A role for the thiol-dependent reductase ERp57 in the assembly of MHC class I molecules

AbstractAn important mammalian defence strategy against intracellular pathogens is the presentation of cytoplasmically derived short peptides by major histocompatibility complex (MHC) class I molecules to cytotoxic T lymphocytes. MHC class I molecules assemble in the endoplasmic reticulum (ER) with chaperones, including calnexin and calreticulin, before binding to the transporter associated with antigen processing (TAP). We show here that the thiol-dependent reductase ERp57 (also known as ER60 protease) is involved in MHC class I assembly. ERp57 co-purified with the rat TAP complex (comprising TAP1 and TAP2), and associated with MHC class I molecules at an early stage in their biosynthesis. This association was sensitive to castanospermine, which inhibits the processing of glycoproteins. Human MHC class I molecules were also found to associate with ERp57. We conclude that ERp57 is a newly identified component of the MHC class I pathway, and that it appears to interact with MHC class I molecules before they associate with TAP

Phosphorylation of Sli15 by Ipl1 is important for proper CPC localization and chromosome stability in <em>Saccharomyces cerevisiae</em>

The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine (sli15-20A) or to acidic residues to mimic constitutive phosphorylation (sli15-20D). Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the 'tension checkpoint'--the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism

KA1-targeted regulatory domain mutations activate Chk1 in the absence of DNA damage

The Chk1 protein kinase is activated in response to DNA damage through ATR-mediated phosphorylation at multiple serine-glutamine (SQ) residues within the C-terminal regulatory domain, however the molecular mechanism is not understood. Modelling indicates a high probability that this region of Chk1 contains a kinase-associated 1 (KA1) domain, a small, compact protein fold found in multiple protein kinases including SOS2, AMPK and MARK3. We introduced mutations into Chk1 designed to disrupt specific structural elements of the predicted KA1 domain. Remarkably, six of seven Chk1 KA1 mutants exhibit constitutive biological activity (Chk1-CA) in the absence of DNA damage, profoundly arresting cells in G2 phase of the cell cycle. Cell cycle arrest induced by selected Chk1-CA mutants depends on kinase catalytic activity, which is increased several-fold compared to wild-type, however phosphorylation of the key ATR regulatory site serine 345 (S345) is not required. Thus, mutations targeting the putative Chk1 KA1 domain confer constitutive biological activity by circumventing the need for ATR-mediated positive regulatory phosphorylation

The multifunctional poly(A)-binding protein (PABP) 1 is subject to extensive dynamic post-translational modification, which molecular modelling suggests plays an important role in co-ordinating its activities

PABP1 [poly(A)-binding protein 1] is a central regulator of mRNA translation and stability and is required for miRNA (microRNA)-mediated regulation and nonsense-mediated decay. Numerous protein, as well as RNA, interactions underlie its multi-functional nature; however, it is unclear how its different activities are co-ordinated, since many partners interact via overlapping binding sites. In the present study, we show that human PABP1 is subject to elaborate post-translational modification, identifying 14 modifications located throughout the functional domains, all but one of which are conserved in mouse. Intriguingly, PABP1 contains glutamate and aspartate methylations, modifications of unknown function in eukaryotes, as well as lysine and arginine methylations, and lysine acetylations. The latter dramatically alter the pI of PABP1, an effect also observed during the cell cycle, suggesting that different biological processes/stimuli can regulate its modification status, although PABP1 also probably exists in differentially modified subpopulations within cells. Two lysine residues were differentially acetylated or methylated, revealing that PABP1 may be the first example of a cytoplasmic protein utilizing a ‘methylation/acetylation switch’. Modelling using available structures implicates these modifications in regulating interactions with individual PAM2 (PABP-interacting motif 2)-containing proteins, suggesting a direct link between PABP1 modification status and the formation of distinct mRNP (messenger ribonucleoprotein) complexes that regulate mRNA fate in the cytoplasm

Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets: identification of mitochondrial fission factor as a new AMPK substrate

AMP-activated protein kinase (AMPK) is a key cellular energy sensor and regulator of metabolic homeostasis. Although it is best known for its effects on carbohydrate and lipid metabolism, AMPK is implicated in diverse cellular processes, including mitochondrial biogenesis, autophagy, and cell growth and proliferation. To further our understanding of energy homeostasis through AMPK-dependent processes, the design and application of approaches to identify and characterise novel AMPK substrates are invaluable. Here, we report an affinity proteomicstrategy for the discovery and validation of AMPK targets using an antibody to isolate proteins containing the phospho-AMPK substrate recognition motif from hepatocytes that had been treated with pharmacological AMPK activators. We identified 57 proteins that were uniquely enriched in the activator-treated hepatocytes, but were absent in hepatocytes lacking AMPK. We focused on two candidates, cingulin and mitochondrial fission factor (MFF), and further characterised/validated them as AMPK-dependent targets by immunoblotting with phosphorylation site-specific antibodies. A small-molecule AMPK activator caused transient phosphorylation of endogenous cingulin at S137 in intestinal Caco2 cells. Multiple splice-variants of MFF appear to express in hepatocytes and we identified a common AMPK-dependent phospho-site (S129) in all the 3 predominant variants spanning the mass range and a short variant-specific site (S146). Collectively, our proteomic-based approach using a phospho-AMPK substrate antibody in combination with genetic models and selective AMPK activators will provide a powerful and reliable platform for identifying novel AMPK-dependent cellular targets

