Yoshiko Akita

A novel phorbol ester receptor/protein kinase, nPKC, distantly related to the protein kinase C family

Protein kinase C (PKC)-related cDNA clones encode an 84 kd protein, nPKC. nPKC contains a cysteine-rich repeat sequence homologous to that seen in conventional PKCs (alpha, beta I, beta II, and gamma), which make up a family of 77-78 kd proteins with closely related sequences. nPKC, when expressed in COS cells, confers increased high-affinity phorbol ester receptor activity to intact cells. Antibodies raised against nPKC identified a 90 kd protein in rabbit brain extract as well as in extracts from COS cells transfected with the cDNA construct. nPKC shows protein kinase activity that is regulated by phospholipid, diacylglycerol, and phorbol ester but is independent of Ca2+. The structural and enzymological characteristics of nPKC clearly distinguish it from conventional PKCs, which until now have been the only substances believed to mediate the various effects of diacylglycerol and phorbol esters. These results suggest an additional signaling pathway involving nPKC.

Structural and functional diversities of a family of signal transducing protein kinases, protein kinase C family; two distinct classes of PKC, conventional cPKC and novel nPKC.

Recent molecular cloning and biochemical experiments on the nature of protein kinase C (PKC) have revealed the existence of two distinct classes of phorbol ester (and diacylglycerol) receptor/protein kinase, conventional PKC (cPKC) and novel PKC (nPKC). Each of these classes contains multiple related molecules expressed in tissues and cells in a type-specific manner. Although nPKC does not show the typical PKC activity ascribable to conventional PKCs and thus was neglected in earlier studies, several lines of evidence suggest that nPKCs are involved in a variety of cell responses to physiological stimuli and phorbol esters. It is possible that in some cases nPKC is the major mediator of the so-called PKC-activators, such as phorbol esters, mezerein, and bryostatins. In addition to the clear difference between cPKC and nPKC, functional diversity among conventional PKCs has also been demonstrated; PKC gamma differs in its competence to mediate the signal toward transcriptional activation through TPA-responsive cis-acting elements from cPKC alpha and nPKC epsilon. The differences between cPKC and nPKC and among the individual members of each of these two classes, and their specific pattern of distribution in tissues and cells, provide a rationale by which to explain the specificity and diversity of cellular responses to external stimuli generating DAG and to phorbol esters. The results presented here also provide a means to dissect the complex signaling pathway in cells and to analyze the molecular basis underlying the signal transduction processes mediated by this family of protein kinases.

Possible role of Ca2+-independent protein kinase C isozyme, nPKCɛ, in thyrotropin- releasing hormone-stimulated signal transduction: Differential down-regulation of nPKCɛ in GH4C1 cells

Abstract Protein kinase C (PKC) molecular species of GH4C1 cells were analyzed after separation by hydroxyapatite column chromatography. A novel Ca2+-independent PKC, nPKC ɛ, was identified together with two conventional Ca2+-dependent PKCs, PKCα and βII by analysis of kinase and phorbol ester-binding activities, immunoblotting using isozyme-specific antibodies, and Northern blotting. These PKCs are down-regulated differently when cells are stimulated by outer stimuli; phorbol esters deplete PKCβII and nPKC ɛ from the cells more rapidly than PKC α, whereas thyrotropin-releasing hormone (TRH) at 200 nM depletes nPKCɛ exclusively with a time course similar to that induced by phorbol esters. However, translocation of PKCα and βII to the membranes is elicited by both TRH and phorbol esters. These results suggest that TRH and phorbol ester activate PKC α and βII differently but that nPKC ɛ is stimulated similarly by both stimuli. Thus, in GH4C1 cells, Ca2+-independent nPKC ɛ may play a crucial role distinct from that mediated by Ca2+-dependent PKC α and βII in a cellular response elicited by both TRH and phorbol esters.

Protein kinase Cε: multiple roles in the function of, and signaling mediated by, the cytoskeleton

Recent studies have established essential roles of protein kinase Cε in signaling pathways controlling various functions of microfilaments and intermediate filaments by modulating multiple cytoskeletal proteins. This review summarizes recent progress in our understanding of the roles of protein kinase Cε in the functions and signaling of microfilaments and intermediate filaments, with a focus mainly on cell–matrix and cell–cell interactions, migrations and contraction, in addition to its relevance in the development of several diseases, such as malignant tumors or cardiac disease.

Molecular interactions between dynamin and G-protein βγ-subunits in neuroendocrine cells

Abstract Dynamin and G-proteins both are guanosine triphosphate (GTP) binding proteins, with dynamin active in cellular membrane trafficking and G-proteins in intracellular signal transduction. Here we demonstrate that dynamin physically and functionally interacts with G-protein βγ-subunits in neuroendocrine GH4C1 cells, on stimulation with thyrotropin-releasing hormone and somatostatin. The interaction appears to be of high affinity and inhibitory on dynamin GTPase activity, mediated by the pleckstrin homology domain and regulated both by the G-protein α-subunit and by guanosine nucleotides. Thus, dynamin may target particular sites for receptor-mediated endocytosis by sharing βγ-subunits with the α subunit of G-proteins in neuroendocrine cells.

