Activation and substrate specificity of the human protein kinase C alpha and zeta isoenzymes.

Protein kinase C (PKC), a class of serine/threonine kinases activated by Ca2+ and/or phospholipids, is involved in a variety of cellular processes such as proliferation, differentiation and secretion. Nine members of the PKC gene family are known; these are differentially expressed in eukaryotic cells and can be divided into two sub-groups: the Ca(2+)-dependent (classical) PKC isoenzymes alpha, beta I, beta II and gamma, and the Ca(2+)-independent neoPKC isoenzymes delta, epsilon, zeta, eta and theta. A detailed biochemical characterisation of these PKC isoenzymes is one prerequisite for the elucidation of their distinct roles within cellular signal transduction. In this study, we report the cloning of a human PKC-zeta cDNA, its expression in recombinant baculovirus-infected insect cells and the partial purification of the PKC-zeta isoenzyme. In comparison to highly purified human PKC alpha, a representative of the classical PKC subgroup, purified PKC zeta was characterised with respect to activator requirement, substrate specificity, proteolytic activation and sensitivity towards PKC inhibitors. In contrast to PKC alpha, PKC zeta exhibits a constitutive kinase activity which is independent of Ca2+, phosphatidylserine and diacylglycerol. Arachidonic acid alone or a combination of gamma-linolenic acid and phosphatidylserine slightly enhance PKC zeta activity. In the presence of the classical PKC activators phosphatidylserine/diacylglycerol, PKC alpha phosphorylates a PKC-alpha pseudosubstrate-derived peptide, an epidermal-growth-factor-receptor-derived peptide, histone III-S and myelin basic protein to an equal extent, whilst PKC zeta phosphorylates only the PKC-alpha-derived peptide. However, arachidonic acid greatly diminishes PKC-alpha activity towards the epidermal-growth-factor-receptor-derived peptide, histone III-S and myelin basic protein, but enhances PKC-zeta activity towards the PKC-alpha-derived peptide. These results indicate a possible modulation of substrate specificity of these two PKC isoenzymes by (the binding of) different activators (to their regulatory domains). In the case of PKC zeta, this finding is strengthened by the fact that the epidermal growth factor receptor-derived peptide, which is not a substrate for the holoenzyme, is significantly phosphorylated by a protein fragment generated by limited proteolysis and comprising only the kinase domain. Furthermore, PKC zeta, in contrast to PKC alpha, is insensitive to PKC inhibitors known to interfere either with the regulatory or the catalytic domain and cannot be activated by phorbol ester treatment of NIH 3T3 cells or insect cells, overexpressing the respective PKC isoenzyme. The potential implications of these findings on the mechanism(s) of activation and the substrate specificity of PKC zeta are discussed.

Activation of purified human protein kinase C alpha and beta I isoenzymes in vitro by Ca2+, phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate.

The increasing number of eukaryotic protein kinase C (PKC) isoenzymes which have been described has raised great interest in potential differences in the cellular expression, the mode of activation and the substrate specificity of these isoenzymes. The last two aspects have mostly been studied with isoenzymes purified from rat or bovine brain or from recombinant-baculovirus-infected insect cells. In this study, we have expressed the human PKC isoenzymes alpha and beta I in recombinant-baculovirus-infected insect cells. The isoenzymes were purified to homogeneity by a four-step procedure which included a reversible Ca(2+)-dependent association/dissociation to and from the endogenous membranes of the lysed insect cells. Characterization of the purified enzymes with respect to ATP requirement and substrate specificity, using the epidermal-growth-factor receptor peptide and histone III-S respectively, revealed no isoenzyme-specific differences. Activation by trypsin or Ca2+ and a variety of different phospholipids and phosphoinositides (in a mixed-micellar assay) gave the following results. Proteolytic cleavage of the PKC isoenzymes by trypsin generated fully activated phospholipid-independent PKC beta I, whereas PKC alpha reached only 50% of the activity obtained in the presence of phospholipids. PKC alpha and beta I showed no difference in their dependence on Ca2+, diacylglycerol (DAG) and phosphatidylserine (PS). Replacement of either DAG or PS by phosphatidylglycerol, cardiolipin, phosphatidylcholine and several phosphoinositides revealed that PtdIns(4,5)P2 can act as a PKC activator similar to DAG, whereas PtdIns can substitute for PS as a cofactor of activation. Thus, at least for the PKC isoenzymes alpha and beta I, a combination of PtdIns and PtdIns(4,5)P2 can fully replace PS and DAG in vitro as the classical activators of PKC.

Purification and characterisation of an initiation-factor-2 kinase from uninduced mouse erythroleukaemia cells.

