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D Fabbro

Publications and source records attributed to D Fabbro.

At least 91 records · Page 5Linked to original sources
PubMed1994

Protein kinase C isozyme expression and down-modulation in growing, quiescent, and transformed renal proximal tubule epithelial cells.

Renal alpha-protein kinase C (PKC) is rapidly down-modulated modulated in animals treated with the renal toxin and tumor promoter, folic acid (Dong et al., Cancer Res., 53: 4542-4549, 1993). To further explore the role of PKC isozymes in renal growth and carcinogenesis, we compared phorbol ester receptor and PKC isozyme content, distribution, and regulation in primary and oncogene-altered rat renal proximal tubule epithelial cells (RPTE) in culture. Immunoblot analysis and RNase protection assays indicated that RPTE expressed at least four PKC isozymes, alpha, delta, epsilon, and zeta. Total phorbol ester receptors were decreased in primary proliferating, E1A-immortalized, and SV40-transformed RPTE compared to primary quiescent RPTE. The decrease in PDBu binding was largely due to a specific decrease in alpha-PKC protein content to approximately 50% of the level in quiescent RPTE. Degradation rates and message levels were compared to determine the mechanism for the decrease in alpha-PKC. Whereas alpha-PKC message levels in quiescent and proliferating primary RPTE were comparable, alpha-PKC degradation was increased in proliferating cells. These results indicate that the decreased alpha-PKC content was due largely to increased turnover. Phorbol ester stimulated the rate of degradation, thus demonstrating a link between degradation rate and PKC activation. These results suggest that the increased basal degradation rate in proliferating and oncogene-altered cells reflects an increase in activity of PKC in these cells.

Animals↗
PubMed1994

Protein kinase C and mammary cell differentiation: involvement of protein kinase C alpha in the induction of beta-casein expression.

Treatment of HC11 mouse mammary epithelial cells with the lactogenic hormones dexamethasone, insulin, and prolactin (DIP) leads to cellular differentiation and production of the milk protein beta-casein. The following experimental evidence suggests the involvement of protein kinase C (PKC) in DIP induced signal transduction. Down-regulation of PKC by 12-O-tetradecanoylphorbol-13-acetate or addition of CGP 41251, a selective inhibitor of PKC, inhibited beta-casein protein expression induced by DIP in HC11 cells. This inhibition occurs at the level of transcription, since the DIP mediated activation of a beta-casein promoter-luciferase reporter construct or of mammary gland specific factor (MGF), an essential transcription factor for beta-casein promoter activity, was also inhibited by CGP 41251. Inhibition or down-regulation of PKC reduced the activation of MGF by prolactin as well. PKC-alpha, the only conventional PKC isoform expressed in HC11 cells, is most likely involved in the DIP induced beta-casein expression. (a) Only PKC-alpha and PKC-epsilon are down-regulated by 12-O-tetradecanoylphorbol-13-acetate whereas PKC-delta and PKC-zeta are not. (b) Of the PKC isoforms expressed in HC11 cells, CGP 41251 inhibits PKC-alpha more potently than PKC-delta, PKC-epsilon, and PKC-zeta. The IC50 for the inhibition of beta-casein synthesis, MGF activation, and beta-casein promoter activity by CGP 41251 correlated well with the IC50 of PKC-alpha inhibition. (c) Finally, only PKC-alpha translocated to membrane fractions after DIP or prolactin treatment. Taken together, these data indicate that PKC-alpha plays an important role in the signaling pathway activated by prolactin during beta-casein induction.

Alkaloids↗
PubMed1993

Effect of salt concentration on TTF-1 HD binding to specific and non-specific DNA sequences.

The Thyroid Transcription factor 1 (TTF-1) recognizes specific DNA sequences by a Homeodomain (TTF-1 HD). The TTF-1 HD DNA-binding properties with both specific and non-specific DNA sequences were investigated. TTF-1 HD exists as a monomer in solution and as a monomer binds DNA. At 75 mM KCl, its relative binding affinity with a specific DNA sequence is about 50 fold higher than with a non-specific DNA sequence. Increase of KCl concentration reduces the apparent binding affinity both to specific and non-specific DNA sequences. However, non-specific binding is more sensitive than specific binding to the increase of salt concentration. When DNA-binding reactions are performed at temperature and salt concentration close to the intracellular environment, TTF-1 HD binds the specific sequence with an affinity at least 1000 fold higher respect to the non-specific sequence.

