Isolation and characterization of two distinct forms of protein kinase C

Protein kinase C (Ca2+- and phospholipid-dependent protein kinase) has been purified from rat brain by a three-step, 18-h procedure resulting in the isolation of milligram quantities of enzyme. Unlike previous preparations from published protocols, which yield a single polypeptide, this procedure yields a protein which consists of a 78/80-kDa doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two polypeptides have been characterized with respect to structure and function and are very similar in both regards. However, the two forms can be distinguished immunologically by polyclonal antisera generated against purified protein kinase C. The 78- and 80-kDa proteins do not appear to be related to one another by proteolytic cleavage or by differential phosphorylation, although the two purified proteins do contain stoichiometric amounts of phosphate. The 78- and 80-kDa polypeptides therefore appear to represent two distinct forms of protein kinase C, thus providing evidence for the existence of multiple isozymes of this key regulatory protein.     

Enhanced expression of a chromatin associated protein tyrosine phosphatase during G0 to S transition

The non-transmembrane protein tyrosine phosphatase, PTP-S, is located predominantly in the cell nucleus in association with chromatin. Here we have analysed the expression of PTP-S upon mitogenic stimulation and during cell division cycle. During liver regeneration after partial hepatectomy, PTP-S mRNA levels increased 16-fold after 6 h (G₁ phase) and declined thereafter. Upon stimulation of serum starved cells in culture with serum, PTP-S mRNA levels increased reaching a maximum during late G₁ phase and declined thereafter. No significant change in PTP-S RNA levels was observed in growing cells during cell cycle. PTP^-S protein levels were also found to increase upon mitogenic stimulation. Upon serum starvation for 72 h, PTP-S protein disappears from the nucleus and is seen in the cytoplasm; after 96 h of serum starvation the PTP-S protein disappears from the nucleus as well as cytoplasm. Refeeding of starved cells for 6 h results in reappearance of this protein in the nucleus. Our results suggest a role of this phosphatase during cell proliferation.     

Monoclonal antibodies specific for type 3 protein kinase C recognize distinct domains of protein kinase C and inhibit in vitro functional activity

Monoclonal antibodies (mAbs) which distinguish Type 3 protein kinase C (PKC) from Types 1 and 2 have been obtained from mice immunized with purified Type 3 PKC from rabbit brain cytosol. Most of these mAbs (seven out of eight) selectively recognize Type 3 versus Types 1 and 2 PKC in both enzyme-linked immunosorbent and immunoblot assays. Trypsin treatment of Type 3 PKC reduced the immunoreactivity with 82-kDa PKC and generated immunoreactive fragments of 45 and 35 kDa. The mAbs can be divided into two classes based on their ability to recognize the 45-kDa catalytic fragment (5/8) or the 35 kDa regulatory domain fragment (3/8). Each of the mAbs inhibits phosphorylation of histone or lipocortin by PKC, although the extent of the inhibition varied. Only those mAbs that recognize the 35-kDa regulatory domain inhibited phorbol ester binding. The inhibition of both kinase and binding activities by this group of mAbs was sensitive to the concentration of phospholipid used in the assay. This functional inhibition suggests that these mAbs may be useful for defining the phospholipid binding domain(s) of Type 3 PKC. The mAbs recognized 82-kDa PKC in a variety of cell types; the presence of smaller molecular weight fragments was not consistently found. Distinct immunofluorescence staining patterns were observed with mAbs directed toward different epitopes, suggesting that there may be heterogeneity in the subcellular localization of PKC. The type specificity of these mAbs will make them valuable tools for studying activation and regulation of Type 3 PKC in cell culture model systems.     

Identification of functionally distinct regions that mediate biological activity of the protein kinase a homolog Tpk2

Kinases regulate key signaling processes that are increasingly implicated in development and disease. Kinase modulators have become important therapeutic tools and often target catalytic domains that are among the most structurally and functionally conserved regions of these enzymes. Such therapies lose efficacy as mutations conferring resistance arise. Because interactions between distinct and often distant regions of kinases can be critical, we took an unbiased genetic approach to identify sites within the protein kinase A homolog Tpk2 that contribute to its biological activity. Because many of these map outside the conserved core, this approach should be broadly useful in identifying new, more kinase-specific therapeutic targets.     