Mouse erythroleukaemia (MEL) cells, which have not been induced into erythroid development, contain a protein kinase (MKu) which phosphorylates the alpha subunit of protein-synthesis-initiation factor 2 (eIF-2 alpha). In this paper, we show that this kinase phosphorylates both eIF-2 alpha and a synthetic peptide based on the phosphorylation site in eIF-2 alpha at Ser51, the target residue for other eIF-2 alpha kinases. Consistent with this, prior treatment of eIF-2 with MKu impaired the exchange of bound GDP for GTP which is catalysed by the exchange factor eIF-2B. Using a modified cell-free translation system, we have shown that MKu inhibits translation, consistent with the above observations concerning the site of phosphorylation and the effect of phosphorylation on eIF-2B-mediated guanine-nucleotide exchange. MKu has been purified and its properties have been compared with those of the haem-controlled repressor eIF-2 alpha kinase (HCR) from rabbit reticulocytes. Its behaviour on gel filtration is similar to that of HCR, while its behaviour on anion exchange resembles that of certain phosphorylated species of HCR. Highly purified preparations of MKu contain a protein with an apparent molecular mass of 98 kDa which comigrates with HCR on SDS/PAGE. This protein undergoes phosphorylation when incubated in the presence of Mg(2+)-ATP, and both this apparent autophosphorylation and the activity of the kinase against eIF-2 alpha are inhibited by the same, low, (10 microM) concentrations of haemin. Phosphorylation of the 98-kDa components present in the MEL-cell kinase preparation and in purified rabbit reticulocyte HCR occurs on serine and threonine residues. Analysis of these phosphoproteins by peptide mapping reveals significant differences in their structures, indicating that they may be closely related, but are certainly not identical.

A 60-kDa protein from rabbit reticulocytes specifically recognizes the capped 5' end of beta-globin mRNA.

The binding of proteins from rabbit reticulocyte lysate to in-vitro-generated beta-globin mRNA and its defined segments was investigated using ultraviolet-cross-linking experiments as well as gel-retardation assays. Under stringent conditions, only three proteins (72, 60 and 50 kDa) were found associated with full-length beta-globin mRNA at different positions. The 72-kDa protein is most likely the poly(A)-binding protein and binds, as expected, to the poly(A) tail, whereas the 50-kDa protein exhibits affinity for the trailer region of beta-globin mRNA. The binding region of the 60-kDa protein is located at the 5' end of beta-globin mRNA. The interaction of this protein is dependent on the presence of the 5' cap structure, as indicated by competition experiments using an uncapped beta-globin-mRNA leader segment. Further competition experiments with beta-globin mRNA, deleted in part in the leader region, suggest that, besides the cap structure, certain sequence elements are necessary for the interaction of the 60-kDa protein and the beta-globin mRNA leader.

Presence of haemin-controlled eIF-2 alpha kinases in both undifferentiated and differentiating mouse erythroleukaemia cells.

In rabbit reticulocytes, globin synthesis is regulated by a haemin-controlled translational inhibitor (HCI) which acts by phosphorylating the alpha-subunit of eukaryotic initiation factor 2 (eIF-2). With purified eIF-2 as substrate, haemin-controlled eIF-2 alpha kinases could be partially purified from cultured mouse erythroleukaemia cells (MEL cells), which can be induced in vivo to erythroid differentiation. The eIF-2 alpha kinases from both uninduced and induced MEL cells are clearly distinct from the double-stranded-RNA-activated eIF-2 alpha kinase described for many mammalian cell types. A rough quantitative estimation indicates that, on a per-cell basis, induced MEL cells contain the same amount of haemin-controlled eIF-2 alpha kinase activity as rabbit reticulocytes, whereas uninduced MEL cells contain about one-tenth as much. As to their chromatographic behavior on CM-Sephadex and DEAE-cellulose and their sensitivity towards physiological concentrations of haemin (5-10 microM), the eIF-2 alpha kinases from MEL cells are indistinguishable from HCI. They differ from HCI with respect to their response towards activating stimuli such as prolonged incubation at 37 degrees C or brief exposure to the thiol reagent N-ethylmaleimide.

Characterization of an inhibitor of protein synthesis initiation from mouse erythroleukemia cells.

This study describes the partial purification of a translational inhibitor from mouse erythroleukemia (MEL) cells. It is present in MEL cells induced to erythroid differentiation and in uninduced cells in approximately equal amounts. The inhibitor blocks initiation but not elongation of in vitro protein synthesis in the rabbit reticulocyte lysate and in extracts prepared from induced or uninduced MEL cells. Nuclease-resistance, heat-sensitivity and the chromatographic behaviour of the inhibitor indicate that it is a protein with a relative molecular mass of approx. (45-70).10(3). The inhibitor has no eIF-2 alpha phosphorylating activity and does not affect the formation of the ternary complex [eIF-2.GTP.Met-tRNAf] nor the binding of Met-tRNAf to the 40 S ribosomal subunit. The inhibitor interferes with the binding of mRNA to the 43 S preinitiation complex, independent of the presence of the m7GTP cap of the mRNA.