Animals↗
PubMed1993

Regulation of protein kinase C isozymes in kidney regeneration.

Tissue damage and repair processes are important factors in renal tumor progression. To determine whether protein kinase C (PKC) is involved in these processes, we characterized PKC isozymes during rat kidney regeneration using 3 models: (a) diffuse cortical hyperplasia and hypertrophy induced by folic acid; (b) focal necrosis of the S3 segments induced by S-(1,2-dichlorovinyl)-L-cysteine; and (c) compensatory renal hypertrophy. Immunoblot analyses demonstrated that 5 PKC isozymes, alpha, beta, delta, epsilon, and zeta, were expressed in rat kidney. Six h after folic acid treatment, phorbol ester receptors were down-modulated. Down-modulation preceded an increase in DNA synthesis which was maximal at 24 h. The reduction in phorbol ester receptors was due largely to a decrease in alpha-PKC. zeta-PKC, which is not a phorbol ester receptor, was also decreased. delta- and epsilon-PKCs were not changed. However, alpha-PKC was not down-modulated during compensatory hypertrophy induced by unilateral nephrectomy. Thus, the observed decrease of alpha-PKC after folic acid treatment is most likely associated with the hyperplastic and not the hypertrophic effects of this renal toxin. These results demonstrate that activation-associated down-modulation of PKC, in particular alpha-PKC, occurs during chemical-induced renal regeneration and suggests a general role for PKC activation in non-phorbol ester tumor promotion.

Animals↗
PubMed1993

Selective regulation of expression of protein kinase C (PKC) isoenzymes in multidrug-resistant MCF-7 cells. Functional significance of enhanced expression of PKC alpha.

The multidrug resistance (MDR) phenotype induces cross-resistance to many chemotherapeutic agents in cancer cells. Protein kinase C (PKC) has been implicated in the regulation of the MDR phenotype. In order to determine the role of specific PKC isoenzymes in regulating the MDR phenotype, the expression and activity of PKC isoenzymes in the human breast cancer cell line, MCF-7-WT, and an MDR subline, MCF-7-MDR, were examined. The MDR phenotype was associated with a 10-fold increase in calcium-dependent PKC activity as well as a 10-fold decrease in calcium-independent activity was due to a selective increase in the activity was due to a selective increase in the expression of PKC alpha as determined by Western blot analysis and hydroxylapatite chromatography. This increase in expression of PKC alpha was regulated at the message level as demonstrated by Northern blot analysis. The decrease in calcium-independent activity was caused by a decrease in the expression of PCK delta and epsilon. The significance of the increase in PKC alpha expression was then demonstrated by a commensurate 11-fold increase in the basal and stimulated phosphorylation of the myristolated alanine-rich C kinase substrate. Phosphorylation of P-glycoprotein, the cellular mediator of the MDR phenotype, was increased > 20-fold in the unstimulated MCF-7-MDR cell line and its phosphorylation was further increased 2-fold in response to phorbol 12-myristate 13-acetate. These changes paralleled the increases in P-glycoprotein pump function and the MDR phenotype underscoring the role for PKC alpha in regulating P-glycoprotein phosphorylation and function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Unphosphorylated alpha-PKC exhibits phorbol ester binding but lacks protein kinase activity in vitro.

Expression of the alpha-isoform of protein kinase C (alpha-PKC) in E. coli yielded the unphosphorylated 74 kD precursor molecule. This precursor form exhibited phospholipid- and calcium-dependent phorbol ester binding but lacked, in contrast to the phosphorylated enzyme, protein kinase activity. In addition, the precursor molecule was found to interact with both threonine and an ATP analogon, which demonstrates that phosphorylation of alpha-PKC is not required for binding of substrates, cofactors, or activators. These results, therefore, suggest that posttranslational phosphorylation of alpha-PKC is not needed for the formation of a functional enzyme-substrate complex but is necessary for the catalytic transfer of phosphate residues from ATP to protein substrates.

Enzyme Precursors↗
PubMed1993

Effects of occupational exposure to mercury and lead on brainstem auditory evoked potentials.

