Shin-ichi Osada

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.

Ras-dependent signal transduction is indispensable but not sufficient for the activation of AP1/Jun by PKC delta.

Modulation of gene expression by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) is thought to be mediated by protein kinase C (PKC), a major cellular receptor for TPA. We confirm this by showing that the overexpression of PKC delta enhances the TPA induction of the TRE‐tk‐CAT reporter gene in NIH3T3 cells. To investigate the mutual relationship between PKC delta‐ and Ras‐dependent signal transduction pathways to a TRE binding transcription factor, AP1/Jun, we constructed constitutively active and dominant negative mutants of PKC delta. Activated Ras induced reporter gene expression in collaboration with overexpressed c‐Jun or JunD, and this induction was insensitive to the dominant negative PKC delta. On the other hand, reporter gene expression induced by the constitutively active PKC delta was severely inhibited by dominant negative Ras, as well as by the dominant negative PKC delta. Thus, Ras activation must be indispensable for PKC delta to activate AP1/Jun. In the absence of overexpressed c‐Jun or JunD, activated Ras was, however, clearly less effective than constitutively active PKC delta which showed full activation of reporter gene expression by itself. This suggests the presence of an additional, Ras‐independent, signaling pathway downstream of PKC delta to activate AP1/Jun. In spite of the remarkable ability of constitutively active PKC delta to activate TRE‐tk‐CAT expression, this mutant suppressed cell growth.

UCN-01, an anti-tumor drug, is a selective inhibitor of the conventional PKC subfamily.

A selective PKC inhibitor, UCN‐01, was shown to exhibit anti‐tumor activity in vitro and in vivo. We investigated UCN‐01 with respect to isozyme‐specific PKC inhibition using purified recombinant or rabbit brain PKC isozymes, cPKCα, β and γ, nPKCδ, ϵ and ν, and aPKCζ. Of the PKC isozymes examined, cPKCα was inhibited by UCN‐01 most effectively (K i = 0.44 nM), suggesting cPKCα is the prime candidate for the physiological target of UCN‐01. The K i values of UCN‐01 estimated from Dixon plots for cPKC isozymes are approximately 1 nM, whereas the K i values for nPKC isozymes are about 20 nM. Moreover, the K i value for aPKCζ is 3.8 μM. Thus, UCN‐01 discriminates between PKC subfamilies. In addition, the inhibitory effects of staurosporine, H7, and calphostin C on aPKCζ were examined and compared with those for cPKCα.

The eta isoform of protein kinase C is localized on rough endoplasmic reticulum.

The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992).

Unique expression pattern of protein kinase C‐θ: high mRNA levels in normal mouse testes and in T‐lymphocytic cells and neoplasms

A 2.2‐kb cDNA that contains the entire coding region of mouse protein kinase C‐θ (PKC‐θ) was cloned from skeletal muscle mRNA using reverse transcription and the polymerase chain reaction (PCR). This clone was used as a probe to study the expression of this PKC isoform in normal and transformed hemopoietic cells and other normal tissues. By far the highest steady‐state level of PKC‐0 MRNA was found as a 2.8‐kb transcript on a Northern blot of poly(A)+ RNA from testes. High levels were also found in skeletal muscle, spleen, T lymphomas and purified normal T lymphocytes, but these tissues and cells expressed two transcripts, 3.3 kb and 3.8 kb. Lower levels of similar size transcripts were found in normal brain, B lymphocytes and B‐lymphocytic tumors and cell lines.

A domain containing the Cdc42/Rac interactive binding (CRIB) region of p65PAK inhibits transcriptional activation and cell transformation mediated by the Ras-Rac pathway

© 1997 Federation of European Biochemical Societies.

