A Role for Protein Kinase Bβ/Akt2 in Insulin-Stimulated GLUT4 Translocation in Adipocytes

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKBbeta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKBalpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKBbeta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKBbeta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKBbeta in insulin-stimulated glucose transport in adipocytes.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

A Novel Role of Protein Tyrosine Kinase2 in Mediating Chloride Secretion in Human Airway Epithelial Cells

Ca²⁺ activated Cl⁻ channels (CaCC) are up-regulated in cystic fibrosis (CF) airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl⁻ secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca²⁺, which not only activates epithelial CaCCs, but also inhibits epithelial Na⁺ hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl⁻ secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca²⁺ and Cl⁻ secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells.     

多聚嘧啶区结合蛋白1通过调控基因的可变剪接促进胆管癌细胞的生长、迁移及侵袭能力

胆管癌是一种起病隐匿、侵袭性强、致死率高的原发性恶性肿瘤。多聚嘧啶区结合蛋白1(polypyrimidine tract-binding protein 1, PTBP1)已被报道,在多种类型肿瘤组织中异常高表达并参与癌症进展,但其在胆管癌中的作用仍未见报道。该研究旨在探讨PTBP1在胆管癌中的生物学功能,并初步解析其分子机制。本文利用公开的癌症基因组图谱(the cancer genome atlas, TCGA)数据,分析了胆管癌及癌旁组织中的PTBP1 mRNA表达水平。结果显示,PTBP1在胆管癌组织中的表达水平显著高于癌旁组织(P < 0.05)。随后,在胆管癌细胞系RBE和HuH28中,通过CCK-8和细胞平板克隆实验,评价了PTBP1对胆管癌细胞生长能力的影响。结果显示,过表达PTBP1可显著促进胆管癌细胞的生长(P < 0.01),而敲低PTBP1显著抑制胆管癌细胞的生长(P < 0.001)。Transwell和Invasion实验结果显示,过表达PTBP1可显著促进胆管癌细胞的迁移和侵袭(P < 0.001),而敲低PTBP1显著抑制胆管癌细胞的迁移和侵袭(P < 0.001)。转录物组测序和通路富集分析结果显示,在胆管癌细胞中,敲低PTBP1后上调表达的基因显著富集于p53信号通路;而下调表达的基因显著富集于胆固醇代谢、Rho GTPase和TGF-β等信号通路。基于上述转录物组测序数据,本文还分析发现,敲低PTBP1可导致一系列基因发生异常的mRNA可变剪接事件,例如参与TGF-β调控的TGIF1及与p53活性相关的GNAS基因等。综上所述,PTBP1可能通过调控一系列基因的可变剪接而影响多个癌症相关的信号通路,从而促进胆管癌的进展。     

Novel Protein Interactions with Endoglin and Activin Receptor-like Kinase 1: Potential Role in Vascular Networks