Constitutive phosphorylation of MDC1 physically links the MRE11–RAD50–NBS1 complex to damaged chromatin

The MRE11–RAD50–Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex accumulates at sites of DNA double-strand breaks (DSBs) in microscopically discernible nuclear foci. Focus formation by the MRN complex is dependent on MDC1, a large nuclear protein that directly interacts with phosphorylated H2AX. In this study, we identified a region in MDC1 that is essential for the focal accumulation of the MRN complex at sites of DNA damage. This region contains multiple conserved acidic sequence motifs that are constitutively phosphorylated in vivo. We show that these motifs are efficiently phosphorylated by caseine kinase 2 (CK2) in vitro and directly interact with the N-terminal forkhead-associated domain of NBS1 in a phosphorylation-dependent manner. Mutation of these conserved motifs in MDC1 or depletion of CK2 by small interfering RNA disrupts the interaction between MDC1 and NBS1 and abrogates accumulation of the MRN complex at sites of DNA DSBs in vivo. Thus, our data reveal the mechanism by which MDC1 physically couples the MRN complex to damaged chromatin

A cardiolipin-activated protein kinase from rat liver structurally distinct from the protein kinases C

A cardiolipin- and protease-activated protein kinase (PAK) has been isolated from cytoplasmic extracts of rat liver. The enzyme (PAK-1) phosphorylates the ribosomal protein S6-(229-239) peptide analogue and can be activated by limited proteolysis. Partial amino acid sequences of tryptic peptides derived from both the purified 116-kDa PAK-1 holoenzyme and its active catalytic fragment reveal that the catalytic domain is most related (50-58% identity) to the protein kinase C family. PAK-1 has protein and peptide substrate specificities distinct from those of known protein kinase C isoforms and is insensitive to inhibition by the protein kinase C-alpha-(19-31) pseudosubstrate peptide. Phosphatidylserine, diacylglycerol, and phorbol ester do not activate PAK-1 toward the S6 peptide substrate. However, other acidic phospholipids, the most effective being cardiolipin, activate PAK-1 to a similar extent as trypsin. The PAK-1 catalytic activities generated through activation by cardiolipin or limited proteolysis were kinetically similar, with K-m values of 3.6 and 3.4 mu M, respectively, for the S6-(229-239) peptide substrate. However, differences were observed in the catalytic activities with protamine sulfate and the glycogen synthase-(1-12) peptide analogue as substrates. It was concluded that PAK-1 is a phospholipid regulated protein kinase with a primary structure, substrate specificity, and mechanism of regulation in vitro distinct from those of any known member of the protein kinase C superfamily

Ebola virus VP35 induces high-level production of recombinant TPL-2–ABIN-2–NF-κB1 p105 complex in co-transfected HEK-293 cells

Activation of PKR (double-stranded-RNA-dependent protein kinase) by DNA plasmids decreases translation, and limits the amount of recombinant protein produced by transiently transfected HEK (human embryonic kidney)-293 cells. Co-expression with Ebola virus VP35 (virus protein 35), which blocked plasmid activation of PKR, substantially increased production of recombinant TPL-2 (tumour progression locus 2)–ABIN-2 [A20-binding inhibitor of NF-κB (nuclear factor κB) 2]–NF-κB1 p105 complex. VP35 also increased expression of other co-transfected proteins, suggesting that VP35 could be employed generally to boost recombinant protein production by HEK-293 cells

Dominant suppression of inflammation via targeted mutation of the mRNA destabilizing protein tristetraprolin

In myeloid cells, the mRNA-destabilizing protein tristetraprolin (TTP) is induced and extensively phosphorylated in response to LPS. To investigate the role of two specific phosphorylations, at serines 52 and 178, we created a mouse strain in which those residues were replaced by nonphosphorylatable alanine residues. The mutant form of TTP was constitutively degraded by the proteasome and therefore expressed at low levels, yet it functioned as a potent mRNA destabilizing factor and inhibitor of the expression of many inflammatory mediators. Mice expressing only the mutant form of TTP were healthy and fertile, and their systemic inflammatory responses to LPS were strongly attenuated. Adaptive immune responses and protection against infection by Salmonella typhimurium were spared. A single allele encoding the mutant form of TTP was sufficient for enhanced mRNA degradation and underexpression of inflammatory mediators. Therefore, the equilibrium between unphosphorylated and phosphorylated TTP is a critical determinant of the inflammatory response, and manipulation of this equilibrium may be a means of treating inflammatory pathologies