d-alpha-tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties.

d-alpha-Tocopherol, but not d-beta-tocopherol, negatively regulates proliferation of vascular smooth muscle cells at physiological concentrations. d-alpha-Tocopherol inhibits protein kinase C (PKC) activity, whereas d-beta-tocopherol is ineffective. Furthermore d-beta-tocopherol prevents the inhibition of cell growth and of PKC activity caused by d-alpha-tocopherol. The negative regulation by d-alpha-tocopherol of PKC activity appears to be the cause and not the effect of smooth muscle cell growth inhibition. d-alpha-Tocopherol does not act by binding to PKC directly but presumably by preventing PKC activation. It is concluded that, in vascular smooth muscle cells, d-alpha-tocopherol acts specifically through a nonantioxidant mechanism and exerts a negative control on a signal transduction pathway regulating cell proliferation.

Cloning and characterization of Lnk, a signal transduction protein that links T-cell receptor activation signal to phospholipase C gamma 1, Grb2, and phosphatidylinositol 3-kinase.

A cDNA encoding a signal transduction protein with a Src homology 2 (SH2) domain and a tyrosine phosphorylation site was cloned from a rat lymph node cDNA library. This protein, which we designate Lnk, has a calculated molecular weight of 33,988. When T lymphocytes were activated by antibody-mediated crosslinking of the T-cell receptor and CD4, Lnk became tyrosine phosphorylated. In activated T lymphocytes, phospholipase C gamma 1, phosphatidylinositol 3-kinase, and Grb-2 coimmunoprecipitated with Lnk. Our results suggest that Lnk becomes tyrosine phosphorylated and links the immediate tyrosine phosphorylation signals of the TCR to the distal phosphatidylinositol 3-kinase, phospholipase C gamma 1 and Ras signaling pathways through its multifunctional tyrosine phosphorylation site.

The versican C-type lectin domain recognizes the adhesion protein tenascin-R.

The core proteins of large chondroitin sulfate proteoglycans contain a C-type lectin domain. The lectin domain of one of these proteoglycans, versican, was expressed as a recombinant 15-kDa protein and shown to bind to insolubilized fucose and GlcNAc. The lectin domain showed strong binding in a gel blotting assay to a glycoprotein doublet in rat brain extracts. The binding was calcium dependent and abolished by chemical deglycosylation treatment of the ligand glycoprotein. The versican-binding glycoprotein was identified as the cell adhesion protein tenascin-R, and versican and tenascin-R were both found to be localized in the granular layer of rat cerebellum. These results show that the versican lectin domain is a binding domain with a highly targeted specificity. It may allow versican to assemble complexes containing proteoglycan, an adhesion protein, and hyaluronan.

Chimeric Na+/H+ exchangers: an epithelial membrane-bound N-terminal domain requires an epithelial cytoplasmic C-terminal domain for regulation by protein kinases.

All cloned members of the mammalian Na+/H+ exchanger gene family encode proteins that consist of two functionally distinct domains: a membrane-bound N terminus and a cytoplasmic C terminus, which are required for ion transport and regulation of transport, respectively. Despite their similarity in structure, three members of this family, designated NHE1, NHE2, and NHE3, exhibit different kinetic mechanisms in response to growth factors and protein kinases. For instance, growth factors stimulate NHE1 by a change in the affinity constant for intracellular H+, K'(Hi+), and regulate NHE2 and NHE3 by a change in Vmax. We have constructed chimeric Na+/H+ exchangers by exchanging the N and C termini among three cloned rabbit Na+/H+ exchangers (NHE1 to NHE3) to determine which domain is responsible for the above Vmax-vs.-K'(H(i)+) effect of the Na+/H+ isoforms. All of the chimeras had functional exchange activity and basal kinetic properties similar to those of wild-type exchangers. Studies with serum showed that the N terminus is responsible for the Vmax-vs.-K'(H(i)+) stimulation of the Na+/H+ exchanger isoforms. Moreover, phorbol 12-myristate 13-acetate and fibroblast growth factor altered Na+/H+ exchange only in chimeras that had an epithelial N-terminal domain matched with an epithelial C-terminal domain. Therefore, the protein kinase-induced regulation of Na+/H+ exchangers is mediated through a specific interaction between the N- and C-termini, whcih is restricted so that epithelial N- and epithelial N-and C-terminal portions of the exchangers are required for regulation.

Signaling by N- and C-terminal sequences of parathyroid hormone-related protein in hippocampal neurons.

