Phosphoinositide 3-kinase inhibitors protect mouse kidney cells from cyclosporine-induced cell death.

The use of cyclosporine has been restricted by its nephrotoxic effects mediated, in part, by reactive oxygen species (ROS). Phosphoinositide 3-kinase, protein kinase B, and extracellular regulated kinase (ERK) pathways are related to survival and cell death and are activated after ROS generation. In this study, we evaluated the effects of cyclosporine on these pathways and their contribution to cyclosporine-induced toxicity. Viability of cells derived from the proximal tubule of transgenic mice was measured with Trypan Blue, ROS generation by a fluorescent probe, while ERK and phosphoinositide 3-kinase/protein kinase B activation were monitored with phospho-specific antibodies. Cyclosporine decreased cell viability and induced ROS generation and ERK and phosphoinositide 3-kinase activation. Both pathways were activated by the epidermal growth factor receptor (EGFR). Antioxidants blocked ERK activation but failed to inhibit protein kinase B phosphorylation or prevent cyclosporine toxicity. ERK inhibition did not protect from cyclosporine-induced cell death. EGFR or phosphoinositide 3-kinase inhibitors protected from cyclosporine-triggered cell death without decreasing ROS. Small interfering RNA against the catalytic subunit of phosphoinositide 3-kinase decreased protein kinase B phosphorylation but did not prevent cyclosporine-mediated cell death. Our results show that EGFR mediates the cytotoxic effects of cyclosporine through an ROS-independent mechanism. Cyclosporine-induced cell death is triggered by a non-classical phosphoinositide 3-kinase and does not require ERK activation.

The carboxy-terminal domain of Grp94 binds to protein kinase CK2 alpha but not to CK2 holoenzyme.

Surface plasmon resonance analysis shows that the carboxy-terminal domain of Grp94 (Grp94-CT, residues 518-803) physically interacts with the catalytic subunit of protein kinase CK2 (CK2 alpha) under non-stressed conditions. A K(D) of 4 x 10(-7) was determined for this binding. Heparin competed with Grp94-CT for binding to CK2 alpha. CK2 beta also inhibited the binding of Grp94-CT to CK2 alpha, and CK2 holoenzyme reconstituted in vitro was unable to bind Grp94-CT. The use of CK2 alpha mutants made it possible to map the Grp94-CT binding site to the four lysine stretch (residues 74-77) present in helix C of CK2 alpha. Grp94-CT stimulated the activity of CK2 alpha wild-type but was ineffective on the CK2 alpha K74-77A mutant.

The C-terminal domain of human grp94 protects the catalytic subunit of protein kinase CK2 (CK2alpha) against thermal aggregation. Role of disulfide bonds.

The C-terminal domain (residues 518-803) of the 94 kDa glucose regulated protein (grp94) was expressed in Escherichia coli as a fusion protein with a His6-N-terminal tag (grp94-CT). This truncated form of grp94 formed dimers and oligomers that could be dissociated into monomers by treatment with dithiothreitol. Grp94-CT conferred protection against aggregation on the catalytic subunit of protein kinase CK2 (CK2alpha), although it did not protect against thermal inactivation. This anti-aggregation effect of grp94-CT was concentration dependent, with full protection achieved at grp94-CT/CK2alpha molar ratios of 4 : 1. The presence of dithiothreitol markedly reduced the anti-aggregation effects of grp94-CT on CK2alpha without altering the solubility of the chaperone. It is concluded that the chaperone activity of the C-terminal domain of human grp94 requires the maintenance of its quaternary structure (dimers and oligomers), which seems to be stabilised by disulphide bonds.

Osmotic stress regulates the stability of cyclin D1 in a p38SAPK2-dependent manner.