The relationship between the bilayer to hexagonal phase transition temperature in membranes and protein kinase C activity

A number of substances affect the activity of protein kinase C. Among uncharged and zwitterionic compounds, those which activate protein kinase C also lower the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine while substances which inhibit protein kinase C raise this transition temperature. Using this criteria, we have identified 3-chloro-5-cholestene, 5-cholan-24-ol and eicosane as new protein kinase C activators and have shown that Z-Ser-Leu-NH₂, Z-Gly-Leu-NH₂, Z-Tyr-Leu-NH₂, cyclosporin A and cholestan-3, 5, 6-triol are protein kinase C inhibitors.     

Expression and properties of two distinct classes of the phorbol ester receptor family, four conventional protein kinase C types, and a novel protein kinase C

Five rabbit cDNAs, encoding four conventional protein kinase Cs (PKCs), alpha, beta I, beta II, and gamma, and a novel PKC-related protein (nPKC epsilon) were transfected into COS cells. Antisera raised against a bacterially synthesized fragment of PKC alpha or nPKC epsilon and against a chemically synthesized peptide of PKC beta I or beta II, specifically identified the corresponding species in the transfected cells. All four PKCs and nPKC epsilon expressed by transfection served as phorbol ester receptors. Phorbol 12,13-dibutyrate (PDBu)-binding activities of all PKCs and nPKC epsilon required phospholipid but not magnesium. The phosphatidylserine requirement for the activity of nPKC epsilon is independent of Ca2+ and similar to that for PKC alpha observed at 0.03 mM Ca2+. Calcium dependence of the binding activity was observed only for the four conventional PKCs. Scatchard plot analysis clearly showed that the dissociation constants of PDBu for all four PKCs were nearly the same (approximately 25 nM) in the presence of Ca2+, and that the value for nPKC epsilon was slightly higher (84 nM) and independent of Ca2+. The latter value is comparable to those observed in several cell types under conditions of Ca2+ chelation. Translocation of conventional PKC alpha to the membranes was induced with phorbol ester in a Ca2+-dependent manner, whereas the PDBu-stimulated translocation of nPKC epsilon did not require Ca2+. These results, together with previous studies on the enzymological characteristics of nPKC epsilon (Ohno, S., Akita, Y., Konno, Y., Imajoh, S., and Suzuki, K. (1988) Cell 53, 731-741), suggest that nPKC epsilon plays an important role in a transmembrane signaling pathway distinct from that involving conventional PKCs.     

Localization of the murine Niemann-Pick C1 protein to two distinct intracellular compartments

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol and other lipids in the lysosomal compartment. In this report, we use subcellular fractionation and microscopy to determine the localization of the murine Niemann-Pick C1 (NPC1) protein. Fractionation of mouse liver homogenates indicates that some NPC1 cosediments with lysosome-associated membrane protein 1 (LAMP1)-containing membranes. However, a significant amount of NPC1 is also found in membranes that do not contain LAMP1. Moreover, fractionation of liver membranes and fibroblasts in the presence of a nonionic detergent showed that a fraction of NPC1 cosediments with caveolin-1 in rafts. Immunofluorescence microscopy of cultured mouse fibroblasts showed that NPC1 is found in two morphologically distinct structures. The first population is characterized by large punctate structures that do not colocalize with major organelle protein markers, but do colocalize with filipin and a small fraction of caveolin-1. Examination of these large NPC1-containing compartments using electron microscopy shows that these structures contain extensive internal membranes. The second population is represented by smaller, more diffuse structures, a fraction of which colocalize with LAMP1-positive compartments. Incubation of fibroblasts with low density lipoprotein (LDL) increases colocalization of NPC1 with LAMP1-containing compartments. This colocalization can be further enhanced by treating fibroblasts with progesterone or chloroquine. The results indicate that NPC1 is associated with an unique vesicular compartment enriched with cholesterol and containing caveolin-1, and that NPC1 cycles to LAMP1-positive compartments, presumably to facilitate the processing of LDL-derived cholesterol.     

c-myc gene expression is stimulated by agents that activate protein kinase C and does not account for the mitogenic effect of PDGF

