Stimulation of CK2-dependent Grp94 phosphorylation by the nuclear localization signal peptide

The nuclear localization signal sequence (NLS) of SV40 Large T antigen is essential and sufficient for the nuclear translocation of the protein. Phosphorylation often modulates the intracellular distribution of signaling proteins. In this study, we investigated effects of the NLS-peptide of Large T antigen on protein phosphorylation. When crude cell lysates were incubated with [γ-³²P]ATP, phosphorylation of several endogenous substrates with molecular masses of 100, 80, 50, and 45 kDa by an endogenous kinase was stimulated by the addition of the wild type NLS-peptide (CPKKKRKVEDP). The mutated NLS-peptide (CPKTKRKVEDP) and the reversed NLS-peptide (PDEVKRKKKPC) are weak in the nuclear localization activity, and they only weakly stimulated phosphorylation of these substrates. The mobility of the 100 kDa phosphoprotein was indistinguishable with that of an endoplasmic reticulum (ER)-resident molecular chaperone glucose-regulated protein 94 (Grp94) belonging to the Hsp90 family, and purified Grp94 was phosphorylated by a kinase in cell lysates in an NLS-dependent fashion. The 100 kDa protein was identified as Grp94 by immunoprecipitation and reconstitution experiments. Purification of the NLS-dependent Grp94 kinase by sequential biochemical column chromatography steps resulted in isolation of two polypeptides with molecular masses of 42 and 27 kDa, which were identified as α and β subunit of protein kinase CK2, respectively, by western blotting analysis and biochemical characterization. Moreover, effect of an excess amount of GTP and V8 peptide mapping showed that the NLS-dependent Grp94 kinase in the cell lysate is identical with CK2. Surprisingly purified CK2 did phosphorylate Grp94 even without the NLS-peptide, suggesting that an additional suppressive factor is required for NLS-dependent phosphorylation of Grp94 by CK2. We suggest a possible general role for CK2-catalyzed phosphorylation in the regulation of NLS-dependent protein nuclear translocation.     

Phosphorylation by CK2 and MAPK enhances calnexin association with ribosomes.

Calnexin was initially identified as an endoplasmic reticulum (ER) type I integral membrane protein, phosphorylated on its cytosolic domain by ER-associated protein kinases. Although the role of the ER luminal domain of calnexin has been established as a constituent of the molecular chaperone machinery of the ER, less is known about the role of the cytosolic phosphorylation of calnexin. Analysis by two-dimensional phosphopeptide maps revealed that calnexin was in vitro phosphorylated in isolated microsomes by casein kinase 2 (CK2) and extracellular-signal regulated kinase-1 (ERK-1) at sites corresponding to those for in vivo phosphorylation. In canine pancreatic microsomes, synergistic phosphorylation by CK2 and ERK-1 led to increased association of calnexin with membrane-bound ribosomes. In vivo, calnexin-associated ERK-1 activity was identified by co-immunoprecipitation. This activity was abolished in cells expressing a dominant-negative MEK-1. Activation of ERK-1 in cells by addition of serum led to a 4-fold increase in ribosome-associated calnexin over unstimulated cells. Taken together with studies revealing calnexin association with CK2 and ERK-1, a model is proposed whereby phosphorylation of calnexin leads to a potential increase in glycoprotein folding close to the translocon.     

The cytosolic protein kinase CK2 phosphorylates cardiac calsequestrin in intact cells

The luminal SR protein CSQ2 contains phosphate on roughly half of the serines found in its C-terminus. The sequence around phosphorylation sites in CSQ2 suggest that the in vivo kinase is protein kinase CK2, even though this enzyme is thought to be present only in the cytoplasm and nucleus. To test whether CSQ2 kinase is CK2, we combined approaches that reduced CK2 activity and CSQ2 phosphorylation in intact cells. Tetrabromocinnamic acid, a specific inhibitor of CK2, inhibited both the CSQ2 kinase and CK2 in parallel across a range of concentrations. In intact primary adult rat cardiomyocytes and COS cells, 24 h of drug treatment reduced phosphorylation of overexpressed CSQ2 by 75%. Down-regulation of CK2α subunits in COS cells using siRNA, produced a 90% decrease in CK2α protein levels, and CK2-silenced COS cells exhibited a twofold reduction in CSQ2 kinase activity. Phosphorylation of CSQ2 overexpressed in CK2-silenced cells was also reduced by a factor of two. These data suggested that CSQ2 in intact cells is phosphorylated by CK2, a cytosolic kinase. When phosphorylation site mutants were analyzed in COS cells, the characteristic rough endoplasmic reticulum form of the CSQ2 glycan (GlcNAc2Man9,8) underwent phosphorylation site dependent processing such that CSQ2-nonPP (Ser to Ala mutant) and CSQ2-mimPP (Ser to Glu mutant) produced apparent lower and greater levels of ER retention, respectively. Taken together, these data suggest CK2 can phosphorylate CSQ2 co-translationally at biosynthetic sites in rough ER, a process that may result in changes in its subsequent trafficking through the secretory pathway.     