A study was performed to examine the effects of industrial exposures to lead and mercury on the brainstem auditory pathway by recording brainstem auditory evoked potentials (BAEPs). BAEPs were recorded from 22 workers exposed to lead, from eight exposed to mercury and from two control groups of age- and sex-matched subjects never exposed to neurotoxic substances. The time between BAEP peaks I and V were evaluated. The mean durations of exposure were 9.3 and 11.7 years for lead and mercury, respectively. Blood lead concentration (PbBc) on the morning of the test day was 47.5 micrograms/dl, whereas urinary mercury content at the end of the previous work day was 325 micrograms/g creatinine. Both mercury and lead exposed workers showed a significant prolongation of wave I-V time. In addition, the interwave time was longer in a lead subgroup with the greatest mean level of lead in blood. These results are consistent with other studies and show that BAEPs may provide a sensitive tool for detecting subclinical central neurotoxicity caused by lead and mercury.

Acoustic Stimulation↗
PubMed1993

The epsilon isoform of protein kinase C is an oncogene when overexpressed in rat fibroblasts.

We have overproduced the Ca(2+)-independent protein kinase C isoform, nPKC epsilon, in Rat 6 embryo fibroblasts, and examined the effects of this novel isoform on cell growth and transformation. As compared to vector control cell lines expressing only the hygromycin resistance gene, the nPKC epsilon overproducing cell lines exhibited a 7-13-fold increase in Ca(2+)-independent enzyme activity. Detailed analysis of seven individual nPKC epsilon overproducing clones indicated that those clones that expressed very high activity displayed a number of disorders in growth control, including: formation of dense foci in monolayer culture, decreased doubling time, increased saturation density, decreased serum requirement, growth in soft agar, and tumor formation in nude mice. These findings are in contrast to previous studies from our laboratory indicating that stable expression of high levels of cPKC beta 1 produced only a partially transformed phenotype (Housey et al., 1988). Taken together, these results provide the first direct evidence that distinct isoforms of PKC can exert different effects on growth control and malignant transformation in the same cell type.

Animals↗
PubMed1992

Effect of tumour-promoting phorbol ester, thrombin and vasopressin on translocation of three distinct protein kinase C isoforms in human platelets and regulation by calcium.

Protein kinase C (PKC) acts in synergy with Ca2+ mobilization for the activation of platelets. Three different PKC subtypes that specifically react with antibodies to alpha- beta- and zeta-PKC have been detected in human platelets. We have compared the subcellular redistribution of these isoforms in platelets after exposure to the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and to two physiological agonists, thrombin and vasopressin. In the presence of PMA, beta-PKC is most rapidly translocated to membranes, followed by zeta-PKC and alpha-PKC [membrane contents of 39 +/- 6, 31 +/- 4 and 24 +/- 4% (means +/- S.E.M.) respectively after 2 min incubation]. In contrast, both thrombin and vasopressin induced a biphasic translocation of PKC isoforms. For both agonists, the first phase of translocation occurred within 1 min and was identical for the three isoforms. However, during the second phase, the translocation of zeta-PKC by thrombin and vasopressin differed [membrane contents (mean +/- S.E.M.) of 24 +/- 3 and 46 +/- 4% respectively after 10 min]. These results suggest a differential activation of zeta-PKC by vasopressin and thrombin. PMA-induced translocation of alpha-PKC was decreased from 278 +/- 27 to 198 +/- 24 (mean +/- S.E.M., P = 0.02; percentage increase over control value) in the presence of 1 mM-EDTA, whereas chelation of intracellular Ca2+ by Quin2-AM does not influence this response. These results suggest that the PMA-induced translocation of alpha-PKC depends on the presence of 1 mM concentration of extracellular Ca2+. In addition, the chelation of either extracellular or intracellular Ca2+ inhibited both vasopressin- and thrombin-induced translocation of all three isoforms, suggesting that Ca2+ is an important requirement for the translocation of alpha-, beta- and zeta-PKC by physiological agonists. In conclusion, the translocation of PKC varies between different isoforms and between different agonists.

Amino Acid Sequence↗
PubMed1992

Alpha-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells.

