Opposite effects of the Hsp90 inhibitor Geldanamycin: induction of apoptosis in PC12, and differentiation in N2A cells.

The inhibitor of the Hsp90 chaperone Geldanamycin has been reported to have several cellular effects, such as inhibition of v-src activity or destabilization of Raf-1 among others. We show now that Geldanamycin treatment induces different phenotypes in different cell lines. In PC12 cells, it triggers apoptosis, whereas in the murine neuroblastoma N2A, it induces differentiation with neurite outgrowth. Geldanamycin effects cannot be mimicked by inhibition of the c-src protein tyrosine kinases, and nerve growth factor does not protect PC12 cells from apoptosis. Mitogen-activated protein kinase activities ERK and JNK are activated differently according to cell type: in PC12 cells JNK is activated, and its inhibition abolishes apoptosis, but not ERK; in N2A cells, both ERK and JNK are activated, but with peak activities at different times.

Isolation and characterization of casein kinase I from Dictyostelium discoideum.

In the present study, the molecular cloning and characterization of a 49-kDa form of casein kinase (CK)I from Dictyostelium discoideum is reported. The predicted amino acid sequence shares 70% identity with the catalytic domain of the mammalian delta and epsilon isoforms, Drosophila CKIepsilon and Schizosaccharomyces pombe Hhp1, and 63% identity with Hrr25, a 57-kDa form of yeast CK involved in DNA repair. D. discoideum CKI (DdCKI) was expressed in vegetative asynchronous cells as well as in differentiated cells, as detected by Northern-blot analysis. The level of DdCKI expression did not change during the cell cycle. Antibodies raised against a truncated version of the protein recognized a 49-kDa protein from D. discoideum extracts. Protein expression paralleled the pattern found for the RNA. The expression of DdCKI in Escherichia coli resulted in an active enzyme that autophosphorylated and phosphorylated casein. Immunofluorescence assays showed that DdCKI was localized in the cytoplasm and nuclei of Dictyostelium cells. The lack of disruptants of the CKI gene suggests that this protein is essential for the vegetative growth of D. discoideum. Overexpression of DdCKI resulted in cells with increased resistance to hydroxyurea, suggesting a potential role for this kinase in DNA repair.

Proteolysis activated protein kinase in Dictyostelium discoideum.

In the search for MBP phosphorylating activities in Dictyostelium discoideum, we have found a proteolysis-activated protein kinase. This activity which is distributed between the soluble and the particulate fractions of the cell, uses MBP and histone as substrate and has a molecular mass of 140 kDa as detected in an 'in situ' assay. This protein kinase has several features shared by the protein kinase C family, such as substrate specificity and sensitivity to proteolysis, but its molecular mass is much larger than that described for the known protein kinase C isoforms. To better characterize this activity we have studied its sensitivity to several protein kinase C inhibitors and activators. This protein kinase is activated neither by phorbol ester nor by phosphatidylserine or Ca2+. The activity is inhibited by staurosporine and PKC zeta pseudosubstrate, but is not affected by the specific protein kinase C inhibitor bisindolylmaleimide. These data lead us to propose that proteolytically activated Dictyostelium protein kinase belongs to the recently described protein kinase C-related family.

Protein kinases in mitochondria of the invertebrate Artemia franciscana.

The information concerning protein kinases in animal mitochondria is scarce and related only to mammals. No data are available for invertebrates. We demonstrate here the presence of casein kinase II (CK II) and cAMP-dependent protein kinase (PKA) in the purified mitochondria of the crustacean Artemia franciscana. Whereas the mitochondrial CK II showed the same characteristics of the cytosolic enzyme, mitochondrial PKA had an apparent Km for its substrate Kemptide 1 order of magnitude lower than that of the cytosolic enzyme. CK II and PKA phosphorylate different sets of proteins in Artemia mitochondria in vitro. The use of an activity gel assay has allowed the detection of additional protein kinases, as yet unidentified, in Artemia mitochondria.

Regulation of ERK2 dephosphorylation in G1-stimulated rat T lymphoblasts.