Wortmannin inhibits the activation of MAP kinase following vasopressin V1 receptor stimulation

Treatment of rat 3Y1 fibroblasts with vasopressin (AVP) results in a transient activation of MAP kinase as potent as with EGF and serum. An antagonist of vasopressin receptor V1, but not an antagonist of V2, inhibited the AVP‐induced activation of MAP kinases, indicating that AVP activates MAP kinases through V1 receptor. Prolonged TPA treatment of cells resulted in partial MAP kinase activation, indicating the presence of PKC‐independent pathway. The pathway was inhibited by wortmannin, an inhibitor of PI3‐kinase. The results suggest that wortmannin‐sensitive molecules such as PI3‐kinase, are involved in the V1 receptor‐mediated activation of the MAP kinase pathway independent of TPA‐sensitive PKC.

YSK1, a novel mammalian protein kinase structurally related to Ste20 and SPS1, but is not involved in the known MAPK pathways

To clarify the upstream regulatory mechanism of mitogen-activated protein kinase (MAPK), we performed the reverse transcriptase-based polymerase chain reaction (RT – PCR) with degenerate primers synthesized based on sequences conserved among the kinase domains of yeast MAPK kinase kinases (MAPKKKs), Ste11, Bck1, and Byr2. We isolated several mammalian cDNA fragments that encode kinase subdomains sharing significant sequence homology with yeast MAPKKKs. Subsequent screening of a HeLa cell cDNA library using one of these cDNA fragments as a probe resulted in the isolation of a full-length cDNA that encodes a novel protein kinase. The catalytic domain sequence of this gene product is closely related to those of budding yeast Sps1 and Ste20 protein kinases. Thus, we call this protein YSK1 (Yeast Sps1/Ste20-related Kinase 1). The transcript of YSK1 was detected in a wide range of tissues and cells. Immunoprecipitated YSK1 shows protein kinase activity. Although YSK1 is significantly similar in its kinase domain to kinases of the yeast and mammalian MAPK pathways, the overexpression of YSK1 did not lead to the activation of the ERK (extracellular signal-regulated kinase) pathway, JNK (c-Jun NH2-terminal kinase)/SAPK (stress-activated protein kinase) pathway, or p38/Mpk2 pathway. These results suggest that YSK1 may be involved in the regulation of a novel intracellular signaling pathway.

Differential expression of protein kinase C genes in cultured mast cells derived from normal and mast-cell-deficient mice and mast cell lines.

We investigated the expression of mRNA of protein kinase C (PKC) isozymes (alpha, beta, gamma, delta, epsilon, xi, and eta) in cultured mast cells (CMC) derived from normal (+/+) mice, CMC derived from genetically mast-cell-deficient (W/W, Wv/Wv, and mi/mi) mice, and murine mast cell lines (IC2, MC9, and P-815) by Northern blotting. In +/+ CMC, abundant expression of PKC delta and moderate expression of PKC alpha and beta was observed, while other PKCs (types gamma, epsilon, xi, and eta) were not detected. In vivo expression of PKC delta was demonstrated in the skin by in situ hybridization. In mast cell lines, the expression pattern of PKC isozymes was similar to that of +/+ CMC, except that the expression of PKC eta was detected in the IC2 cell line. The expression levels of PKC delta in CMC derived from c-kit-deficient mutants, W/W, Wv/Wv, and mi/mi, were lower than that of +/+ mice. These results indicate that PKC delta is the main isozyme in various types of murine mast cells and also suggest that the reduced level of PKC delta expression in mutant mice may be caused by a deficit in the signal transduction system through c-kit receptor.

Evidence for a new, high-molecular weight isoform of protein kinase C in rat hippocampus

We describe a new form of protein kinase C (PKC) with a molecular weight of 97 kDa, higher than the known forms of vertebrate PKC. This putative new high-molecular weight isoform, which we are calling PKC (HMW), is increased in the membrane fraction either upon application of phorbol esters or with afferent synaptic stimulation of Schaffer collaterals in hippocampal slices. The protein cross-reacts on immunoblot with affinity-purified polyclonal antiserum raised against a peptide derived from the carboxy-terminus of PKC eta; it does not cross-react, however, with antiserum against the amino-terminal region of PKC eta. In the tissues examined, PKC(HMW) is localized primarily in brain, in contrast to PKC eta, which is found predominantly in lung and skin.