Tumor promotion by depleting cells of protein kinase C delta.

Tumor-promoting phorbol esters activate, but then deplete cells of, protein kinase C (PKC) with prolonged treatment. It is not known whether phorbol ester-induced tumor promotion is due to activation or depletion of PKC. In rat fibroblasts overexpressing the c-Src proto-oncogene, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced anchorage-independent growth and other transformation-related phenotypes. The appearance of transformed phenotypes induced by TPA in these cells correlated not with activation but rather with depletion of expressed PKC isoforms. Consistent with this observation, PKC inhibitors also induced transformed phenotypes in c-Src-overexpressing cells. Bryostatin 1, which inhibited the TPA-induced down-regulation of the PKCdelta isoform specifically, blocked the tumor-promoting effects of TPA, implicating PKCdelta as the target of the tumor-promoting phorbol esters. Consistent with this hypothesis, expression of a dominant negative PKCdelta mutant in cells expressing c-Src caused transformation of these cells, and rottlerin, a protein kinase inhibitor with specificity for PKCdelta, like TPA, caused transformation of c-Src-overexpressing cells. These data suggest that the tumor-promoting effect of phorbol esters is due to depletion of PKCdelta, which has an apparent tumor suppressor function.     

Carbachol stimulates TYR phosphorylation and association of PKCdelta and PYK2 in pancreas

Carbachol treatment resulted in increased phosphorylation on tyrosine of PKCdelta immunoprecipitated from rat pancreatic acinar cells. The Ca2+-dependent tyrosine kinase PYK2 coimmunoprecipitated with PKCdelta from carbachol-exposed cells and also exhibited increased tyrosine phosphorylation. Tyrosine phosphorylation of both PKCdelta and PYK2 was concentration-dependent with respect to carbachol, and rapid, reaching maximal levels by 5 min of treatment. Exposure of acinar cells to phorbol myristate acetate (PMA), a phorbol ester activator of PKCdelta, also resulted in increased phosphorylation of PKCdelta and PYK2 isolated using anti-PKCdelta immunoprecipitation. These results are suggestive of a physical and functional interaction between PKCdelta and PYK2 following muscarinic stimulation in the pancreatic acinar cell.     

PKCdelta and MAPK mediate G(1) arrest induced by PMA in SKBR-3 breast cancer cells

The effects of activating endogenous protein kinase C (PKC) on cell proliferation and the cell cycle were investigated by treating the breast cancer cell line SKBR-3 with phorbol 12-myristate 13 acetate (PMA). This inhibited cell growth in a concentration-dependent manner, causing a marked arrest of cells in G(1). Pre-treatment with GF109203X completely blocked the antiproliferative effect of PMA, and pre-treatment with the PKCdelta inhibitor rottlerin partially blocked it. Infecting SKBR-3 cells with an adenovirus vector containing wild-type PKCdelta, WTPKCdeltaAdV, had similar effects on PMA. Infecting the cells with a dominant-negative PKCdeltaAdV construct blocked the growth inhibition induced by PMA. Downstream of PKC, PMA treatment inhibited extracellular signal-regulated kinase mitogen-activated protein kinase phosphorylation, up-regulated c-jun NH(2)-terminal kinase phosphorylation, and inhibited retinoblastoma (Rb) phosphorylation. These results strongly implicated PKC (mainly PKCdelta) in the G(1) arrest induced by PMA and suggested PKC as a target for breast cancer treatment.     

