber formation. Importantly, this actin rearrangement was prevented by the CaM inhibitor, but not by the PKC inhibitor and is thus in a good agreement with the signaling cascade of the mGluR1-FAK pathway. These results suggest that the Ca(2+)/CaM signaling and its downstream FAK tyrosine phosphorylation play an important role in cellular function of mGluR1.