Parathyroid hormone-related protein (PTHrP) is synthesized in the brain, and a single type of cloned receptor for the N-terminal portion of PTHrP and PTH is present in the central nervous system. Nothing is known about the physiological actions or signaling pathways used by PTHrP in the brain. Using cultured rat hippocampal neurons, we demonstrate that N-terminal PTHrP[1-34] and PTH[1-34] signal via cAMP and cytosolic calcium transients. The cAMP response showed strong acute (< or = 6 h) homologous and heterologous desensitization after preincubation with PTHrP or PTH. In contrast, the acute calcium response did not desensitize after preincubation with PTHrP; in fact, preincubation dramatically recruited additional responsive neurons. Unexpectedly, C-terminal PTHrP[107-139], which does not bind or activate the cloned PTH/PTHrP receptor, signaled in neurons via cytosolic calcium but not cAMP. Although some neurons responded to both PTHrP[1-34] and PTHrP[107-139], others responded only to PTHrP[1-34]. We conclude that certain hippocampal neurons exhibit dual signaling in response to PTHrP[1-34] and that some neurons have a receptor for C-terminal PTHrP that signals only via cytosolic calcium.

Role of phospholipids containing docosahexaenoyl chains in modulating the activity of protein kinase C.

It is known that the phospholipids of the brain cells of fish are altered during cold adaptation. In particular, the 1-monounsaturated 2-polyunsaturated phosphatidylethanolamines (PEs) increase 2- to 3-fold upon adaptation to cold. One of the most striking changes is in the 18:1/22:6 species of PE. We determined how this lipid affected the bilayer-to-hexagonal-phase transition temperature of 16:1/16:1 PE. We found that it was more effective in lowering this transition temperature than were other, less unsaturated, PE species. In addition, it was not simply the presence of the 18:1/22:6 acyl chains which caused this effect, since the 18:1/22:6 species of phosphatidylcholine had the opposite effect on this transition temperature. Zwitterionic substances that lower the bilayer-to-hexagonal-phase transition temperature often cause an increase in the activity of protein kinase C (PKC). Indeed, the 18:1/22:6 PE caused an increase in the rate of histone phosphorylation by PKC which was greater than that caused by other, less unsaturated, PEs. The 18:1/22:6 phosphatidylcholine had no effect on this enzyme. The stimulation of the activity of PKC by the 18:1/22:6 PE is a consequence of this lipid's increasing the partitioning of PKC to the membrane.

Tyrosine phosphorylation of protein kinase C-delta in response to the activation of the high-affinity receptor for immunoglobulin E modifies its substrate recognition.

The delta isoform of protein kinase C is phosphorylated on tyrosine in response to antigen activation of the high-affinity receptor for immunoglobulin E. While protein kinase C-delta associates with and phosphorylates this receptor, immunoprecipitation of the receptor revealed that little, if any, tyrosine-phosphorylated protein kinase C-delta is receptor associated. In vitro kinase assays with immunoprecipitated tyrosine-phosphorylated protein kinase C-delta showed that the modified enzyme had diminished activity toward the receptor gamma-chain peptide as a substrate but not toward histones or myelin basic protein peptide. We propose a model in which the tyrosine phosphorylation of protein kinase C-delta regulates the kinase specificity toward a given substrate. This may represent a general mechanism by which in vivo protein kinase activities are regulated in response to external stimuli.

Protein kinase C chimeras: catalytic domains of alpha and beta II protein kinase C contain determinants for isotype-specific function.

Protein kinase C (PKC) is involved in the proliferation and differentiation of many cell types. In human erythroleukemia (K-562) cells, the PKC isoforms alpha and beta II play distinct functional roles. alpha PKC is involved in phorbol 12-myristate 13-acetate-induced cytostasis and megakaryocytic differentiation, whereas beta II PKC is required for proliferation. To identify regions within alpha and beta II PKC that allow participation in these divergent pathways, we constructed chimeras in which the regulatory and catalytic domains of alpha and beta II PKC were exchanged. These PKC chimeras can be stably expressed, exhibit enzymatic properties similar to native alpha and beta II PKC in vitro, and participate in alpha and beta II PKC isotype-specific pathways in K-562 cells. Expression of the beta/alpha PKC chimera induces cytostasis in the same manner as overexpression of wild-type alpha PKC. In contrast, the alpha/beta II PKC chimera, like wild-type beta II PKC, selectively translocates to the nucleus and leads to increased phosphorylation of the nuclear envelope polypeptide lamin B in response to bryostatin-1. Therefore, the catalytic domains of alpha and beta II PKC contain determinants important for alpha and beta II PKC isotype function. These results suggest that the catalytic domain represents a potential target for modulating PKC isotype activity in vivo.

