Disappearance of Ca2+-sensitive, phospholipid-dependent protein kinase activity in phorbol ester-treated 3T3 cells

doi:10.1016/S0006-291X(84)80213-2

Biochemical and Biophysical Research Communications

Volume 120, Issue 3, 16 May 1984, Pages 1053-1059

https://doi.org/10.1016/S0006-291X(84)80213-2 Get rights and content

Summary

Treatment of intact 3T3 cells with biologically active phorbol esters causes a progressive decline in the total activity of Ca²⁺-sensitive, phospholipid-dependent protein kinase (protein kinase C) measured in cell-free, detergent-solubilized, extracts. The effect is dose-dependent and reversible; protein kinase C activity reappears when the phorbol esters are removed from the medium.

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Cancer is among the main causes of death worldwide showing an increasing incidence and a growing mortality in our aging population. Natural products have long been used in popular anticancer treatment. Nowadays, some compounds of natural sources or their semisynthetic derivatives have been used in the clinic as anticancer drugs. Moreover, a huge number of natural metabolites have shown to interact with diverse cellular targets involved in carcinogenesis. The discovery of the natural tumor-promoting phorbol esters (PEs) extracted from the seed oil of the Croton tiglium, as protein kinase C (PKC) activators opened a new research area on the role of PKC proteins in cell growth and tumor promotion. The PKC is a family of serine/threonine kinases composed by 10 structurally and functionally related isoforms divided into three subgroups: classical, novel, and atypical PKCs. Some of the PKC isoforms have been widely implicated in tumor suppression, while other isoforms have been involved in tumor development and invasion. Thus, PKC family isoforms are widely recognized therapeutic targets in anticancer drug development. Several natural compounds have been identified either as inhibitors or activators of the PKC isoforms and used to modulate the PKC activity in tumor cells. Besides the aforementioned PEs that are tigliane diterpenes, the polyoxygenated macrocyclic lactones bryostatins, the staurosporine alkaloids, and the ingenene macrocyclic diterpenes are among the most studied natural PKC modulators. In addition, the daphnane tricyclic diterpenes, the abietane diterpenoids as carnosol and coleon U, the phenolic compounds as curcumin and resveratrol, and the flavonoids as quercetin, have more recently been studied as PKC modulators. Despite this, only two of these compounds reached the clinic, ingenol mebutate and bryostatin 1, in combination with paclitaxel.
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In this current study, we employed this method as an indicator of PKC activation. However, since phorbol ester activation results in a complete loss of most PKC isoforms until the drug is removed (Rodriguez-Pena and Rozengurt, 1984), extended phorbol ester treatment has been used to generate PKC deficient cells (Matthies et al., 1987). Given the dearth of PKC isoform-specific inhibitors, this could be a potentially excellent method for us in our future studies of PKC modulation of NMDAR function.
Protein kinase C (PKC) is a family of serine/threonine kinases comprised of 10 isoforms. Although commercial antibodies are available for all 10 isoforms, the specificity of these antibodies has been questioned. We have identified immunoblot conditions in which commercially purchased PKC antibodies are specific for their respective isoform. We then used these conditions to determine that PKC isoforms α, βI, βII, δ, ε, γ, λ, θ, and ζ are present in rat primary cultured cerebellar granule cells (CGCs) 6–14 days in vitro (DIV). This PKC profile is identical to that observed in cerebellar homogenates taken from 6-, 14- and 21-day-old rats. Western blot analysis indicated that the classical and the atypical PKC isoforms were more prevalent in the cytosolic subcellular fraction compared to the particulate fraction under basal conditions. Immunoreactivity for the novel isoforms tended to be higher in the particulate fraction under basal conditions. Phorbol 12-myristate 13-acetate (PMA) treatment resulted in translocated immunoreactivity from the cytosolic to the particulate fraction for all of the classical and novel PKC isoforms, but not for the atypical isoforms. However, the degree of translocation as well as the speed of translocation varied among the isoforms. The stability of the individual isoforms after PMA-induced activation also varied among the isoforms. Differences in these parameters were dependent upon culture batches and PKC isoform groups. We have identified experimental conditions in which reproducible results can be obtained with primary cultured CGCs in the study of PKC. We discuss possible solutions for problems encountered when utilizing primary cultured neurons to study PKC-mediated signal transduction.
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Despite acutely inhibiting adenylate cyclase, prolonged activation of Gα_i/o-coupled receptors leads to a subsequent heterologous sensitization of adenylate cyclase responsiveness. Recently, protein kinase signaling and phosphorylation have been implicated in the sensitization of adenylate cyclase type 6 (AC6). To examine the sensitization specifically of AC6, we constructed human embryonic kidney cells (HEK293) cells stably expressing AC6 and the Gα_i/o-coupled D_2L dopamine receptor. In contrast to observations in δ-opioid-expressing Chinese hamster ovary (CHO) cells that express endogenous AC6 and AC7, neither protein kinase C (PKC) nor tyrosine kinase inhibitors attenuated D_2L receptor-mediated sensitization of AC6. Inhibition of Raf1 modestly inhibited the magnitude of D_2L receptor-induced sensitization of AC6; however, activation of PKC robustly enhanced D_2L receptor-mediated AC6 sensitization in a Raf1-dependent manner. These data indicate that, although PKC and Raf1 are not required for sensitization, activation of the PKC-Raf1 pathway robustly potentiated D_2L receptor-mediated sensitization of AC6.
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Inhibitory effect of IFN-τ on phorbol ester (PdBu)-induced PGF_2α secretion was hypothesized to be manifested by the regulation of protein kinase C (PKC) in bovine endometrial (BEND) cells. Following 12 h stimulation with PdBu, cells were unresponsive to freshly added PdBu. Pretreatment of cells with a PKC inhibitor abolished PGF_2α secretion in response to PdBu. Therefore, PdBu induction of PGF_2α secretion is through activation of PKC. The α, ɛ, ι and λ isotypes of the PKC family were identified by Western blotting. Cells were then treated with medium alone (control), PdBu or PdBu + IFN-τ for 3 or 6 h. The PdBu-induced secretion of PGF_2α was suppressed by IFN-τ. At 3 and 6 h, PKCα and PKCɛ were detected both in the cytosolic and membrane fractions of unstimulated cells. There was a clear reduction of PKCα in the cytoplasm induced by PdBu and PdBu + IFN-τ at 3 and 6 h. The total abundance (cytoplasm and membrane fractions) of PKCα was lower in the PdBu + IFN-τ than PdBu alone. These temporal responses indicate a PKCα responsiveness of BEND cells to PdBu and PDBu + INF-τ with some evidence that IFN-τ causes a slight but detectable reduction in PKCα when added with PdBu. However, IFN-τ-induced decrease in the total abundance of PKCα was not enough to affect negatively the translocation of the PKCα to the membrane. Therefore, IFN-τ's ability to suppress secretion of PGF_2α is unlikely due to an interference with the PdBu-induced activity of PKC.

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Disappearance of Ca2+-sensitive, phospholipid-dependent protein kinase activity in phorbol ester-treated 3T3 cells

Summary

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Cell. Biochem.

Adv. Cancer Res.

Disappearance of Ca²⁺-sensitive, phospholipid-dependent protein kinase activity in phorbol ester-treated 3T3 cells