The mechanism by which an agonist, binding to a cell surface receptor, exerts an effect on events in the nucleus is not known. We have previously shown (Leach, K. L., Ruff, V. A., Wright, T. M., Pessin, M. S., and Raben, D. M. (1991) J. Biol. Chem. 266, 3215-3221) that alpha-thrombin treatment of IIC9 cells results in increased levels of cellular 1,2-diacylglycerol (DAG) and activation of protein kinase C (PKC). Here, we have examined whether changes in nuclear PKC and nuclear DAG also are induced following alpha-thrombin treatment. IIC9 cells were treated with 500 ng/ml alpha-thrombin, and nuclei were then isolated. Western blot analysis using isozyme-specific antibodies demonstrated the presence of PKC alpha, but not PKC epsilon or zeta in the nuclei of cells treated with either phorbol 12-myristate 13-acetate or alpha-thrombin. The increase in nuclear PKC alpha levels was accompanied by a 10-fold increase in nuclear PKC specific activity and stimulated phosphorylation of at least six nuclear proteins. The rise in nuclear PKC levels occurred rapidly and reached a maximum at 30-60 s, which was followed by a decline back to the control level over the next 15 min. In addition, alpha-thrombin treatment resulted in an immediate rise in DAG mass levels in the nuclear fractions. Kinetic analysis indicated that a maximum increase in DAG levels occurred 2.5-5 min after the addition of alpha-thrombin and remained elevated for at least 30 min. In cells labeled with [3H]myristic acid, alpha-thrombin treatment induced an increase in radiolabeled nuclear diglycerides, suggesting that the stimulated nuclear DAGs are derived, at least in part, from phosphatidylcholine. Our results suggest that increases in both nuclear DAG levels and PKC activity following alpha-thrombin treatment may play a role in mediating thrombin-induced nuclear responses such as changes in gene expression and cellular proliferation.

Animals↗
PubMed1992

Expression of four protein kinase C isoforms in rat fibroblasts. Distinct subcellular distribution and regulation by calcium and phorbol esters.

Protein kinase C (PKC), the major receptor for tumor-promoting phorbol esters, consists of a family of at least eight distinct lipid-regulated enzymes. How the various PKC isozymes are regulated in vivo and how they couple to particular cellular responses is largely unknown. We have examined the expression and regulation of PKC isoforms in R6 rat embryo fibroblasts. Northern and Western blot analyses indicate that these cells express four PKC isoforms, cPKC alpha, nPKC epsilon, nPKC delta, and nPKC zeta; of which nPKC epsilon and nPKC delta are the most abundant. In agreement with the simultaneous presence of cPKC and nPKC isozymes, both Ca(2+)-dependent and -independent PKC activities were detected in extracts of these cells. cPKC alpha and nPKC zeta were predominantly localized in the cytosol when subcellular fractionation was carried out in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. When cell lysis was carried out in the presence of Ca2+, greater than 50% of cPKC alpha redistributed to the particulate fraction, whereas nPKC zeta remained in the cytosol. In contrast to cPKC alpha and nPKC zeta, 60-80% of nPKC epsilon and nPKC delta were located in a Ca(2+)-insensitive, membrane-bound form. Treatment of R6 cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA), resulted in the translocation of all four PKC isozymes to the membrane fraction, and the subsequent down-regulation of cPKC alpha, nPKC zeta, and nPKC delta, nPKC epsilon, however, was only partially down-regulated in response to long-term TPA exposure. Overproduction of exogenous cPKC beta I in R6 cells conferred partial resistance of nPKC delta to TPA-induced down-regulation and potentiated the resistance of nPKC epsilon to down-regulation. These results demonstrate that the multiple isoforms of PKC which coexist within a single cell type are differentially regulated by extra- and intracellular stimuli and may thereby influence growth control and transformation via distinct mechanisms.

Animals↗
PubMed1992

Expression of four protein kinase C isoforms in rat fibroblasts. Differential alterations in ras-, src-, and fos-transformed cells.