Rat T lymphoblasts arrested in the G1 phase of the cell cycle by interleukin-2 (IL-2) deprivation can be forced to proceed to the S phase when they are stimulated with IL-2 or the phorbol ester phorbol 12,13-dibutyrate (PDBu). When PDBu is used as a stimulus, extracellular regulated kinase 2 (ERK2) is activated by threonine and tyrosine phosphorylation by the dual-specificity kinase MEK. Here we have studied the regulation of ERK2 dephosphorylation as a mechanism for inactivation of this kinase. In vivo inhibition of ERK2 dephosphorylation observed after preincubation with translation or transcription inhibitors (cycloheximide or actinomycin, respectively) indicates the involvement of at least one inducible phosphatase, the best candidate for which is the dual-specificity phosphatase PAC-1. Other noninducible phosphatases must act as well, however, because sodium orthovanadate is a more effective dephosphorylation blocker than cycloheximide. In addition, the okadaic acid effect in ERK2 dephosphorylation indicates that Ser/Thr phosphatases are also involved, directly and/or indirectly.

Raf-1 and ERK2 kinases are required for phorbol 12,13-dibutyrate-stimulated proliferation of rat lymphoblasts. ERK2 activation precedes Raf-1 hyperphosphorylation.

Rat lymphoblasts are arrested in the G1 phase of the cell cycle and can be promoted to proceed up to the S phase, when they are stimulated by phorbol ester. In this work, we have studied some details of the phorbol 12,13-dibutyrate (PBu2)-stimulated proliferation. We show that in response to PBu2 at least four different protein kinase C (PKC) isoforms translocate to the membrane. A specific PKC zeta antibody recognizes two bands of 75 and 82 kDa. These two activities are separated using a Mono Q chromatography and we show that p75 is the classical PKC zeta isoform, while p82 might be a related isoform which is PBu2 sensitive. Our data show that there is a correlation between the ability of PBu2 to promote mitogenesis and to activate ERK2 kinase, suggesting that ERK2 kinase might be the limiting step of the process. We also show that ERK kinase activation precedes Raf-1 kinase hyperphosphorylation, suggesting that Raf-1 kinase activation is not required for ERK kinase activation. This idea was checked using a Raf-1 kinase antisense (AS) oligonucleotide. The results obtained with the Raf-1 AS oligonucleotide indicate that this serine/threonine kinase is dispensable for ERK kinase activation, but needed for the PBu2 mitogenic signaling even as late as 7 h after the delivery of the signal.

Chemotactic stimulation of aggregation-stage Dictyostelium cells induces rapid changes in energy metabolism, as measured by succinic thiokinase phosphorylation.

In crude mitochondrial fractions of the cellular slime mold Dictyostelium discoideum, a 38-kDa protein can be detected in phosphorylation assays under autophosphorylation conditions in SDS polyacrylamide gels. p38 can be phosphorylated in vitro using either ATP or GTP as phosphoryl donors. After stimulation of aggregation competent cells with the chemoattractant cAMP, p38 phosphorylation pattern changes rapidly. Caffeine, a known inhibitor of cAMP relay in D. discoideum inhibits cAMP induced changes in p38 phosphorylation. The rapid changes in p38 phosphorylation after cAMP stimulation reflect changes in energy metabolism and these changes are most likely mediated by changes in internal calcium concentrations. The mitochondrial localization and other data presented on the characterization of this protein led us to the conclusion that p38 is the alpha subunit of succinic thiokinase. Data showing a correlation between in-vitro p38 phosphorylation and the metabolic state of the cells at the moment of the cell lysis are included.

Purification of a soluble casein kinase II from Dictyostelium discoideum lacking the beta subunit: regulation during proliferation and differentiation.

A type II casein kinase has been purified from the soluble fraction of Dictyostelium discoideum vegetative cells. The enzyme has been purified 370 fold and behaves catalytically as casein kinase type II, in the sense that it utilizes GTP as well as ATP as phosphoryl donors, it is inhibited by low heparin concentrations and phosphorylates a specific peptide for CK II. It is a tetramer of 38 kDa-subunits with catalytic activity and ability to autophosphorylate in vitro. The comparison of this activity with the nuclear enzyme previously purified from the same organism indicates that both have the same molecular structure. Both enzymes have antigenic determinants in common with casein kinase II from bovine thymus, suggesting a high degree of conservation during evolution. Studies on the activity of this enzyme during early differentiation, and in the transition from quiescence to proliferation shows an increase in specific activity suggesting a crucial role for the enzyme in this organism.