Binding of mRNA by an oligopeptide containing an evolutionarily conserved sequence from RNA binding proteins.

Several proteins with an affinity to RNA contain a conserved sequence of 8 amino acids which is postulated as being important for RNA binding. An oligopeptide of 11 amino acids containing this sequence is shown to bind 32P-labelled globin mRNA in a filter binding assay. High concentrations of heparin compete for this binding. 10 other peptides with different sequences do not exhibit affinity to RNA in this assay. These results support the relevance of the conserved peptide sequence in the binding of proteins to RNA.

Differential effect of hemin-controlled eIF-2 alpha kinases from mouse erythroleukemia cells on protein synthesis.

Cultured mouse erythroleukemia (MEL) cells can be induced to erythroid differentiation by a variety of chemical agents. This differentiation process is marked by the onset of globin mRNA and hemoglobin synthesis. In rabbit reticulocytes, globin synthesis is regulated by a hemin-controlled translational inhibitor (HCI) which acts via phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2). From both uninduced and induced MEL cells, hemin-controlled eIF-2 alpha kinases have been partially purified. They resemble HCI with respect to their chromatographic behaviour and their sensitivity towards physiological concentrations of hemin (5-10 microM). Further purification on phosphocellulose, however, reveals that the eIF-2 alpha kinase from uninduced MEL cells is chromatographically distinct from HCI, whilst the eIF-2 alpha kinase activity from induced MEL cells represents a mixture of the former and the HCI-type eIF-2 alpha kinase. The latter inhibits protein synthesis in a fractionated system from rabbit reticulocytes which is free of, but sensitive to, HCI, whereas the eIF-2 alpha kinase from uninduced MEL cells does not show any inhibitory activity. This observation is supported by the finding that induced MEL cells respond in vivo to iron depletion with a shut-off of protein synthesis (as do rabbit reticulocytes), whilst uninduced MEL cells do not.

The phosphorylation of eukaryotic initiation factor 2: a principle of translational control in mammalian cells.

In eukaryotic cells, protein biosynthesis is controlled at the level of polypeptide chain initiation. During the initiation process, eukaryotic initiation factor 2 (eIF-2) catalyzes the binding of Met-tRNAf and GTP to the 40S ribosomal subunit. In a later step, eIF-2 is released from the ribosomal initiation complex, most likely as an eIF-2.GDP complex, and another initiation factor termed eIF-2B is necessary to recycle eIF-2 by displacing GDP by GTP. In rabbit reticulocytes, inhibition of protein synthesis is accompanied by the phosphorylation of the alpha-subunit of eIF-2, a process that does not render eIF-2 inactive, but prevents it from being recycled by eIF-2B. First described in rabbit reticulocytes as inhibitors of translation, two distinct eIF-2 alpha kinases are known: the haemin-controlled kinase (termed HCI) and the double-stranded RNA-activated kinase (termed DAI). eIF-2 alpha phosphorylation appears to be a reversible control mechanism since corresponding phosphatases have been described. Recent reports indicate a correlation between eIF-2 alpha phosphorylation and the inhibition of protein synthesis in several mammalian cell types under a range of physiological conditions. In this review, the physical and functional features of the known eIF-2 alpha kinases are described with respect to their role in mammalian cells and the mode of activation by cellular signals. Furthermore, the possible impact of the eIF-2/eIF-2B ratio and of the subcellular compartmentation of these factors (and the eIF-2 alpha kinases) on mammalian protein synthesis is discussed.

A (re)initiation-dependent cell-free protein-synthesis system from mouse erythroleukemia cells.

Cultured mouse erythroleukemia cells (MEL cells) can be induced in vivo to erythroid differentiation which is marked by the onset of globin mRNA and haemoglobin synthesis. When these cells are briefly exposed to hypertonic growth medium prior to lysis, the resulting post-mitochondrial supernatants show a high in vitro protein-synthesis activity. Amino acid incorporation is linear up to 60 min; more than 80% of this is due to (re)initiation, as shown by the inhibition with edeine. Extracts from induced cells reach only a third of overall incorporation as compared to extracts from uninduced cells. This reduction of the protein-synthesizing capacity is also observed in vivo. Polyacrylamide gel electrophoresis shows that extracts from uninduced cells faithfully translate their endogenous mRNA, whereas in extracts from induced cells, non-globin protein synthesis is reduced and globin is preferentially synthesized. Haemin (40 microM) as well as purified eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes enhance amino acid incorporation in both kinds of extracts, which suggests that both uninduced and induced MEL cells contain a haemin-controlled eIF-2 alpha kinase. This system should be useful for studying the mechanisms controlling protein synthesis in a nucleated differentiating cell.