CD45 negatively regulates monocytic cell differentiation by inhibiting phorbol 12-myristate 13-acetate-dependent activation and tyrosine phosphorylation of protein kinase Cdelta

The protein-tyrosine phosphatase CD45 is expressed on all monocytic cells, but its function in these cells is not well defined. Here we report that CD45 negatively regulates monocyte differentiation by inhibiting phorbol 12-myristate 13-acetate (PMA)-dependent activation of protein kinase C (PKC) delta. We found that antisense reduction of CD45 in U937 monocytic cells (CD45as cells) increased by 100% the ability of PMA to enlarge cell size, increase cell cytoplasmic process width and length, and induce surface expression of CD11b. In addition, reduction in CD45 expression caused the duration of peak PMA-induced MEK and extracellular signal-regulated kinase (ERK) 1/2 activity to increase from 5 min to 30 min while leading to a 4-fold increase in PMA-dependent PKCdelta activation. Importantly, PMA-dependent tyrosine phosphorylation of PKCdelta was also increased 4-fold in CD45as cells. Finally, inhibitors of MEK (PD98059) and PKCdelta (rottlerin) completely blocked PMA-induced monocytic cell differentiation. Taken together, these data indicate that CD45 inhibits PMA-dependent PKCdelta activation by impeding PMA-dependent PKCdelta tyrosine phosphorylation. Furthermore, this blunting of PKCdelta activation leads to an inhibition of PKCdelta-dependent activation of ERK1/2 and ERK1/2-dependent monocyte differentiation. These findings suggest that CD45 is a critical regulator of monocytic cell development.     

PKCdelta-dependent cleavage and nuclear translocation of annexin A1 by phorbol 12-myristate 13-acetate.

Annexin A1 (ANX-1), a calcium-dependent, phospholipid binding protein, is known to be involved in diverse cellular processes, including regulation of cell growth and differentiation, apoptosis, and inflammation. The mitogen phorbol 12-myristate 13-acetate (PMA) induces expression and phosphorylation of ANX-1. However, the roles of ANX-1 in PMA-induced signal transduction is unknown. Here, we study the cellular localization of ANX-1 in the PMA-induced signal transduction process. We have found that PMA induces the cleavage of ANX-1 in human embryonic kidney (HEK) 293 cells, and that the cleaved form of ANX-1 translocates to the nucleus. The PMA-induced nuclear translocation of ANX-1 was inhibited by the protein kinase C (PKC)delta-specific inhibitor rottlerin, indicating that PKCdelta plays a role in nuclear translocation of the cleaved ANX-1. We propose a novel mechanism of PMA-induced translocation of ANX-1 to the nucleus that may participate in the regulation of cell proliferation and differentiation.     

Phorbol 12-myristate 13-acetate induces epidermal growth factor receptor transactivation via protein kinase Cdelta/c-Src pathways in glioblastoma cells

Both the epidermal growth factor receptor (EGFR) and protein kinase C (PKC) play important roles in glioblastoma invasive growth; however, the interaction between the EGFR and PKC is not well characterized in glioblastomas. Treatment with EGF stimulated global phosphorylation of the EGFR at Tyr(845), Tyr(992), Tyr(1068), and Tyr(1045) in glioblastoma cell lines (U-1242 MG and U-87 MG). Interestingly, phorbol 12-myristate 13-acetate (PMA) stimulated phosphorylation of the EGFR only at Tyr(1068) in the two glioblastoma cell lines. Phosphorylation of the EGFR at Tyr(1068) was not detected in normal human astrocytes treated with the phorbol ester. PMA-induced phosphorylation of the EGFR at Tyr(1068) was blocked by bisindolylmaleimide (BIM), a PKC inhibitor, and rottlerin, a PKCdelta-specific inhibitor. In contrast, Go 6976, an inhibitor of classical PKC isozymes, had no effect on PMA-induced EGFR phosphorylation. Furthermore, gene silencing with PKCdelta small interfering RNA (siRNA), siRNA against c-Src, and mutant c-Src(S12C/S48A) and treatment with a c-Src inhibitor (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine) abrogated PMA-induced EGFR phosphorylation at Tyr(1068). PMA induced serine/threonine phosphorylation of Src, which was blocked by both BIM and rottlerin. Inhibition of the EGFR with AG 1478 did not significantly alter PMA-induced EGFR Tyr(1068) phosphorylation, but completely blocked EGF-induced phosphorylation of the EGFR. The effects of PMA on MAPK phosphorylation and glioblastoma cell proliferation were reduced by BIM, rottlerin, the MEK inhibitor U0126, and PKCdelta and c-Src siRNAs. Taken together, our data demonstrate that PMA transactivates the EGFR and increases cell proliferation by activating the PKCdelta/c-Src pathway in glioblastomas.     