The role of the phosphoinositide turnover-protein kinase C pathway in mediating PDGF-stimulated c-myc expression and cell proliferation was studied. Both direct activators of kinase C (e.g. phorbol ester analogues) and hormones that activate kinase C via receptor-mediated phosphoinositide turnover (e.g. PDGF, bradykinin, or vasopressin) elicited a rapid increase in c-myc mRNA expression. Desensitization of the kinase C pathway by prolonged exposure to phorbol abolished the induction of c-myc by subsequent phorbol challenge and attenuated c-myc induction by PDGF and bradykinin, but did not affect PDGF-stimulated mitogenesis. Bradykinin and phorbol esters stimulated the same magnitude of c-myc expression as PDGF but elicited less than one-tenth the PDGF-induced mitogenic response. We conclude that stimulation of c-myc expression is a common response to a diverse group of agents that elicit phosphoinositide turnover and activate protein kinase C, and that neither activation of protein kinase C nor enhanced c-myc expression is sufficient for the mitogenic action of PDGF.     

Intracellular pH controls growth factor-induced ribosomal protein S6 phosphorylation and protein synthesis in the G0→G1 transition of fibroblasts

Mitogen-induced intracellular alkalinization mediated by activation of a Na+/H+ antiporter is a common feature of eukaryotic cells stimulated to divide. A Chinese hamster fibroblast mutant (PS120) lacking Na+/H+ antiport activity (Pouysségur et al., Proc natl acad sci US 81 (1984) 4833) [42] possesses an intracellular pH (pHi) 0.2-0.3 units lower than the wild type (CCL39) and requires a more alkaline pHout (pHo) for growth. Here, we show that serum-stimulated ribosomal protein S6 phosphorylation, protein synthesis activation and DNA synthesis re-initiation are pH-regulated events that display a similar threshold pHo value (6.60) in CCL39 cells. pH-Dependencies for initiation of all three events are shifted toward higher pHo values in the mutant PS120, indicating that growth factor-induced alkalinization has a permissive effect on the pleiotypic response. However, cytoplasmic alkalinization per se is insufficient to trigger S6 phosphorylation, polysome formation, and subsequent DNA synthesis. Transient exposure to a non-permissive pHo (6.5) inhibits both the rate of leucine incorporation into proteins and the progression through the G1 phase of the cell cycle. In contrast, cells committed to DNA synthesis are unaltered by the acidic pHo. These observations suggest that pHi by controlling the rate of protein synthesis play a determinant role in the control of cell division.     

Deoxyadenosine blockade of G0 to G1 transition in lymphocytes: Possible involvement of protein kinases

Immunodeficiency in adenosine deaminase deficiency has been attributed to the lymphotoxicity of deoxyadenosine that accumulates to high levels in patients. To gain insight into the mechanism of deoxyadenosine toxicity, we investigated the dose-response and time course of its toxic effects on concanavalin A-stimulated mouse splenic lymphocytes by thymidine incorporation and flow cytometry. Deoxyadenosine at a level as low as 0.3 μM inhibited the progression of G₀. In contrast, higher concentrations of the nucleoside, i.e., in the range of 1 to 3 μM, were needed to block transition of the stimulated lymphocytes from G₀ to G₁. The inhibition of their S entry and progression required even higher concentrations. Furthermore, staurosporine, a potent inhibitor of protein kinases, was found to potentiate the toxicity of deoxyadenosine in mitogen-stimulated lymphocytes. Calcium mobilization in mitogen-activated lymphocytes was inhibited by deoxyadenosine. Our data suggest that, while ribonucleotide reductase inhibition by dATP could explain the blockade of S entry and progression by deoxyadenosine in cycling lymphocytes or leukemic cells, more important effects of this compound on antigen-activated lymphocytes occur at the early G₀ phase. A possible mechanism of deoxyadenosine lethality is its inhibition of protein phosphorylation. © 1996 Wiley-Liss, Inc.     