Decreased protein and mRNA expression of ER stress proteins GRP78 and GRP94 in HepG2 cells over-expressing CYP2E1

CYP2E1 causes oxidative stress mediated cell death; the latter is one mechanism for endoplasmic reticulum (ER) stress in the cell. Unfolded proteins accumulate during ER stress and ER resident proteins GRP78 and GRP94 protect cells against ER dysfunction. We examined the possible role of GRP78 and GRP94 as protective factors against CYP2E1-mediated toxicity in HepG2 cells expressing CYP2E1 (E47 cells). E47 cells expressed high levels of CYP2E1 protein and catalytic activity which is associated with increased ROS generation, lipid peroxidation and the elevated presence of ubiquinated and aggregated proteins as compared to control HepG2 C34 cells which do not express CYP2E1. The mRNA and protein expression of GRP78 and GRP94 were decreased in E47 cells compared to the C34 cells, which may explain the accumulation of ubiquinated and aggregated proteins. Expression of these GRP proteins was induced with the ER stress agent thapsigargin in E47 cells, and E47 cells were more resistant to the toxicity caused by thapsigargin and calcimycin, possibly due to this upregulation and also because of the high expression of GSH and antioxidant enzymes in E47 cells. Antioxidants such as trolox and N-acetylcysteine increased GRP78 and GRP94 levels in the E47 cells, suggesting that CYP2E1- derived oxidant stress was responsible for down regulation of these GRPs in the E47 cells. Thapsigargin mediated toxicity was decreased in cells treated with the antioxidant trolox indicating a role for oxidative stress in this toxicity. These results suggest that CYP2E1 mediated oxidative stress downregulates the expression of GRP proteins in HepG2 cells and oxidative stress is an important mechanism in causing ER dysfunction in these cells.     

Brefeldin A, thapsigargin, and AIF4- stimulate the accumulation of GRP78 mRNA in a cycloheximide dependent manner, whilst induction by hypoxia is independent of protein synthesis.

The glucose regulated proteins (GRPs) are major structural components of the endoplasmic reticulum (ER) and are involved in the import, folding, and processing of ER proteins. Expression of the glucose regulated proteins (GRP78 and GRP94) is greatly increased after cells are exposed to stress agents (including A23187 and tunicamycin) which inhibit ER function. Here, we demonstrate that three novel inhibitors of ER function, thapsigargin (which inhibits the ER Ca(2+)-ATPase), brefeldin A (an inhibitor of vesicle transport between the ER and Golgi) and AIF4-, (which inhibits trimeric G-proteins), can increase the expression of both GRP78 and 94. The common characteristic shared by activators of GRP expression is that they disrupt some function of the ER. The increased levels of GRPs may be a response to the accumulation of aberrant proteins in the ER or they may be increased in response to structural/functional damage to the ER. The increased accumulation of GRP78 mRNA after exposure of cells to either thapsigargin, brefeldin A, AIF4-, A23187, or tunicamycin can be blocked by pre-incubation in cycloheximide. In contrast, accumulation of GRPs after exposure to hypoxia was independent of cycloheximide. In addition, the protein kinase inhibitor genistein blocked the thapsigargin induced accumulation of GRP78 mRNA, whereas the protein phosphatase inhibitor okadaic acid caused increased accumulation of GRP78 mRNA. The data indicates that there are at least 2 mechanisms for induced expression of GRPs, one of which involves a phosphorylation step and requires new protein synthesis (e.g., thapsigargin, A23187) and one which is independent of both these steps (hypoxia).     