We report here that different cell stresses regulate the stability of cyclin D1 protein. Exposition of Granta 519 cells to osmotic shock, oxidative stress, and arsenite induced the post-transcriptional down-regulation of cyclin D1. In the case of osmotic shock, this effect was completely reversed by the addition of p38(SAPK2)-specific inhibitors (SB203580 or SB220025), indicating that this effect is dependent on p38(SAPK2) activity. Moreover, the use of proteasome inhibitors prevented this down-regulation. Thus, osmotic shock induces proteasomal degradation of cyclin D1 protein by a p38(SAPK2)-dependent pathway. The effect of p38(SAPK2) on cyclin D1 stability might be mediated by direct phosphorylation at specific sites. We found that p38(SAPK2) phosphorylates cyclin D1 in vitro at Thr(286) and that this phosphorylation triggers the ubiquitination of cyclin D1. These results link for the first time a stress-induced MAP kinase pathway to cyclin D1 protein stability, and they will help to understand the molecular mechanisms by which stress transduction pathways regulate the cell cycle machinery and take control over cell proliferation.

Differential action of nerve growth factor and phorbol ester TPA on rat synaptosomal PKC isoenzymes.

The subcellular redistribution of protein kinase C family members (alpha, beta, gamma, delta, epsilon and zeta isoforms) was examined in response to treatment with 12-O-tetradecanoyl-phorbol-13 acetate (TPA) or nerve growth factor (NGF) in a synaptosomal-enriched P2 fraction from rat brain. Treatment with TPA affected members of the classical-PKC family (alpha, beta and gamma), resulting in a final loss of total protein of each isoenzyme. The kinetics of changes of members of the novel-PKC family are different, the delta isoform being translocated, but not down-regulated, while the epsilon isoform showing only a slight diminishing of immunoreactivity in the soluble and particulate fractions. The atypical-PKC zeta isoform was not translocated in response to TPA. Incubation with NGF induced a loss of immunoreactivity of the cytosolic alpha, beta and epsilon isoforms, but the membrane fractions of these isoforms were not appreciably affected. In contrast, a marked translocation from cytosol to membrane was observed in the case of the gamma and delta isoforms. The zeta isoform presented a slight translocation from the particulate fraction to the soluble fraction. Thus, the results show that the effects of TPA and NGF on PKC isoforms are not coincident in synaptosomes, the 6 isoform being activated and not down-regulated by both treatments, whereas the gamma isoform is only down-regulated in the case of TPA, but presents sustained translocation with NGF, indicating that PKC isoform-specific degradation pathways exist in synaptic terminals. The effects of NGF on PKC isoforms coexist with an increase in NGF-induced polyphosphoinositide hydrolysis, suggesting the participation of phospholipases.

Tumour suppressor protein p53 released by nuclease digestion increases at the onset of rat liver regeneration.

BACKGROUND/AIMS: Protein kinase CK2 (CK2) increases when cells are committed to proliferate, as in liver regeneration. This enzyme phosphorylates the tumour suppressor protein p53, whose expression controls the levels of many other cell cycle proteins. The aim of this study was to determine if CK2 was affected by p53. METHODS: Male Sprague-Dawley rats (200-250 g) were subjected to either partial hepatectomy or laparotomy and the levels and subcellular distribution of p53 were studied, following the approach used earlier for CK2. The levels of both proteins were also studied in the human cell lines HL-60 (devoid of p53) and HepG2 (with normal p53 levels) and in fibroblasts from transgenic p53-deficient mice (p53-/-) or homozygous for wild-type p53 (p53+/+). Computer-assisted search was used to detect p53 consensus sequences in genes for CK2 subunits Binding of p53 protein to some of these sequences was assayed by electrophoretic mobility shift assay. RESULTS: Rat liver p53 protein was present mainly in the fraction extracted from intact nuclei by nucleases (S1) and showed a transient increase at 6 h post partial hepatectomy, as observed previously with nuclear CK2. The human CK2a gene presents the consensus sequence for trans-activation by p53 and specific binding of p53 protein to some of these sequences was detected in vitro. Total CK2a was higher in HepG2 than in HL-60 cells but total CK2 and its cytosolic/ nuclear distribution was similar in mice (p53+/+) fibroblasts and (p53-/-) fibroblasts. CONCLUSIONS: p53 is present in the nuclease-extracted S1 fraction from liver cells, as described for CK2, and undergoes similar changes at the beginning of rat liver regeneration. However, the data on cultured cells suggest that the expression of CK2 and its subcellular localization are p53-independent events.