Evidence that bacteria induce translocation of protein kinase C activity in human polymorphonuclear leukocytes

Particulate fraction associated protein kinase activity was studied in human polymorphonuclear leukocytes stimulated by bacteria. Staphylococcus aureus was found to increase particulate fraction associated protein kinase C activity in a time and concentration dependent manner. The increase comprised both the phospholipid dependent and independent kinase activity and was augmented by addition of serum. Similar observations were done using Staphylococcus epidermidis and Klebsiellae pneumoniae. However, Escherichia coli only increased the phospholipid independent kinase activity in the particulate fraction, which suggests the presence of protease activity.     

Mitogen-induced preferential synthesis of proteins during the G0 to S phase transition in human lymphocytes

We have followed the induction of protein synthesis in mitogen-activated human peripheral blood mononuclear cells during the transition from quiescence, or G0, through the prereplicative phase and into first S phase. Doses of mitogens optimal for proliferative response preferentially enhance the synthesis of a subset of intracellular proteins during the approximately 24-h lag interval. The mitogenic lectin phytohemagglutinin (PHA) and OKT3, a mitogenic monoclonal antibody to the CD3 component of the T cell antigen receptor, preferentially enhance bands of the same molecular weight in one-dimensional SDS-PAGE. The proteins are low detergent soluble (0.1% Triton X-100) "cytoplasmic" cellular components and some have been identified as single spots on two-dimensional gels. Bands of 51 and 66 kDa are induced early in lag phase (4 h after stimulation) but are transiently synthesized, decreasing later in lag phase. The majority of the mitogen-induced proteins, 39, 51, 55, 60, 73, and 95 kDa are enhanced by mid lag phase (12 h after stimulation). With the exception of the 55-kDa band, five of these proteins are clearly enhanced in T cells purified after mitogen stimulation. The same five bands show sustained synthesis in actively cycling cells 42-48 h after stimulation and are major synthesized proteins, and corresponding bands are synthesized in a transformed T cell line, MOLT-4. Two of the proteins in this group that are most prominently synthesized during the lag interval have been previously identified as the heat shock proteins, HSP 90 (95-kDa band) and HSC 70 (73-kDa band). We speculate that this group of five proteins, including HSP 90 and HSC 70, may be coordinately expressed in actively replicating T cells and may have some common structural or functional role in sustaining the replicative state.     

Evidence that protein kinase C (PKC) participates in the meiosis I to meiosis II transition in mouse oocytes.

Oocytes from LTXBO mice exhibit a delayed entry into anaphase I and frequently enter interphase after the first meiotic division. This unique oocyte model was used to test the hypothesis that protein kinase C (PKC) may regulate the meiosis I-to-meiosis II transition. PKC activity was detected in LTXBO oocytes at prophase I and increased with meiotic maturation, with the highest (P < 0.05) activity observed at late metaphase I (MI). Treatment of late MI-stage oocytes with the PKC inhibitor, bisindolylmaleimide I (BIM), transiently reduced (P < 0.05) M-phase-promoting factor (MPF) activity and promoted (P < 0.05) progression to metaphase II (MII), while mitogen-activated protein kinase (MAPK) activity remained elevated during the MI-to-MII transition. Confocal microscopy analysis of LTXBO oocytes during this transition showed PKC-delta associated with the meiotic spindle and then with the chromosomes at MII. Inhibition of PKC activity also prevented untimely entry into interphase, but only when PKC activity was reduced in oocytes before the progression to MII and thus indicates that the transition into interphase is directly associated with the delayed triggering of anaphase I. Moreover, the defect(s) that initiate activation occur upstream of MAPK, as suppression of PKC activity failed to prevent activation by Mos(tm1Ev)/ Mos(tm1Ev) LTXBO oocytes expressing no detectable MAPK activity. In summary, PKC participates in the regulatory mechanisms that delay entry into anaphase I in LTXBO oocytes, and the disruption promotes untimely entry into interphase. Thus, loss of regulatory control over PKC activity during oocyte maturation disrupts the critical MI-to-MII transition, leading to a precocious exit from meiosis.     

The A- and B-type cyclins of Drosophila are accumulated and destroyed in temporally distinct events that define separable phases of the G2-M transition.