Endoglin and activin receptor-like kinase 1 are specialized transforming growth factor-beta (TGF-β) superfamily receptors, primarily expressed in endothelial cells. Mutations in the corresponding ENG or ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT1 and HHT2 respectively). To discover proteins interacting with endoglin, ACVRL1 and TGF-β receptor type 2 and involved in TGF-β signaling, we applied LUMIER, a high-throughput mammalian interactome mapping technology. Using stringent criteria, we identified 181 novel unique and shared interactions with ACVRL1, TGF-β receptor type 2, and endoglin, defining potential novel important vascular networks. In particular, the regulatory subunit B-beta of the protein phosphatase PP2A (PPP2R2B) interacted with all three receptors. Interestingly, the PPP2R2B gene lies in an interval in linkage disequilibrium with HHT3, for which the gene remains unidentified. We show that PPP2R2B protein interacts with the ACVRL1/TGFBR2/endoglin complex and recruits PP2A to nitric oxide synthase 3 (NOS3). Endoglin overexpression in endothelial cells inhibits the association of PPP2R2B with NOS3, whereas endoglin-deficient cells show enhanced PP2A-NOS3 interaction and lower levels of endogenous NOS3 Serine 1177 phosphorylation. Our data suggest that endoglin regulates NOS3 activation status by regulating PPP2R2B access to NOS3, and that PPP2R2B might be the HHT3 gene. Furthermore, endoglin and ACVRL1 contribute to several novel networks, including TGF-β dependent and independent ones, critical for vascular function and potentially defective in HHT.Transforming growth factor-β (TGF-β)¹ superfamily ligands, including TGF-βs, activins and bone morphogenic proteins (BMPs), regulate several pathways essential for vascular development and function (1). Responses to these ligands are controlled by type I and II serine kinase receptors, coreceptors and signaling SMAD intermediates. Endothelial cells express the coreceptor, endoglin, and the specialized type I receptor, ACVRL1 (activin receptor-like kinase 1 or ALK1); both molecules are critical for regulation of angiogenesis and vasomotor function by TGF-β superfamily ligands (2, 3).Mutations in ENG and ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT), types 1 and 2, respectively (4). HHT affects 1 in 5000–8000 people worldwide and is characterized by arteriovenous malformations (AVMs) in multiple organs, potentially leading to severe hemorrhages and strokes (4). Haploinsufficiency is the underlying cause of HHT, indicating that reduced levels of functional endoglin or ACVRL1 (ALK1) proteins predispose to endothelial dysfunction and AVMs (5). Although the mechanisms responsible for AVMs remain unclear, the elucidation of how members of the TGF-β superfamily and their molecular networks regulate vascular integrity is vital for future treatments of HHT.We have demonstrated that endoglin interacts with endothelial nitric oxide synthase (NOS3 or eNOS) and regulates its activation (2). NOS3 is a Ca⁺² and calmodulin-regulated enzyme that produces NO● in response to humoral and mechanical stimuli via dynamic interactions with various allosteric regulators such as heat shock protein 90 (HSP90). NOS3 is also regulated by dynamic changes in its phosphorylation status. For example, effects of the vascular endothelial growth factor (VEGF) on angiogenesis, vascular permeability and vasomotor tone are mediated in part through Akt-dependent phosphorylation of NOS3 Ser1177 and by increased NOS3-HSP90 association (6). Although phosphorylation of NOS3 Ser1177 is indicative of agonist-induced activation, it is preceded by dephosphorylation at Thr495. TGF-β1 and -β3 but not -β2 responses can sensitize NOS3 for activation by inducing dephosphorylation at Thr495, and therefore contribute to NOS3 activation and NO-dependent vasorelaxation (7). Endoglin regulates TGF-β1 and -β3 but not -β2 responses, and is required for their induction of NOS3 Thr495 dephosphorylation (7, 8).In the vascular endothelium of HHT patients and in Eng and Alk1 heterozygous mice, impaired association of NOS3 with HSP90 renders the enzyme uncoupled, causing production of superoxide (●O₂⁻) instead of NO● (2, 3, 9) and leading to endothelial damage. Interestingly, TGF-β1 and -β3 do not induce phosphorylation at NOS3 Ser1177, yet NOS3 activation in response to TGF-β1 is abolished in endoglin-deficient cells, impairing vasomotor function (3). ACVRL1 (or ALK1) also interacts with NOS3, and its reduced levels in endothelial cells similarly cause NOS3-derived oxidative stress (3, 9).In view of the crucial roles of endoglin and ACVRL1 in the development and maintenance of the normal vasculature and the definite contribution of their mutated state to HHT, we used the LUMIER high-throughput technology (10) to identify novel protein interactions and molecular networks for these predominantly endothelial receptors. We included TGFBR2 to further define TGF-β protein networks potentially important for vascular function, and attempt to distinguish the TGF-β networks from those associated with BMP9/BMP10 and mediated by ACVRL1 in a complex with BMPR2 and endoglin (11, 12).One of identified proteins interacting with all three receptors was protein phosphatase 2A (PP2A), implicated in multiple pathways. PP2A is a holoenzyme with one structural subunit (PPP2R1A or PPP2R1B) associated with one catalytic subunit (PPP2CA or PPP2CB) and one of 19 regulatory B subunits, the latter conferring specificity to the enzyme by recruiting interacting proteins (13, 14). Of interest, PP2A interacts with NOS3 to regulate Ser1177 phosphorylation and NO● production (15). However, the mechanisms governing recruitment of PP2A to NOS3 and the contribution of TGF-β/BMP receptor complexes are unknown. Recently, the human PPP2R2B gene coding for PPP2R2B protein (also known as PP2A-Bβ regulatory subunit) was mapped to chromosome 5q31-q32, in an interval in linkage disequilibrium with the HHT3 locus (16, 17). We now report that PPP2R2B interacts with the ACVRL1/TGFBR2/endoglin complex and that endoglin governs NOS3 phosphorylation and activation status by hindering PP2A access to NOS3 via the PPP2R2B subunit. Loss of endoglin leads to constitutive reduction in NOS3 phosphorylation and likely to changes in several networks with consequent endothelial dysfunction.     