Heterogeneous nuclear ribonucleoprotein A2 interacts with protein kinase CK2.

The catalytic subunit of protein kinase CK2 (CK2alpha) was found associated with heterogeneous nuclear ribonucleoprotein particles (hnRNPs) that contain the core proteins A2 and C1-C2. High levels of CK2 activity were also detected in these complexes. Phosphopeptide patterns of hnRNP A2 phosphorylated in vivo and in vitro by protein kinase CK2 were similar, suggesting that this kinase can phosphorylate hnRNPA2 in vivo. Binding experiments using human recombinant hnRNP A2, free human recombinant CK2alpha or CK2beta subunits, reconstituted CK2 holoenzyme and purified native rat liver CK2 indicated that hnRNP A2 associated with both catalytic and regulatory CK2 subunits, and that the interaction was independent of the presence of RNA. However, the capability of hnRNP A2 to bind to CK2 holoenzyme was lower than its binding to the isolated subunits. These data indicate that the association of CK2alpha with CK2beta interferes with the subsequent binding of hnRNP A2. HnRNP A2 inhibited the autophosphorylation of CK2beta. This effect was stronger with reconstituted human recombinant CK2 than with purified native rat liver CK2.

Association of protein kinase CK2 with eukaryotic translation initiation factor eIF-2 and with grp94/endoplasmin.

Protein kinase CK2 forms complexes with some protein substrates what may be relevant for the physiological control of this protein kinase. In previous studies in rat liver cytosol we had detected that the trimeric form of eukaryotic translation initiation factor 2 (eIF-2) co-eluted with protein kinase CK2. We have now observed that the ratio between eIF-2 and cytosolic CK2 contents in testis, liver and brain is quite similar, being eIF-2 levels about 5-fold higher than those of CK2. Furthermore eIF-2 was present in liver samples immunoprecipitated with anti-CK2alpha/alpha' antibodies, confirming the existence of complexes containing both proteins. Nonetheless, these complexes would represent only a fraction of total cytosolic CK2 and eIF-2. We had also observed that rat liver membrane glycoproteins obtained through chromatography on wheat-germ lectin-Sepharose contain CK2 activity which copurifies with grp94/endoplasmin. We have now confirmed that this activity was due to the presence of protein kinase CK2 as evidenced by immunodetection with antibodies against CK2alpha/alpha'. The fractions enriched in grp94/endoplasmin and CK2 also contained another 55-kDa polypeptide (p55) phosphorylated by CK2 which has been identified as calreticulin by N-terminal sequencing. Calreticulin and grp94/endoplasmin could be partially resolved from CK2 by chromatography on heparin-agarose and almost completely on ConA-Sepharose. However, phosphorylation of immunoprecipitated grp94/endoplasmin was enhanced by its preincubation with purified CK2 prior to immunoprecipitation, what confirms the easy reassociation between these proteins. The association of protein kinase CK2 with eIF-2 and with grp94/endoplasmin may serve to locate the enzyme in the cellular machinery involved in protein synthesis and folding, and reinforces the possible involvement of CK2 in these processes.

Multiple forms of protein kinase CK2 present in leukemic cells: in vitro study of its origin by proteolysis.

Human recombinant CK2 subunits were incubated for different times with the two main cytosolic proteases m-calpain and 20 S proteasome. Both, m-calpain in a calcium dependent manner and the 20 S proteasome, were able to degrade CK2 subunits in vitro. In both cases, CK2alpha' was more resistant to these proteases than CK2alpha. When these proteases were assayed on the reconstituted (alpha2beta2 holoenzyme), a 37 kDa alpha-band, analogous to that observed in AML extracts, was generated which was resistant to further degradation. No degradation was observed when the 26 S proteasome was assayed on free subunits. Studies with CK2alpha deletion mutants showed that m-calpain and the 20 S proteasome acted on the C-terminus end of CK2alpha. These results pointed to cytosolic proteases as agents involved in the control of the amount of free CK2 subunits within the cell, which becomes evident when CK2 is overexpressed as in AML cells.