We show that the sequence of Drosophila cyclin B has greater identity with B-type cyclins from other animal phyla than with Drosophila cyclin A, suggesting that the two cyclins have distinct roles that have been maintained in evolution. Cyclin A is not detectable in unfertilized eggs and is present at low levels prior to cellularization of the syncytial embryo. In contrast, the levels of cyclin B remain uniformly high throughout these developmental stages. In cells within cellularized embryos and the larval brain, cyclin A accumulates to peak levels in prophase and is degraded throughout the period in which chromosomes are becoming aligned on the metaphase plate. The degradation of cyclin B, on the other hand, does not occur until the metaphase-anaphase transition. In cells arrested at c-metaphase by treating with microtubule destabilizing drugs to prevent spindle formation, cyclin A has been degraded in the arrested cells, whereas cyclin B is maintained at high levels. These observations suggest that cyclin A has a role in the G2-M transition that is independent of spindle formation, and that entry into anaphase is a key requirement for the degradation of cyclin B.     

Convergence and divergence of stress-induced mitogen-activated protein kinase signaling pathways at the level of two distinct mitogen-activated protein kinase kinases

Plants respond to biotic and abiotic stresses by inducing overlapping sets of mitogen-activated protein kinases (MAPKs) and response genes. To define the mechanisms of how different signals can activate a common signaling pathway, upstream activators of SIMK, a salt stress- and pathogen-induced alfalfa MAPK, were identified. Here, we compare the properties of SIMKK, a MAPK kinase (MAPKK) that mediates the activation of SIMK by salt stress, with those of PRKK, a distantly related novel MAPKK. Although both SIMKK and PRKK show strongest interaction with SIMK, SIMKK can activate SIMK without stimulation by upstream factors. In contrast, PRKK requires activation by an upstream activated MAPKK kinase. SIMKK mediates pathogen elicitor signaling and salt stress, but PRKK transmits only elicitor-induced MAPK activation. Of four tested MAPKs, PRKK activates three of them (SIMK, MMK3, and SAMK) upon elicitor treatment of cells. However, PRKK is unable to activate any MAPK upon salt stress. In contrast, SIMKK activates SIMK and MMK3 in response to elicitor, but it activates only SIMK upon salt stress. These data show that (1) MAPKKs function as convergence points for stress signals, (2) MAPKKs activate multiple MAPKs, and (3) signaling specificity is obtained not only through the inherent affinities of MAPKK-MAPK combinations but also through stress signal-dependent intracellular mechanisms.     

Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1 μM) and the Src inhibitor PP2 (10 μM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca²⁺ transients were reduced by imatinib and/or PP2. Ca²⁺ transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca²⁺ transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCα catalytic activity and PKCα co-immunoprecipitated with Bcr/Abl.Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca²⁺ influx was reduced by complexing extracellular Ca²⁺ with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca²⁺ transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.     

Targeted deletion of protein kinase C lambda reveals a distribution of functions between the two atypical protein kinase C isoforms

Protein kinase C lambda (PKClambda) is an atypical member of the PKC family of serine/threonine kinases with high similarity to the other atypical family member, PKCzeta. This similarity has made it difficult to determine specific roles for the individual atypical isoforms. Both PKClambda and PKCzeta have been implicated in the signal transduction, initiated by mediators of innate immunity, that culminates in the activation of MAPKs and NF-kappaB. In addition, work from invertebrates shows that atypical PKC molecules play a role in embryo development and cell polarity. To determine the unique functions of PKClambda, mice deficient for PKClambda were generated by gene targeting. The ablation of PKClambda results in abnormalities early in gestation with lethality occurring by embryonic day 9. The role of PKClambda in cytokine-mediated cellular activation was studied by making mouse chimeras from PKClambda-deficient embryonic stem cells and C57BL/6 or Rag2-deficient blastocysts. Cell lines derived from these chimeric animals were then used to dissect the role of PKClambda in cytokine responses. Although the mutant cells exhibited alterations in actin stress fibers and focal adhesions, no other phenotypic differences were noted. Contrary to experiments using dominant interfering forms of PKClambda, mutant cells responded normally to TNF, serum, epidermal growth factor, IL-1, and LPS. In addition, no abnormalities were found in T cell development or T cell activation. These data establish that, in vertebrates, the two disparate functions of atypical PKC molecules have been segregated such that PKCzeta mediates signal transduction of the innate immune system and PKClambda is essential for early embryogenesis.