IL-4-dependent CD86 expression requires JAK/STAT6 activation and is negatively regulated by PKCdelta

CD86 expression is up-regulated in activated monocytes and macrophages by a mechanism that is not clearly defined. Here, we report that IL-4-dependent CD86 expression requires activation of ERK1/2 and JAK/STAT6 but is negatively regulated by PKCdelta. PMA differentiated U937 monocytic cells when stimulated with IL-4 increased CD11b and CD86 expression by 52- and 98-fold, respectively. PMA+IL-4 treatment also induced a synergistic enhancement of ERK1/2 activation when compared to the effects of PMA and IL-4 alone. Use of the mitogen or extracellular kinase (MEK) inhibitor, PD98059, completely blocked up-regulation of CD11b and CD86 demonstrating the importance of MEK-activated ERK1/2. JAK inhibition with WHI-P154-abrogated IL-4-dependent CD11b and CD86 up-regulation and inhibited STAT6 tyrosine phosphorylation. Importantly, CD11b and CD86 expression were not reliant on IL-4-dependent activation of phosphatidylinositol 3'-kinase (PI 3-kinase). Blockade of PKCdelta activation with rottlerin prevented CD11b expression but lead to a 75- and 213-fold increase in PMA and PMA+IL-4-dependent CD86 expression, respectively. As anticipated, increasing PKCdelta activity with anti-sense reduction of CD45 increased CD11b expression and reduced CD86 expression. Likewise, rottlerin prevented nuclear localization of activated PKCdelta. We conclude from these data that IL-4-dependent CD11b expression relies predominantly on enhanced activation of ERK1/2, while IL-4-dependent CD86 expression utilizes the JAK/STAT6 pathway.     

Novel phosphorylation site markers of protein kinase C delta activation

Protein kinase C delta (PKCdelta) is a Ser/Thr kinase which regulates numerous cellular processes, including proliferation, differentiation, migration and apoptosis. Here, we demonstrate that PKCdelta undergoes in vitro autophosphorylation at three sites within its V3 region (S299, S302, S304), each of which is unique to this PKC isoform and evolutionarily conserved. We demonstrate that S299 and S304 can be phosphorylated in mammalian cells following phorbol ester stimulation and that S299-phosphorylated PKCdelta is localised to both the plasma and nuclear membranes. These data indicate that PKCdelta is phosphorylated upon activation and that phospho-S299 represents a useful marker of the activated enzyme.     

Protein kinase CbetaI interacts with the beta1-adrenergic signaling pathway to attenuate lipolysis in rat adipocytes

We have shown previously that insulin attenuates beta1-adrenergic receptor (beta1-AR)-mediated lipolysis via activation of protein kinase C (PKC) in rat adipocytes. This antilipolysis persists after removal of insulin and is independent of the phosphodiesterase 3B activity, and phorbol 12-myristate 13-acetate (PMA) could substitute for insulin to produce the same effect. Here, we attempted to identify the PKC isoform responsible for antilipolysis. Isolated adipocytes were treated with high and low concentrations of PMA for up to 6 h to degrade specific PKC isoforms. In the PMA-treated cells, the downregulation profiles of PKC isoforms alpha and betaI, but not betaII, delta, epsilon, or zeta, correlated well with a decrease of lipolysis-attenuating effect of PMA. After rats fasted for 24 h, adipocyte expression of PKC isoform alpha increased, while expression of PKCdelta decreased. Fasting did not change the potency of PMA to attenuate lipolysis, however. The lipolysis-attenuating effect of PMA was blocked by the PKCbetaI/betaII inhibitor LY 333531, but not by the PKCbetaII inhibitor CGP 53353 or the PKCdelta inhibitor rottlerin. These data suggest that PKCbetaI interacts with beta1-AR signaling and attenuates lipolysis in rat adipocytes.     