Protein kinase CK2 is altered in insulin-resistant genetically obese (fa/fa) rats.

Hepatic insulin receptor levels in 6-week-old obese (fa/fa) rats were about 2-fold lower than those from lean (Fa/-) rats, which agrees with their insulin-resistant state. Nuclear protein kinase CK2 activity and protein content in livers from obese (fa/fa) rats were similar to those of lean (Fa/-) animals but the cytosolic levels were reduced to half, due to a decrease in the 39-kD)a catalytic subunit. Marked increases in activity, due to rises in the 44-kDa and 39-kDa catalytic subunits, were seen in the 16000 x g sediments (M1) from insulin-resistant rats, with moderate changes in the 100000xg sediments (M2). The increase in CK2 binding to M1 did not require increases in the molecular chaperone grp94, which was unaltered in insulin-resistant rats.

Substrates for protein kinase CK2 in insulin receptor preparations from rat liver membranes: identification of a 210-kDa protein substrate as the dimeric form of endoplasmin.

Chromatography of extracts from rat liver membranes on wheat-germ lectin-Sepharose resulted in a partial resolution of the insulin receptor from other phosphorylatable proteins. Among the latter, a protein (p210, with an apparent M(r) of 210 kDa on SDS/PAGE under nonreducing conditions) was found to be phosphorylated by protein kinase CK2 on Thr and Ser residues. Under reducing conditions p210 was resolved into two phosphopolypeptides with apparent M(r) of 95 and 105 kDa. Neither the 95-kDa nor the 105-kDa polypeptides were recognized by antibodies against the beta-subunit of the insulin receptor. Both polypeptides gave identical phosphopeptide maps after protease V8 digestion and contained the same N-terminal amino acid sequence. This sequence coincided with that of endoplasmin, and both polypeptides as well as p210 were recognized by antibodies against this protein. This shows that p210 corresponds to the dimeric form of rat liver endoplasmin. DEAE-Sepharose chromatography of p210 preparations removed most other contaminating proteins and revealed the presence of a protein kinase activity that coeluted with p210. This protein kinase possessed the properties (substrate specificity and inhibition by heparin) that are characteristic of the protein kinase CK2 enzymes. Furthermore, phosphoamino acid analysis and phosphopeptide maps of the 95/105-kDa polypeptides phosphorylated either by the endogenous protein kinase or by exogenous protein kinase CK2 gave similar results. The phosphorylation of p210/endoplasmin by protein kinase CK2 and its coelution gives support to the involvement of this protein kinase in membrane-associated processes.

Differential effect of alkyl chain-modified ether lipids on protein kinase C autophosphorylation and histone phosphorylation.

Analogues of 1-O-octadecyl-2-O-methyl-rac-glycerol-3-phosphocholine (ET-18-OMe), containing a carbonyl group at different positions in the alkyl chain and/or a pentylammonium group in sn-3 of glycerol, were evaluated as inhibitors of protein kinase C (PKC; EC 2.7.1.37). The presence of a carbonyl group in the alkyl chain of Et-18-OMe had a dual role in decreasing the inhibitory effect on histone phosphorylation and activating this reaction at low concentrations of compound. The optimal stimulatory effect was observed with the compound having the carbonyl function in C-7 of the alkyl chain. In contrast, all of these compounds were only inhibitors of PKC autophosphorylation, its potency decreasing progressively with the distance between the carbonyl group and the sn-1 position of glycerol. Replacement of the phosphocholine group of ET-18-OMe by a pentamethylene trimethylammonium group maintained the inhibitory effect on histone phosphorylation and autophosphorylation of PKC, and the simultaneous introduction of a ketone group in C-7 of the alkyl chain did not decrease any of these effects. The effects of all these analogues on PKC autophosphorylation, but not on histone phosphorylation, correlated quite well with their known antiproliferative activity on human tumor cell lines and membranolytic activity.