Endoplasmic reticulum stress-inducible protein GRP94 is associated with an Mg2+-dependent serine kinase activity modulated by Ca2+ and GRP78/BiP

The 94-kDa glucose-regulated protein (GRP94) is a glycoprotein in the endoplasmic reticulum (ER). It has been characterized as a Ca²⁺-binding protein and a molecular chaperone. In this report we show that highly purified GRP94 exhibits an active Mg²⁺-dependent serine kinase activity (termed 94-kinase). The 94-kinase can be recovered from ER membrane fractions and is able to phosphorylate both the constitutive and stress-induced forms of GRP94, correlating with their induction kinetics. The 94-kinase activity is distinct from casein kinase II. In contrast to the heat-stable, Ca²⁺-dependent autophosphorylation activity recently reported for GRP94, the labile 94-kinase activity is inhibited by Ca²⁺. We determined that the phosphopeptide map of in vitro phosphorylated GRP94 by the 94-kinase resembles that of the in vivo phosphorylated GRP94. Further, the 94-kinase activity can be specifically stimulated by GRP78, a coregulated protein in the ER known to interact with GRP94. J. Cell. Physiol. 170:115–129, 1997. © 1997 Wiley-Liss, Inc.     

Dimerization and release of molecular chaperone inhibition facilitate activation of eukaryotic initiation factor-2 kinase in response to endoplasmic reticulum stress

Phosphorylation of eukaryotic initiation factor-2 (eIF2) by pancreatic eIF2 kinase (PEK), induces a program of translational expression in response to accumulation of malfolded protein in the endoplasmic reticulum (ER). This study addresses the mechanisms activating PEK, also designated PERK or EIF2AK3. We describe the characterization of two regions in the ER luminal portion of the transmembrane PEK that carry out distinct functions in the regulation of this eIF2 kinase. The first region mediates oligomerization between PEK polypeptides, and deletion of this portion of PEK blocked induction of eIF2 kinase activity. The second characterized region of PEK facilitates interaction with ER chaperones. In the absence of stress, PEK associates with ER chaperones GRP78 (BiP) and GRP94, and this binding is released in response to ER stress. ER luminal sequences flanking the transmembrane domain are required for GRP78 interaction, and deletion of this portion of PEK led to its activation even in the absence of ER stress. These results suggest that this ER chaperone serves as a repressor of PEK activity, and release of ER chaperones from PEK when misfolded proteins accumulate in the ER induces gene expression required to enhance the protein folding capacity of the ER.     

The stress response in Chinese hamster ovary cells. Regulation of ERp72 and protein disulfide isomerase expression and secretion

Expression of the glucose-regulated proteins (GRPs), GRP78 and GRP94, is induced by a variety of stress conditions including treatment of cells with tunicamycin or the calcium ionophore A23187. The stimulus for induction of these resident endoplasmic reticulum (ER) proteins appears to be accumulation of misfolded or underglycosylated protein within the ER. We have studied the induction of mRNAs encoding two other resident ER proteins, ERp72 and protein disulfide isomerase (PDI), during the stress response in Chinese hamster ovary cells. ERp72 shares amino acid sequence homology with PDI within the presumed catalytic active sites. ERp72 mRNA and, to a lesser degree, PDI mRNA were induced by treatment of Chinese hamster ovary cells with tunicamycin or A23187. These results identify ERp72 as a member of the GRP family. Stable high level overproduction of ERp72 or PDI from recombinant expression vectors did not alter the constitutive or induced expression of other GRPs. High level overexpression resulted in secretion of the overproduced protein specifically but not other resident ER proteins. This suggests that the ER retention mechanism is mediated by more specific interactions than just KDEL sequence recognition.     

DHA induces apoptosis by altering the expression and cellular location of GRP78 in colon cancer cell lines