Regulation of the Interaction between Protein Kinase C-related Protein Kinase 2 (PRK2) and Its Upstream Kinase, 3-Phosphoinositide-dependent Protein Kinase 1 (PDK1)

The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by ³²P_i revealed phosphorylation at two sites, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do not apply for other AGC kinases.The regulation of protein function by phosphorylation and dephosphorylation is a key mechanism of intracellular signaling pathways in eukaryotic organisms. Protein phosphorylation is catalyzed by protein kinases, which are themselves often regulated by phosphorylation (1). The specificity of protein kinases is essential for their cellular functions. In some groups of protein kinases, the specificity is achieved by means of “docking interactions.” Protein kinase docking interactions involve a recognition site on the kinase or a flanking domain that is different from the active site. The most notable example, MAP kinases, uses a docking interaction to specifically recognize substrates, upstream kinases, and phosphatases. Despite the large amount of data on protein kinase docking interactions, e.g. in the MAP kinase field, there is very little information on how these essential interactions are regulated (2–4).3-Phosphoinositide-dependent protein kinase 1 (PDK1)³ belongs to the AGC family of protein kinases and is the activation loop kinase for several other AGC kinases (5). A key feature of the AGC kinase family members except PDK1 is the presence of a C-terminal extension (CT) to the catalytic core that contains a conserved hydrophobic motif (HM) harboring a phosphorylation site. In many AGC kinases, the HM mediates a docking interaction with PDK1. For example, p90 ribosomal S6 kinase (RSK), p70 S6 kinase (S6K) and serum- and glucocorticoid-induced protein kinase (SGK) interact with PDK1 upon phosphorylation of the HM site (6–9). The phosphorylated HM binds to a HM-binding pocket in the catalytic core of PDK1 that was originally termed the PIF-binding pocket (6, 10).Besides its role in the docking of substrates to PDK1, the HM/PIF-binding pocket was also identified as a ubiquitous and key regulatory site in likely all AGC kinases (7, 11). Thus, in AGC kinases studied up to now, the HM/PIF-binding pocket serves as an intramolecular docking site for the phosphorylated HM. In summary, the HM has a dual function in AGC kinase activation, (i) mediating the intermolecular interaction with PDK1 and (ii) acting as an intramolecular allosteric activator that stabilizes the active conformation of the kinase domain via binding to the HM/PIF-binding pocket.The CT of AGC kinases additionally contains a second regulatory phosphorylation site traditionally termed the “turn motif” (TM), and more recently the zipper (Z) site. The Z/TM phosphate interacts with a binding site within the kinase domain, acting like a zipper which serves to support the intramolecular binding of the phosphorylated HM to the HM/PIF-binding pocket (12). Hence, AGC kinases are synergistically activated by phosphorylation at the activation loop, the HM, and the Z/TM sites.Protein kinase C-related protein kinases (PRKs) (13) (also named PAK for protease-activated kinase (14–16) and PKN for protein kinase N (17)) represent a subfamily of AGC kinases. So far, three PRK isoforms were identified, PRK1, PRK2, and PKN3, which are effectors of the small GTP-binding protein Rho. PRKs, as well as the Rho-associated kinases (ROCKs), are considered to be the protein kinases that mediate the phosphorylation events downstream of Rho activation and both can be inhibited by the highly specific protein kinase inhibitor Y27632 (18). The most notable role described for PRK2 is the control of entry into mitosis and exit from cytokinesis (19). In addition, PRK2 phosphorylates the hepatitis C virus (HCV) RNA polymerase (20). In support of a function in HCV RNA replication, PRK2 inhibitors like Y27632 suppress HCV replication (21).The N-terminal region of PRK2 possesses three Rho effector (HR1) domains (13), a pseudosubstrate region that is thought to have an autoinhibitory function (22) and a C2-like domain, which is a potential binding site for lipid activators. The C-terminal region of PRK2 harbors the HM that mediates the docking interaction with the HM/PIF-binding pocket in its upstream kinase PDK1 (10, 23). Interestingly, PRKs and also atypical protein kinase Cs (PKCs, PKCζ, and PKCι/λ), contain an acidic residue instead of a phosphorylatable amino acid at the site equivalent to the HM phosphorylation site in other AGC kinases. Therefore, the molecular events that regulate the interaction of PRK2 and PKCζ with PDK1 must be different from the mechanism characterized for S6K, SGK, and RSK.In the present work we extended and refined the model of docking interaction between PRK2 and PDK1 and characterized C-terminal regions of PRK2 that participate in the regulation of this interaction. The work sheds light on the common as well as specific mechanisms that operate in the regulation of PDK1 docking interaction with its different substrates.     