Rat liver pp49, a protein that forms complexes with protein kinase CK2, is composed of the beta and the gamma subunits of translation initiation factor eIF-2.

Comparison of the amino acid sequences of two peptides derived from proteolysis of rat liver pp49 identified it as composed of the beta-subunit and the gamma-subunit of eukaryotic initiation factor-2 (eIF-2). Partial purification of rat liver eIF-2 showed that its trimeric form (alpha beta gamma) co-eluted with protein kinase CK2. Heat-inactivated preparations of the trimeric form of eIF-2 inhibited CK2, increasing its Km for beta-casein, as observed with pp49. The form of eIF-2 that lacks the beta-subunit had no effect on CK2. These data indicate that the beta gamma subunits of eIF-2 may complex with CK2 and modulate its activity.

Changes in the activity of nuclear protein kinase CK2 during rat liver regeneration.

Protein kinase CK2 has been found in two nuclear fractions obtained after treatment of purified rat liver nuclei with nucleases (S1 fraction) and subsequently with 1.6 M NaCl (S2 fraction). In both fractions three isoforms of the alpha subunit were identified. Two of them corresponded to the classical alpha and alpha' subunits, whereas the identity of the third one (alpha 3) remains unknown. In the S1 fraction two peaks of CK2 activity were detected at 6 h (5.5 fold) and 24 h (1.9 fold) after partial hepatectomy, whereas no significant changes were found in the S2 fraction. At 6 h after laparatomy a much lower increase of CK2 in S1 fraction was also detected (2.5 fold). The increases in CK2 activity found at 6 h after hepatectomy or laparatomy were accompanied with rises in the amount of the alpha subunit.

Phosphorylation of the Goodpasture antigen by type A protein kinases.

Collagen IV is the major component of basement membranes. The human alpha 3 chain of collagen IV contains an antigenic domain called the Goodpasture antigen that is the target for the circulating immunopathogenic antibodies present in patients with Goodpasture syndrome. Characteristically, the gene region encoding the Goodpasture antigen generates multiple alternative products that retain the antigen amino-terminal region with a five-residue motif (KRGDS). The serine therein appears to be the major in vitro cAMP-dependent protein kinase phosphorylation site in the isolated antigen and can be phosphorylated in vitro by two protein kinases of approximately 50 and 41 kDa associated with human kidney plasma membrane, suggesting that it can also be phosphorylated in vivo. Consistent with this, the Goodpasture antigen is isolated from human kidney in phosphorylated and non-phosphorylated forms and only the non-phosphorylated form is susceptible to phosphorylation in vitro. Since this motif is exclusive to the human alpha 3(IV) chain and includes the RGD cell adhesion motif, its phosphorylation might play a role in pathogenesis and influence cell attachment to basement membrane.

Protein kinase CKII: possible regulation by interaction with protein substrates.

Rat liver cytosolic CKII shows heterogeneity resulting from association of the alpha/alpha'-subunits with the beta-subunit or with a phosphorylatable protein of 49 kDa (pp49). Preparations of pp49 were resolved into several spots by two dimensional analysis which might be derived from different degrees of phosphorylation. pp49 was phosphorylated in vitro by purified rat liver CKII and to a lower extent by purified rat brain protein kinase C. In all cases, phosphorylation of pp49 occurred exclusively on Ser. Phosphopeptide maps of phosphorylated pp49 confirmed that the phosphorylation by CKII or PKC takes place in different sites. Prior phosphorylation of pp49 by protein kinase C had no significant influence on the increase of the Km value for beta-casein of CKII, caused by pp49. A tryptic peptide from pp49 has been recently sequenced and antibodies against it had been raised. The antibodies were able to recognize pp49 in rat liver extracts as well as in HL-60 extracts what leads us to presume that this kind of interaction might exist in other species and tissues.