n-3 polyunsaturated fatty acids exert growth-inhibitory and pro-apoptotic effects in colon cancer cells. We hypothesized that the anti-apoptotic glucose related protein of 78kDa (GRP78), originally described as a component of the unfolded protein response in endoplasmic reticulum (ER), could be a molecular target for docosahexaenoic acid (DHA) in these cells. GRP78 total and surface overexpression was previously associated with a poor prognosis in several cancers, whereas its down-regulation with decreased cancer growth in animal models. DHA treatment induced apoptosis in three colon cancer cell lines (HT-29, HCT116 and SW480), and inhibited their total and surface GRP78 expression. The cell ability to undergo DHA-induced apoptosis was inversely related to their level of GRP78 expression. The transfection of the low GRP78-expressing SW480 cells with GRP78-GFP cDNA significantly induced cell growth and inhibited the DHA-driven apoptosis, thus supporting the essential role of GRP78 in DHA pro-apoptotic effect. We suggest that pERK1/2 could be the first upstream target for DHA, and demonstrate that, downstream of GRP78, DHA may exert its proapoptotic role by augmenting the expression of the ER resident factors ERdj5 and inhibiting the phosphorylation of PKR-like ER kinase (PERK), known to be both physically associated with GRP78, and by activating caspase-4. Overall, the regulation of cellular GRP78 expression and location is suggested as a possible route through which DHA can exert pro-apoptotic and antitumoral effects in colon cancer cells.     

Determination of a putative phosphate-containing peptide in calreticulin.

Calreticulin is an abundant endo/sarcoplasmic reticulum (ER/SR) protein that may carry out multiple functions inside cells. Except for calreticulin, all of the major ER/SR Ca2+-binding proteins are substrates for protein kinase CK2 in vitro, which led us to hypothesize that native calreticulin might exist in the phosphorylated form. To investigate this possibility, we purified calreticulin from cardiac microsomes and verified its identity by immunoblot analysis and sequencing of tryptic peptides. Purified calreticulin, like cardiac calsequestrin, contained endogenous phosphate as determined by a Malachite green assay for phosphate. Previous analyses of cardiac calsequestrin have localized phosphate to a single tryptic peptide containing serine phosphate on sites phosphorylated by protein kinase CK2. Using a similar procedure, we analyzed calreticulin tryptic peptides with Malachite green, localizing phosphate binding to a single calreticulin peptide 367LKEEEEDKK. As this peptide contains no phosphorylatable residues, our results suggest that calreticulin may tightly bind phosphate or a phosphate-containing molecule at this site.     

Effects of cyclic nucleotide dependent protein kinases on the endoplasmic reticulum Ca2+ pump of bovine pulmonary artery

This paper describes the stimulation by cyclic nucleotide dependent protein kinases on the Ca2+ uptake by isolated endoplasmic reticulum (ER) vesicles from the bovine main pulmonary artery. This ER fraction has previously been shown to be highly enriched in phospholamban, a protein kinase substrate that has been well characterized in cardiac sarcoplasmic reticulum (SR), where its phosphorylation is accompanied by an increased rate of Ca2+ uptake. As previously observed for the phosphorylation of phospholamban, the stimulation of the rate of Ca uptake was as high with cGMP dependent protein kinase as with cAMP dependent protein kinase. The effect of phosphorylation of the ER membranes from smooth muscle on the Ca2+ uptake was smaller than that seen in cardiac SR, and it was only observed if albumin was included during the isolation of the membranes. This relatively small effect is probably not due to a lower ratio of phospholamban to Ca2(+)-transport enzyme in the ER membranes as compared to cardiac SR. Several alternative explanations are discussed.     

Overexpression of GRP78 mitigates stress induction of glucose regulated proteins and blocks secretion of selective proteins in Chinese hamster ovary cells.

GRP78 is a resident protein of the endoplasmic reticulum (ER) and a member of the glucose regulated protein (GRP) family. Many secretion incompetent proteins are found in stable association with GRP78 and are retained in the ER. Some proteins which are destined for secretion transiently associate with GRP78. To further increase our understanding of the role of GRP78 in secretion, we have stably overexpressed GRP78 in Chinese hamster ovary (CHO) cells and examined the effect on protein secretion and the stress response. GRP78 overexpressing cells treated with tunicamycin or A23187 exhibited a reduced induction of endogenous GRP78 and GRP94 mRNAs compared to wild-type CHO cells. This suggests that GRP78 overexpression either alleviates the stress or is directly involved in signaling stress-induced expression of GRPs. Transient expression of secreted proteins was used to measure secretion efficiency in the GRP78 overexpressing cells. Secretion of von Willebrand factor and a mutant form of factor VIII, two proteins which transiently associate with GRP78, was reduced by GRP78 overexpression. In contrast, secretion of M-CSF, which was not detected in association with GRP78, was unaffected. This indicates that elevated levels of GRP78 may increase stable association and decrease the secretion efficiency of proteins which normally transiently associate with GRP78. These results indicate that one function of GRP78 is selective protein retention in the ER.