A Novel cdc2-Related Protein Kinase Expressed in the Nervous System

Abstract: We report the cloning and characterization of a cDNA encoding a cdc2-related protein kinase, named PFTAIRE, that is expressed primarily in the postnatal and adult nervous system. We have demonstrated by in situ hybridization and indirect immunofluorescence that several populations of terminally differentiated neurons and some neuroglia expressed PFTAIRE mRNA and protein. In neurons, PFTAIRE protein was localized in the nucleus and cytoplasm of cell bodies. The anatomical, cellular, and ontogenic patterns of PFTAIRE expression in the nervous system differed from those of p34^cdc2 and cdk5, which are expressed in brain and several other mitotic tissues. Proteins of ～58–60 kDa coprecipitated specifically with PFTAIRE from cytosolic protein preparations of adult mouse brain and transfected cells. These proteins appeared to be the major endogenous substrates associated with this kinase activity. The temporal and spatial expression patterns of PFTAIRE in the postnatal and adult nervous system suggest that PFTAIRE kinase activity may be associated with the postmitotic and differentiated state of cells in the nervous system and that its function may be distinct from those of p34^cdc2 and cdk5.     

The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis

Mitochondrial translation involves a complex interplay of ancient bacteria-like features and host-derived functionalities. Although the basic components of the mitochondrial translation apparatus have been recognized, very few protein factors aiding in recruiting ribosomes on mitochondria-encoded messenger RNA (mRNAs) have been identified in higher plants. In this study, we describe the identification of the Arabidopsis (Arabidopsis thaliana) MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) protein, a new member of the Pentatricopeptide Repeat family, and show that it is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. We demonstrate that mtl1 mutant plants fail to accumulate the Nad7 protein, even though the nad7 mature mRNA is produced and bears the same 5′ and 3′ extremities as in wild-type plants. We next observed that polysome association of nad7 mature mRNA is specifically disrupted in mtl1 mutants, indicating that the absence of Nad7 results from a lack of translation of nad7 mRNA. These findings illustrate that mitochondrial translation requires the intervention of gene-specific nucleus-encoded PPR trans-factors and that their action does not necessarily involve the 5′ processing of their target mRNA, as observed previously. Interestingly, a partial decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that MTL1 is also involved in group II intron splicing. However, this second function appears to be less essential for nad7 expression than its role in translation. MTL1 will be instrumental to understand the multifunctionality of PPR proteins and the mechanisms governing mRNA translation and intron splicing in plant mitochondria.Translation is the fundamental process decoding the genetic message present on mRNAs into proteins. In plant cells, mRNA translation occurs in the cytoplasm but also in two organelles, mitochondria and plastids. Because of their prokaryotic origin, the translation machineries operating in these two organelles share many characteristics with the bacterial translation apparatus (Bonen, 2004; Barkan, 2011). However, most of these bacteria-like features have been modified throughout evolution, and current organellar translation systems cooperate with numerous nucleus-encoded eukaryotic trans-factors. The divergence from bacteria is particularly obvious in plant mitochondria, notably because mitochondrial mRNAs lack the typical Shine and Dalgarno (SD) motif in their 5′ leaders and alternative start codons other than AUG are often used to initiate translation (Bonen, 2004). Proteomic and bioinformatic analyses allowed the identification of most proteins and RNA factors forming the core of the plant mitochondrial translation machinery, including translation initiation and elongation factors as well as ribosomal proteins (Bonen, 2004; Bonen and Calixte, 2006). However, the dynamics of this machinery remains largely obscure. In particular, nothing is known about the recruitment of mitochondrial ribosomes on 5′ untranslated regions