Molecular mechanisms of protein kinase C-induced apoptosis in prostate cancer cells

Protein kinase C (PKC) isozymes, a family of serine-threonine kinases, are important regulators of cell proliferation and malignant transformation. Phorbol esters, the prototype PKC activators, cause PKC translocation to the plasma membrane in prostate cancer cells, and trigger an apoptotic response. Studies in recent years have determined that each member of the PKC family exerts different effects on apoptotic or survival pathways. PKCdelta, one of the novel PKCs, is a key player of the apoptotic response via the activation of the p38 MAPK pathway. Studies using RNAi revealed that depletion of PKCdelta totally abolishes the apoptotic effect of the phorbol ester PMA. Activation of the classical PKCalpha promotes the dephosphorylation and inactivation of the survival kinase Akt. Studies have assigned a pro-survival role to PKCepsilon, but the function of this PKC isozyme remains controversial. Recently, it has been determined that the PKC apoptotic effect in androgen-dependent prostate cancer cells is mediated by the autocrine secretion of death factors. PKCdelta stimulates the release of TNFalpha from the plasma membrane, and blockade of TNFalpha secretion or TNFalpha receptors abrogates the apoptotic response of PMA. Molecular analysis indicates the requirement of the extrinsic apoptotic cascade via the activation of death receptors and caspase-8. Dissecting the pathways downstream of PKC isozymes represents a major challenge to understanding the molecular basis of phorbol ester-induced apoptosis.     

Diacylglycerol (DAG)-lactones, a new class of protein kinase C (PKC) agonists, induce apoptosis in LNCaP prostate cancer cells by selective activation of PKCalpha

Phorbol esters, the archetypical (PKC) activators, induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. In this study we evaluate the effect of a novel class of PKC ligands, the diacylglycerol (DAG)-lactones, as inducers of apoptosis in LNCaP cells. These unique ligands were designed using novel pharmacophore- and receptor-guided approaches to achieve highly potent DAG surrogates. Two of these compounds, HK434 and HK654, induced apoptosis in LNCaP cells with much higher potency than oleoyl-acetyl-glycerol or phorbol 12,13-dibutyrate. Moreover, different PKC isozymes were found to mediate the apoptotic effect of phorbol 12-myristate 13-acetate (PMA) and HK654 in LNCaP cells. Using PKC inhibitors and dominant negative PKC isoforms, we found that both PKCalpha and PKCdelta mediated the apoptotic effect of PMA, whereas only PKCalpha was involved in the effect of the DAG-lactone. The PKCalpha selectivity of HK654 in LNCaP cells contrasts with similar potencies in vitro for binding and activation of PKCalpha and PKCdelta. Consistent with the differences in isoform dependence in intact cells, PMA and HK654 show marked differences in their abilities to translocate PKC isozymes. Both PMA and HK654 induce a marked redistribution of PKCalpha to the plasma membrane. On the other hand, unlike PMA, HK654 translocates PKCdelta predominantly to the nuclear membrane. Thus, DAG-lactones have a unique profile of activation of PKC isozymes for inducing apoptosis in LNCaP cells and represent the first example of a selective activator of a classical PKC in cellular models. An attractive hypothesis is that selective activation of PKC isozymes by pharmacological agents in cells can be achieved by differential intracellular targeting of each PKC.     

Activation of acid sphingomyelinase by protein kinase Cdelta-mediated phosphorylation