In the accompanying study (Borner, C.B., Guadagno, S. N., and Weinstein, I. B. (1992) J. Biol. Chem. 267, 12892-12899) we found that R6 embryo fibroblasts express four isoforms of PKC, cPKC alpha, nPKC epsilon, nPKC delta, and nPKC zeta whose subcellular distribution, activation, and down-regulation are differentially regulated. Furthermore, we demonstrated that overproduction of an exogenous cPKC beta I isoform in these cells (R6-PKC3) altered the TPA-induced down-regulation of nPKC delta and nPKC epsilon. In this paper we show that transformation of R6 or R6-PKC3 cells with a variety of different oncogenes results in differential alterations in expression of individual PKC isoforms. R6 or R6-PKC3 cells transformed by an activated c-H-ras oncogene displayed a marked increase in the expression of both cPKC alpha and nPKC delta, decreased expression of nPKC epsilon, and no change in the expression of nPKC zeta. These alterations occurred at both the mRNA and protein levels but did not significantly affect the subcellular distribution of any of the four isoforms. Studies using actinomycin D and nuclear run-off assays indicated that the increased expression of cPKC alpha in ras-transformed cells was due to increased de novo transcription rather than increased mRNA stability. Qualitatively similar, but less extensive changes in the expression of the four PKC isoforms were seen in v-fos-transformed R6-PKC3 cells. Decreased expression of nPKC epsilon was also seen in the v-src-transformed R6- and R6-PKC3 lines; however, the cellular level of cPKC beta I appeared to be a limiting factor in mediating the effects of v-src on the increased expression of cPKC alpha and nPKC delta. Interestingly, no major changes in the levels of expression of any of the four PKC isoforms were found when R6 cells were transformed by myc, neu/erb-B2, or mos oncogenes. These results demonstrate that transformation of R6 cells by the oncogenes ras, src, and fos differentially alter the expression of three isoforms of PKC in the same host cell, and they suggest that individual isoforms may play distinct roles in mediating cellular transformation by specific oncogenes.

Alkaloids↗
PubMed1992

Immunocharacterization of delta- and zeta-isoenzymes of protein kinase C in rat renal mesangial cells.

The isoforms of protein kinase C (PKC) present in rat mesangial cells were identified by immunoblot analysis with antibody raised against isotype-specific peptides. In addition to the previously observed alpha- and epsilon-subspecies, mesangial cells also express the delta- and zeta-isoenzymes of PKC. On exposure to phorbol 12,13-dibutyrate (PDB) a complete depletion of PKC-delta is observed within 8 h. Removal of PDB results in a recovery of PKC-delta. In contrast, PKC-zeta is unaffected by addition or removal of PDB.

Amino Acid Sequence↗
PubMed1992

Expression and partial characterization of rat protein kinase C-delta and protein kinase C-zeta in insect cells using recombinant baculovirus.

Expression of rat protein kinase C-delta (PKC-delta) and PKC-zeta in insect cells using recombinant baculovirus resulted in the production of proteins with a molecular size of approximately 76 kD and 78 kD, respectively, as determined by immunoblotting with subtype-specific antisera. Although the PKC-zeta cDNA encoded for 592 amino acids, a 76 kD protein was also generated by in vitro transcription/translation. Extracts of cells expressing PKC-delta were able to bind phorbol ester to levels comparable to extracts of cells expressing PKC-alpha. No phorbol ester binding was, however, detected in insect cell extracts expressing PKC-zeta. However, similar levels of protein kinase activity were detected in lysates of cells expressing PKC-delta or PKC-zeta when protamine sulfate was used as exogenous substrate. Compared to protamine sulfate, both, myelin basic protein (MBP) or histone, were poor substrates for PKC-delta and PKC-zeta. In contrast to PKC-zeta, the PKC-delta enzyme activity phosphorylated MBP or histone in a phosphatidylserine-(PS)/diacylglycerol(DG)-dependent manner, albeit not to the same extent as PKC-alpha. Lack of stimulation of the enzyme activity of PKC-zeta by PS/DG, was confirmed by endogenous phosphorylation of insect cell proteins by PKC-zeta, whereas several insect cell proteins were phosphorylated by PKC-delta in a PS/DG-dependent manner, including a protein of 78 kD. Our data demonstrate that the 76 kD PKC-zeta, in contrast to PKC-delta, is unable to bind phorbol esters and displays a protein kinase activity that is independent of PS or PS/DG. In addition, staurosporine was about 2-4 order of magnitudes less effective in inhibiting the protein kinase activities of PKC-delta and PKC-zeta when compared to PKC-alpha.

Alkaloids↗
PubMed1992

Selective redistribution of protein kinase C isozymes by thapsigargin and staurosporine.