Characterization of the hepatic insulin receptor undergoing internalization through clathrin-coated vesicles and endosomes.

Administration of insulin to rats caused a transient increase in the amount of hepatic insulin receptor present in clathrin-coated vesicles and endosomes. However, the total 'in vitro' insulin stimulated tyrosine kinase activity of the receptor present in endosomes did not vary when expressed per mg of protein and decreased when expressed per beta-subunit content. A decrease in the endogenous phospho tyrosine content of the receptor beta-subunit was observed in endosomes in response to insulin. This indicates that a fraction of the internalized receptor is dephosphorylated in endosomes, which renders it unable to become stimulated by insulin 'in vitro'.

Phosphorylation of maize RAB-17 protein by casein kinase 2.

The maize gene RAB-17, which is responsive to abscisic acid, encodes a basic glycine-rich protein containing, in the middle part of its sequence, a cluster of 8 serine residues followed by a putative casein kinase 2-type substrate consensus sequence. This protein was found to be highly phosphorylated in vivo. Here, we show that RAB-17 protein is a real substrate for casein kinase 2. RAB-17 protein is phosphorylated in vitro by casein kinase 2 isolated from rat liver cytosol and from maize embryos. A maximum of 4 mol of phosphate were incorporated per mol of RAB-17 protein following incubation with casein kinase 2. Phosphopeptide mapping experiments show that the peptide phosphorylated by casein kinase 2 in vitro is identical to that derived from the protein phosphorylated in vivo. Purification by high performance liquid chromatography and partial sequencing of the phosphopeptide indicate that it corresponds to the region of the protein (residues 56-89) containing the cluster of serine residues. Our results indicate that RAB-17 is phosphorylated by casein kinase 2 or a kinase with a similar specificity and that phosphorylation takes place in the serine cluster region of the protein both in vitro and in vivo.

Heterogeneity of rat liver cytosol casein kinase 2. Association between the alpha/alpha' -subunits of casein kinase 2 and the phosphorylatable protein pp49.

Casein kinase 2 activity could be resolved into three peaks by chromatography on DEAE-Sepharose. The peak eluted at high salt concentrations (casein kinase 2b) showed molecular and kinetic properties typical of the heterotetramer composed of alpha-(or alpha'-) and beta-subunits. In contrast, the peak that was eluted at low salt concentrations (casein kinase 2a) contained no beta-subunit but a phosphorylatable protein of 49 kDa (pp49), in addition to the alpha/alpha'-subunits. The presence of alpha/alpha'/alpha"-subunits in preparations of casein kinases 2a and 2b was confirmed by immunological assays. Casein kinase 2a had low specific activity and a very high apparent Km for beta-casein. The peak eluted at intermediate ionic strength contained the alpha/alpha'-subunits and variable amounts of beta-subunit and pp49, and had kinetic properties intermediate between those of casein kinases 2a and 2b. Experiments based on heat inactivation, inhibition by low concentrations of heparin and ability to use GTP as substrate suggested that phosphorylation of pp49 was catalysed by the alpha/alpha'-subunits of casein kinase 2. No similarities were observed in the phosphopeptide maps of pp49 and beta-subunit. These results show that the alpha/alpha'-subunits of rat liver cytosol casein kinase 2 can form complexes not only with the beta-subunit but also with pp49, and that the complexes containing pp49 have a reduced affinity for the exogenous protein substrate beta-casein.

Dissociation of the hepatic insulin receptor favours its phosphorylation by casein kinase 2.

The alpha beta heterodimeric form of untreated hepatic insulin receptor was a substrate for casein kinase 2, whereas the alpha 2 beta 2 heterotetramer was not. On the contrary, autophosphorylation was detected only in the heterotetramer. Dissociation of the receptor by treatment with dithiothreitol decreased its autophosphorylation but favoured phosphorylation of its beta-subunit by casein kinase 2.