Involvement of protein kinase C ɛ in thyrotropin-releasing hormone-stimulated phosphorylation of the myristoylated alanine-rich C kinase substrate in rat pituitary clonal cells

We have shown previously that novel protein kinase Cε (nPKCε) plays a key role in the basal and thyrotropin‐releasing hormone (TRH)‐stimulated prolactin (PRL) secretion in rat pituitary GH4C1 cells (Akita et al., J. Biol. Chem. 1994, 269, 4653—4660). Here we examined the region downstream of nPKCε activation in order to understand the molecular mechanism by which nPKCε mediates TRH‐induced signal transduction. Exposure of GH4C1 cells to TRH causes a stimulation of the phosphorylation of a p80 (Mr ˜ 80 000, pI ˜ 4.3) and two p19 (p19a and b; Mr ˜ 19 000, pI ˜ 5.6 and 5.5, respectively). Phorbol ester, a potent activator of protein kinase C (PKC), also enhances these phosphorylations, whereas bisindolylmaleimide I, a specific inhibitor of PKC, clearly inhibits the phosphorylation of p80. p80 and p19 were identified as myristoylated alanine‐rich C kinase substrate (MARCKS) and stathmin, respectively, as assessed by their two‐dimensional gel electrophoretic profiles and their stabilities to heat and acid treatment. In nPKCε‐overexpressing stable clones, the phosphorylated level of MARCKS but not stathmin was high in the resting state, and enhanced and sustained upon TRH stimulation, correlating with the increased activation of nPKCε. TRH stimulates the release of MARCKS from the membrane/cytoskeletal fraction to the cytosol fraction. These results, taken together with previous data concerning PRL secretion, suggest that MARCKS, a regulatory component of the cytoskeletal architecture, is the major substrate of nPKCε in vivo, and that its phosphorylation may regulate TRH‐stimulated PRL secretion.

Protein kinase C epsilon phosphorylates keratin 8 at Ser8 and Ser23 in GH4C1 cells stimulated by thyrotropin-releasing hormone.

Protein kinase C ε (PKCε) is activated by thyrotropin‐releasing hormone (TRH), a regulator of pituitary function in rat pituitary GH4C1 cells. We analyzed the downstream mechanism after PKCε activation. Exposure of GH4C1 cells to TRH or a phorbol ester increased the phosphorylation of three p52 proteins (p52a, p52b and p52c) and decreased the phosphorylation of destrin and cofilin. GF109203X, an inhibitor of protein kinases including PKC, inhibited phosphorylation of the p52 proteins by TRH stimulation. Peptide mapping, amino‐acid sequencing, and immunochemical studies indicated that p52a, p52b, and p52c are the differentially phosphorylated isoforms of keratin 8 (K8), an intermediate filament protein. The unphosphorylated K8 (p52n) localized exclusively to the cytoskeleton, whereas the phosphorylated forms (especially p52c), which are increased in TRH‐stimulated cells, localized mainly to the cytosol. K8 phosphorylation was enhanced in PKCε‐overexpressing clones, and purified recombinant PKCε directly phosphorylated K8 with a profile similar to that observed in TRH‐stimulated cells. PKCε and K8 colocalized near the nucleus under basal conditions and were concentrated in the cell periphery and cell–cell contact area after TRH stimulation. MS analyses of phospho‐K8 and K8‐synthesized peptide (amino acids 1–53) showed that PKCε phosphorylates Ser8 and Ser23 of K8. Phosphorylation of these sites is enhanced in TRH‐stimulated cells and PKCε‐overexpressing cells, as assessed by immunoblotting using antibodies to phospho‐K8. These results suggest that K8 is a physiological substrate for PKCε, and the phosphorylation at Ser8 and Ser23 transduces, at least in part, TRH–PKCε signaling in pituitary cells.

Direct evidence that the kinase activity of protein kinase C is involved in transcriptional activation through a TPA-responsive element.

In order to examine the involvement of protein kinase C (PKC) in the transcriptional activation of genes by TPA (12‐O‐tetradecanoyl phorbol 13‐acetate) we have constructed a series of PKC expression plasmids. Transient expression of an active fragment of PKC in rat fibroblasts resulted in the transcriptional activation of a TRE (TPA‐responsive element)‐CAT chimeric gene which contains various repetitions of collagenase TREs. These provide the first direct evidence that kinase activity of PKC is involved in TPA‐induced transcriptional activation through TRE.

The second phase activation of protein kinase C delta at late G1 is required for DNA synthesis in serum-induced cell cycle progression.

Background: Cell lines that stably over‐express protein kinase C (PKC) δ frequently show a decrease in growth rate and saturation density, leading to the hypothesis that PKCδ has a negative effect on cell proliferation. However, the mode of PKCδ activation, the cell cycle stage requiring PKCδ activity, and the exact role of PKCδ at that stage remains unknown.

Protein kinase Cε: the mitochondria‐mediated signaling pathway

Mitochondria, which are the cellular energy plants, also act as the integration center of cellular signaling pathways. Apoptosis is a well‐known pathway in which mitochondria are involved. Protein kinase Cε has been classified as a novel type of protein kinase C and is involved in many cellular events regulating mitochondrial function. Much evidence has accumulated regarding the relationships between mitochondria‐mediated apoptosis and protein kinase Cε. Therefore, by focusing on these relationships, in particular the anti‐apoptotic effects of protein kinase Cε on mitochondrial function, we highlight the importance and significance of protein kinase Cε in cell survival and death.