Protein kinase C (PKC) is the major cellular receptor for tumor promoting phorbol esters. Phorbol esters activate alpha-, beta-, delta- and epsilon-PKCs in GH4C1 rat pituitary cells and cause their redistribution from a soluble to a particulate fraction. We have now characterized the effect of several non-phorbol ester tumor promoters on PKC isozyme distribution in GH4C1 cells. The incomplete tumor promoter mezerein caused redistribution of alpha-, beta-, delta- and epsilon-PKCs. Thus, it did not display partial agonist activity. The phosphatase inhibitor okadaic acid did not cause redistribution of any isozyme. The calcium ATPase inhibitor thapsigargin and the ser/thr kinase inhibitor staurosporine caused redistribution of epsilon-PKC and, to a lesser extent, delta-PKC. Although the mechanism of the selective effect on delta- and epsilon-PKCs is not yet known, these data clearly demonstrate that their subcellular distribution can be regulated by a pathway that does not influence alpha- and beta-PKCs. Phorbol ester activation of epsilon-PKC was associated with appearance of a more slowly migrating immunoreactive band in the particulate fraction. Both epsilon-PKC forms accumulated phosphate during phorbol ester treatment. The phosphorylated forms of epsilon-PKC were preferentially recovered in the particulate fraction. Although staurosporine caused redistribution, it prevented the phorbol dibutyrate (PDBu)-mediated appearance of the upper band of the doublet and the increased phosphorylation of both bands. The PDBu-mediated redistribution of alpha- and beta-PKCs was not inhibited by staurosporine, even though staurosporine effectively inhibited PKC catalytic activity. Therefore, catalytic activity is not required for redistribution.

Alkaloids↗
PubMed1992

Phorbol ester inhibits erythropoietin production in human hepatoma cells (Hep G2).

Using the human hepatoma cell line Hep G2, we have studied a possible role of protein kinase C (PKC) activity for regulation of erythropoietin (EPO) production. During a 72-h incubation, EPO production by the cells was stimulated sevenfold by exposure to low oxygen tension (1%) and threefold by exposure to cobaltous chloride (100 microM). The phorbol ester phorbol 12-myristate-13 acetate (PMA) led to a concentration-dependent inhibition of basal and stimulated EPO formation (ED50 10 nM). This decrease of EPO production, which was apparent already after 1 h of incubation with PMA, reached its maximal effect after 24 h and held on for 72 h. It was paralleled by an inhibition of the increase of EPO mRNA levels in response to stimulation. A 24-h preincubation of the cells with PMA (100 nM) virtually blunted the effect of hypoxia on EPO formation. Recovery of EPO synthesis after removal of PMA took 48-72 h. The effect of PMA on EPO production was mimicked by phorbol 12,13-dibutyrate (ED50 1 microM) but not by 4 alpha-phorbol 12,13-didecanoate. The synthetic diacylglycerol analogues oleolyl-acetylglycerol and dioctanoylglycerol (2-200 microM) also had no effect on either basal or stimulated EPO production. Treatment with PMA caused a translocation of the alpha-isoenzyme of PKC from the cytosol to the membrane after 1 h and a disappearance of the membrane-bound form after 24 h of incubation. Staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, two structurally different inhibitors of PKC activity, inhibited basal and stimulated EPO production with ED50 values of 9 nM and 50 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Hormone- and phorbol ester-activated protein kinase C isozymes mediate a reorganization of the actin cytoskeleton associated with prolactin secretion in GH4C1 cells.

TRH regulates PRL secretion and synthesis in GH4C1 rat pituitary cells. TRH responses are associated with activation of protein kinase C (PKC) isozymes and elevation of cytosolic calcium. To determine which PKC isozymes are involved in TRH-directed responses, we evaluated the effect of TRH on GH cell alpha-, beta-, delta-, and epsilon-PKC isozymes. Immunoblot analysis demonstrated that TRH caused rapid redistribution of all isozymes to a Triton X-100-insoluble (i.e. cytoskeletal) fraction. Corollary immunocytofluorescence studies demonstrated that redistributed PKCs accumulate in cell peripheries. Exocytosis involves reorganization of the cytoskeleton, therefore, each of the GH cell PKCs is appropriately located to phosphorylate proteins important for cytoskeleton organization. To determine the relative contributions of calcium and PKC signal transduction pathways in mediating TRH responses, the effects of potassium depolarization (which increases cytosolic calcium) and phorbol dibutyrate (which activates all PKC isozymes without increasing calcium) were compared. The data indicate that TRH-mediated reorganization of vinculin proceeds via a calcium-mediated pathway, whereas fragmentation of actin filaments proceeds via a PKC-dependent pathway. Selective down-modulation of epsilon-PKC with prolonged TRH-treatment was used to demonstrate that epsilon-PKC is not necessary for certain TRH-stimulated biological responses.

Actins↗