12(S)-hydroxyeicosatetraenoic acid and 13(S)-hydroxyoctadecadienoic acid regulation of protein kinase C-alpha in melanoma cells: role of receptor-mediated hydrolysis of inositol phospholipids.

Protein kinase C (PKC) isoenzymes are essential components of cell signaling. In this study, we investigated the regulation of PKC-alpha in murine B16 amelanotic melanoma (B16a) cells by the monohydroxy fatty acids 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and 13(S)-hydroxyoctadecadienoic acid [13(S)-HODE]. 12(S)-HETE induced a translocation of PKC-alpha to the plasma membrane and focal adhesion plaques, leading to enhanced adhesion of B16a cells to the matrix protein fibronectin. However, 13(S)-HODE inhibited these 12(S)-HETE effects on PKC-alpha. A receptor-mediated mechanism of action for 12(S)-HETE and 13(S)-HODE is supported by the following findings. First, 12(S)-HETE triggered a rapid increase in cellular levels of diacylglycerol and inositol trisphosphate in B16a cells. 13(S)-HODE blocked the 12(S)-HETE-induced bursts of both second messengers. Second, the 12(S)-HETE-increased adhesion of B16a cells to fibronectin was sensitive to inhibition by a phospholipase C inhibitor and pertussis toxin. Finally, a high-affinity binding site (Kd = 1 nM) for 12(S)-HETE was detected in B16a cells, and binding of 12(S)-HETE to B16a cells was effectively inhibited by 13(S)-HODE (IC50 = 4 nM). In summary, our data provide evidence that regulation of PKC-alpha by 12(S)-HETE and 13(S)-HODE may be through a guanine nucleotide-binding protein-linked receptor-mediated hydrolysis of inositol phospholipids.

Identification of human cyclin-dependent kinase 8, a putative protein kinase partner for cyclin C.

Metazoan cyclin C was originally isolated by virtue of its ability to rescue Saccharomyces cerevisiae cells deficient in G1 cyclin function. This suggested that cyclin C might play a role in cell cycle control, but progress toward understanding the function of this cyclin has been hampered by the lack of information on a potential kinase partner. Here we report the identification of a human protein kinase, K35 [cyclin-dependent kinase 8 (CDK8)], that is likely to be a physiological partner of cyclin C. A specific interaction between K35 and cyclin C could be demonstrated after translation of CDKs and cyclins in vitro. Furthermore, cyclin C could be detected in K35 immunoprecipitates prepared from HeLa cells, indicating that the two proteins form a complex also in vivo. The K35-cyclin C complex is structurally related to SRB10-SRB11, a CDK-cyclin pair recently shown to be part of the RNA polymerase II holoenzyme of S. cerevisiae. Hence, we propose that human K35(CDK8)-cyclin C might be functionally associated with the mammalian transcription apparatus, perhaps involved in relaying growth-regulatory signals.

Close association of the alpha subunits of Gq and G11 G proteins with actin filaments in WRK1 cells: relation to G protein-mediated phospholipase C activation.

A selective polyclonal antibody directed toward the C-terminal decapeptide common to the alpha subunits of Gq and G11 G proteins (G alpha q/G alpha 11) was prepared and used to investigate the subcellular distribution fo these proteins in WRK1 cells, a rat mammary tumor cell line. In immunoblots, the antibody recognized purified G alpha q and G alpha 11 proteins and labeled only two bands corresponding to these alpha subunits. Functional studies indicated that this antibody inhibited vasopressin- and guanosine 5'-[alpha-thio]triphosphate-sensitive phospholipase C activities. Immunofluorescence experiments done with this antibody revealed a filamentous labeling corresponding to intracytoplasmic and perimembranous actin-like filament structures. Colocalization of G alpha q/G alpha 11 and F-actin filaments (F-actin) was demonstrated by double-labeling experiments with anti-G alpha q/G alpha 11 and anti-actin antibodies. Immunoblot analysis of membrane, cytoskeletal, and F-actin-rich fractions confirmed the close association of G alpha q/G alpha 11 with actin. Large amounts of G alpha q/G alpha 11 were recovered in the desmin- and tubulin-free F-actin-rich fraction obtained by a double depolymerization-repolymerization cycle. Disorganization of F-actin filaments with cytochalasin D preserved G alpha q/G alpha 11 and F-actin colocalization but partially inhibited vasopressin- and fluoroaluminate-sensitive phospholipase C activity, suggesting that actin-associated G alpha q/G alpha 11 proteins play a role in signal transduction.