in the absence of the SD motif and about the recognition of the correct translation initiation codon by the small ribosomal subunit. The high degree of sequence divergence among 5′ leaders of mitochondrial genes suggests a ribosome recruitment mechanism involving gene-specific cis-sequences and trans-factors (Hazle and Bonen, 2007; Choi et al., 2012). Up to now, only two proteins belonging to the Pentatricopeptide Repeat (PPR) family have been found to promote mitochondrial translation in higher plants (Uyttewaal et al., 2008b; Manavski et al., 2012). How they facilitate translation is still unclear, as for the few characterized PPR proteins shown to participate in plastid translation (Fisk et al., 1999; Schmitz-Linneweber et al., 2005; Cai et al., 2011; Zoschke et al., 2012, 2013). The plastid PENTATRICOPEPTIDE REPEAT PROTEIN10 (PPR10) protein of maize (Zea mays) is the only one for which the function has been elucidated at the molecular level. It was shown that, upon binding, PPR10 impedes the formation of a stem-loop structure in the 5′ leader of the ATP synthase subunit c (atpH) mRNA, permitting the recruitment of ribosomes through the liberation of an SD motif (Prikryl et al., 2011).PPR proteins represent a large family of RNA-binding proteins that has massively expanded in terrestrial plants (Barkan and Small, 2014). Most eukaryotes encode a handful of these proteins, whereas plant nuclear genomes express over 400 PPR proteins that are almost exclusively predicted to target mitochondria and/or plastids (Lurin et al., 2004; O’Toole et al., 2008). This family of proteins is characterized by the succession of tandem degenerate motifs of approximately 35 amino acids (Small and Peeters, 2000; Lurin et al., 2004). Based on the length of these repeats, the PPR family has been divided into two groups of roughly equal size in higher plants. P-type PPR proteins contain only successions of canonical 35-amino acid repeats (P), whereas PLS PPR proteins are composed of sequential repeats of P, short (S), and long (L) PPR motifs. P-type PPR proteins were shown to participate in various aspects of organellar RNA processing, whereas PLS PPR proteins have been almost exclusively associated with C-to-U RNA editing (for review, see Barkan and Small, 2014; Hammani and Giegé, 2014). Recent crystal structures showed that PPR motifs adopt an antiparallel helix-turn-helix fold whose repetition forms a solenoid-like structure (Ringel et al., 2011; Howard et al., 2012; Ban et al., 2013; Yin et al., 2013; Coquille et al., 2014; Gully et al., 2015). PPR tracks organize highly specific interaction domains that were shown to associate with single-stranded RNAs (Schmitz-Linneweber et al., 2005; Beick et al., 2008; Uyttewaal et al., 2008a; Williams-Carrier et al., 2008; Pfalz et al., 2009; Cai et al., 2011; Hammani et al., 2011; Prikryl et al., 2011; Khrouchtchova et al., 2012; Manavski et al., 2012; Zhelyazkova et al., 2012; Ke et al., 2013; Yin et al., 2013). The mechanism of sequence-specific RNA recognition by PPR proteins was recently uncovered, and combinations involving amino acid 6 of one motif and amino acid 1 of the subsequent motif correlate strongly with the identity of the RNA base to be bound (Barkan et al., 2012; Takenaka et al., 2013; Yagi et al., 2013).Besides those involved in RNA editing, few mitochondria-targeted PPR proteins have been characterized to date. Thus, our knowledge of the mechanisms governing the production and the expression of mitochondrial RNAs in higher plants is very limited. In this analysis, we describe the function of a novel mitochondria-targeted PPR protein of Arabidopsis (Arabidopsis thaliana) called MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1). Genetic and biochemical analyses indicate that MTL1 is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. Effectively, the Nad7 protein does not accumulate to detectable levels in mtl1 mutants, and this absence correlates with a lack of association of nad7 mature mRNA with mitochondrial polysomes. Interestingly, a partial but significant decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that the MTL1 protein is also involved in group II intron splicing. Since the decrease in splicing was only partial, this second function of MTL1 appears less essential for nad7 expression than its role in translation.     