Dictyostelium discoideum acidic ribosomal phosphoproteins: identification and in vitro phosphorylation.

Four acidic phosphoproteins from the ribosomes of the slime mold Dictyostelium discoideum have been identified and partially characterized. These proteins are selectively released from ribosomal particles by salt/ethanol washes, have low molecular weight and acidic pI, and tend to aggregate in solution to form homodimers. These features correspond to proteins of different origins that have been included in the conserved family of eukaryotic A-ribosomal proteins, and, therefore, we have named them Dictyostelium ribosomal proteins A1, A2, A3 and A4. We also demonstrate that Dictyostelium ribosomal A-proteins are specifically phosphorylated in vitro by a type II casein kinase previously identified in Dictyostelium. Isoelectric focusing separation has permitted us to identify four proteins (or P-proteins) that may consist of the phosphorylated forms of A-proteins. A-proteins from Dictyostelium and yeast do not present immunological cross-reactivity. Dictyostelium A-proteins contain, therefore, some specific features in their amino acid sequence that distinguish them from other members of the conserved eukaryotic A-protein family; this conclusion is coherent with data deduced from the nucleotide sequence of cDNA clones encoding two Dictyostelium A-proteins (P1 and P2) which we have recently reported.

Characterization of three casein kinases type I from Dictyostelium discoideum.

Three different casein kinases type I have been characterized and partially purified from vegetative cells of Dictyostelium discoideum. The enzymes have been classified as type I because they are excluded from DEAE cellulose columns and do not utilize GTP as phosphoryl donor. We have named these activities as casein kinases IA, IB and IC respectively, according to the elution profile on phosphocellulose chromatography. The three activities differ in: the sensitivity to heparin inhibition; the salt optimum for activity and the amino acids phosphorylated, using casein as substrate. Experiments carried out in conditions that favor autophosphorylation indicate that casein kinase IB could have a 53 kDa subunit, susceptible to autophosphorylation in vitro.

A phospholipid-stimulated protein kinase from Dictyostelium discoideum.

A protein kinase with unusual characteristics has been found in Dictyostelium discoideum. This kinase can use histone H1 as exogenous substrate, and the activity is stimulated by phospholipids, but not by Ca2+. This enzyme has been partially purified by using chromatography on DEAE-cellulose DE-52, spermine-agarose and phosphatidylserine-polyacrylamide. The protein kinase activity is very labile, even in the presence of protease inhibitors, making further purification difficult. In the activity-containing fractions, an endogenous protein of 140 kDa is labelled in vitro with [gamma-32P]ATP under conditions in which intramolecular rather than intermolecular reactions are favoured. This protein is labelled only in the presence of phospholipids, but not of Ca2+. We propose that the 140 kDa phosphoprotein might be the autophosphorylated enzyme.

Regulation of diacylglycerol kinase in the transition from quiescence to proliferation in Dictyostelium discoideum.

Diacylglycerol kinase and phosphatidylinositol kinase were examined in stationary phase D. discoideum amoeba induced to synchronously proliferate by dilution into fresh medium. Membrane bound diacylglycerol kinase activity showed a rapid and transitory 3-5 fold increase in the preproliferative interphase while phosphatidylinositol kinase activity was kept quite constant during the same period. The changes in diacylglycerol kinase activity seem to be due to a translocation of the enzyme from the soluble to the particulate cell compartments.

Changes in the plasma membrane ATPase activity in relationship to cell proliferation in Dictyostelium.

The plasma membrane ATPase activity of Dictyostelium amoebae increases ca 2.5 fold from non dividing stationary phase cells to synchronously growing cells. This increase in ATPase activity takes place during the three hours lag period that precede the cell division after diluting stationary cells into fresh medium and is prevented by cycloheximide. No differences in the Km for ATP or in the optimal pH for activity were observed in kinetic studies carried out with purified plasma membranes from stationary and proliferating cells.