The product of the ataxia-telangiectasia group D complementing gene, ATDC, interacts with a protein kinase C substrate and inhibitor.

Ataxia-telangiectasia (AT) is an autosomal recessive human genetic disease characterized by immunological, neurological, and developmental defects and an increased risk of cancer. Cells from individuals with AT show sensitivity to ionizing radiation, elevated recombination, cell cycle abnormalities, and aberrant cytoskeletal organization. The molecular basis of the defect is unknown. A candidate AT gene (ATDC) was isolated on the basis of its ability to complement the ionizing radiation sensitivity of AT group D fibroblasts. Whether ATDC is mutated in any AT patients is not known. We have found that the ATDC protein physically interacts with the intermediate-filament protein vimentin, which is a protein kinase C substrate and colocalizing protein, and with an inhibitor of protein kinase C, hPKCI-1. Indirect immunofluorescence analysis of cultured cells transfected with a plasmid encoding an epitope-tagged ATDC protein localizes the protein to vimentin filaments. We suggest that the ATDC and hPKCI-1 proteins may be components of a signal transduction pathway that is induced by ionizing radiation and mediated by protein kinase C.

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

Regulated assembly of tight junctions by protein kinase C.

We have previously shown that protein phosphorylation plays an important role in the sorting and assembly of tight junctions. We have now examined in detail the role of protein kinases in intercellular junction biogenesis by using a combination of highly specific and broad-spectrum inhibitors that act by independent mechanisms. Our data indicate that protein kinase C (PKC) is required for the proper assembly of tight junctions. Low concentrations of the specific inhibitor of PKC, calphostin C, markedly inhibited development of transepithelial electrical resistance, a functional measure of tight-junction biogenesis. The effect of PKC inhibitors on the development of tight junctions, as measured by resistance, was paralleled by a delay in the sorting of the tight-junction protein, zona occludens 1 (ZO-1), to the tight junction. The assembly of desmosomes and the adherens junction were not detectably affected, as determined by immunocytochemical analysis. In addition, ZO-1 was phosphorylated subsequent to the initiation of cell-cell contact, and treatment with calphostin C prevented approximately 85% of the phosphorylation increase. Furthermore, in vitro measurements indicate that ZO-1 may be a direct target of PKC. Moreover, membrane-associated PKC activity more than doubled during junction assembly, and immunocytochemical analysis revealed a pool of PKC zeta that appeared to colocalize with ZO-1 at the tight junction. A preformed complex containing ZO-1, ZO-2, p130, as well as 330- and 65-kDa phosphoproteins was detected by coimmunoprecipitation in both the presence and absence of cell-cell contact. Identity of the 330- and 65-kDa phosphoproteins remains to be determined, but the 65-kDa protein may be occludin. The mass of this complex and the incorporation of ZO-1 into the Triton X-100-insoluble cytoskeleton were not PKC dependent.

A C-terminally-anchored Golgi protein is inserted into the endoplasmic reticulum and then transported to the Golgi apparatus.

Unlike conventional membrane proteins of the secretory pathway, proteins anchored to the cytoplasmic surface of membranes by hydrophobic sequences near their C termini follow a posttranslational, signal recognition particle-independent insertion pathway. Many such C-terminally-anchored proteins have restricted intracellular locations, but it is not known whether these proteins are targeted directly to the membranes in which they will ultimately reside. Here we have analyzed the intracellular sorting of the Golgi protein giantin, which consists of a rod-shaped 376-kDa cytoplasmic domain followed by a hydrophobic C-terminal anchor sequence. Unexpectedly, we find that giantin behaves like a conventional secretory protein in that it inserts into the endoplasmic reticulum (ER) and then is transported to the Golgi. A deletion mutant lacking a portion of the cytoplasmic domain adjacent to the membrane anchor still inserts into the ER but fails to reach the Golgi, even though this mutant has a stable folded structure. These findings suggest that the localization of a C-terminally-anchored Golgi protein involves at least three steps: insertion into the ER membrane, controlled incorporation into transport vesicles, and retention within the Golgi.