Role of Calmodulin-Calmodulin Kinase II,cAMP/Protein Kinase A and ERK 1/2 on Aeromonas hydrophila-Induced Apoptosis of Head Kidney Macrophages

The role of calcium (Ca²⁺) and its dependent protease calpain in Aeromonas hydrophila-induced head kidney macrophage (HKM) apoptosis has been reported. Here, we report the pro-apoptotic involvement of calmodulin (CaM) and calmodulin kinase II gamma (CaMKIIg) in the process. We observed significant increase in CaM levels in A. hydrophila-infected HKM and the inhibitory role of BAPTA/AM, EGTA, nifedipine and verapamil suggested CaM elevation to be Ca²⁺-dependent. Our studies with CaM-specific siRNA and the CaM inhibitor calmidazolium chloride demonstrated CaM to be pro-apoptotic that initiated the downstream expression of CaMKIIg. Using the CaMKIIg-targeted siRNA, specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKIIg signalling to be critical for apoptosis of A. hydrophila-infected HKM. Inhibitor studies further suggested the role of calpain-2 in CaMKIIg expression. CaMK Kinase (CaMKK), the other CaM dependent kinase exhibited no role in A. hydrophila-induced HKM apoptosis. We report increased production of intracellular cAMP in infected HKM and our results with KN-93 or KN-92 implicate the role of CaMKIIg in cAMP production. Using siRNA to PKACA, the catalytic subunit of PKA, anti-PKACA antibody and H-89, the specific inhibitor for PKA we prove the pro-apoptotic involvement of cAMP/PKA pathway in the pathogenicity of A. hydrophila. Our inhibitor studies coupled with siRNA approach further implicated the role of cAMP/PKA in activation of extracellular signal-regulated kinase 1 and 2 (ERK 1/2). We conclude that the alteration in intracellular Ca²⁺ levels initiated by A. hydrophila activates CaM and calpain-2; both pathways converge on CaMKIIg which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM.     

A Novel,Universally Active C-terminal Protein Degradation Signal Generated by Alternative Splicing

Proteome integrity is crucial for cellular homeostasis and adaptation to stress conditions such as hypoxia. One mechanism for rapid adaptation of the proteome in response to changing environmental signals is alternative splicing. In addition to generating different protein isoforms, alternative splicing is also capable of controlling total protein levels by the regulated synthesis of non-productive mRNA isoforms. The hypoxia-induced isoform E of the tumor suppressor MAX is produced by retention and translation of the last intron. This leads to an alternative C-terminus that harbors a potent C-degron, the isoE degron. Strikingly, the isoE degron represents a universal protein degradation signal that is not only functional in mammalian cells, but also in yeast and even in bacteria. Essential for efficient protein decay is a conserved (F/W)xxW motif. Degradation of isoE tagged proteins is mediated by the proteasome in eukaryotes and Lon protease in bacteria. Thus, the isoE degron is a broadly applicable and highly efficient tool in protein analyses.