Although important for cellular stress signaling pathways, the molecular mechanisms of acid sphingomyelinase (ASMase) activation remain poorly understood. Previous studies showed that treatment of MCF-7 mammary carcinoma cells with the potent protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate (PMA), induces a transient drop in sphingomyelin concomitant with an increase in cellular ceramide levels (Becker, K. P., Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., and Hannun, Y. A. (2005) J. Biol. Chem. 280, 2606-2612). Here we show that PMA selectively activates ASMase and that ASMase accounts for the majority of PMA-induced ceramide. Pharmacologic inhibition and RNA interference experiments indicated that the novel PKC, PKCdelta, is required for ASMase activation. Immunoprecipitation experiments revealed the formation of a novel PKCdelta-ASMase complex after PMA stimulation, and PKCdelta was able to phosphorylate ASMase in vitro and in cells. Using site-directed mutagenesis, we identify serine 508 as the key residue phosphorylated in response to PMA. Phosphorylation of Ser(508) proved to be an indispensable step for ASMase activation and membrane translocation in response to PMA. The relevance of the proposed mechanism of ASMase regulation is further validated in a model of UV radiation. UV radiation also induced phosphorylation of ASMase at serine 508. Moreover, when transiently overexpressed, ASMase(S508A) blocked the ceramide formation after PMA treatment, suggesting a dominant negative function for this mutant. Taken together, these results establish a novel direct biochemical mechanism for ASMase activation in which PKCdelta serves as a key upstream kinase.     

B-cell receptor- and phorbol ester-induced NF-kappaB and c-Jun N-terminal kinase activation in B cells requires novel protein kinase C's

Antigen receptor signaling is known to activate NF-kappaB in lymphocytes. While T-cell-receptor-induced NF-kappaB activation critically depends on novel protein kinase C theta (PKCtheta), the role of novel PKCs in B-cell stimulation has not been elucidated. In primary murine splenic B cells, we found high expression of the novel PKCs delta and epsilon but only weak expression of the theta isoform. Rottlerin blocks phorbol ester (phorbol myristate acetate [PMA])- or B-cell receptor (BCR)-mediated NF-kappaB and c-Jun N-terminal kinase (JNK) activation in primary B and T cells to a similar extent, suggesting that novel PKCs are positive regulators of signaling in hematopoietic cells. Mouse 70Z/3 pre-B cells have been widely used as a model for NF-kappaB activation in B cells. Similar to the situation in splenic B cells, rottlerin inhibits BCR and PMA stimulation of NF-kappaB in 70Z/3 cells. A derivative of 70Z/3 cells, 1.3E2 cells, are defective in NF-kappaB activation due to the lack of the IkappaB kinase (IKKgamma) protein. Ectopic expression of IKKgamma can rescue NF-kappaB activation in response to lipopolysaccharides (LPS) and interleukin-1beta (IL-1beta), but not to PMA. In addition, PMA-induced activation of the mitogen-activated protein kinase JNK is blocked in 1.3E2 cells, suggesting that an upstream component common to both pathways is either missing or mutated. Analysis of various PKC isoforms revealed that exclusively PKCtheta was absent in 1.3E2 cells while it was expressed in 70Z/3 cells. Stable expression of either novel PKCtheta or -delta but not classical PKCbetaII in 1.3E2 IKKgamma-expressing cells rescues PMA activation of NF-kappaB and JNK signaling, demonstrating a critical role of novel PKCs for B-cell activation.     

Tumor-promoting phorbol esters stimulate the proliferation of interleukin-3 dependent cells

To determine whether activation of protein kinase C is involved in the proliferation of interleukin-3 (IL-3) -dependent cells, we examined the effect of tumor-promoting phorbol esters on the in vitro proliferation of the IL-3-dependent cell lines FD and DA-1. The viability of FD and DA-1 cells cultured for 24 hours in 100 nM phorbol myristate acetate (PMA) and 10% FCS was similar to that of cells cultured in 25% WEHI-3 conditioned medium as a source of IL-3, and 10% FCS. FD cells failed to proliferate in concentrations of FCS of up to 50%, while DA-1 cell proliferation was not markedly influenced by FCS. By contrast, PMA promoted the proliferation of FD and DA-1 cells in the absence of FCS and enhanced their proliferation in the presence of 10% FCS, 60- and 20-fold, respectively. Stimulation of proliferation was achieved with as little as 10 nM PMA and was maximal at 100 nM PMA. Low concentrations (0.05-0.1%) of WEHI-3 CM promoted the proliferative response of FD and DA-1 cells to PMA, but at concentrations of WEHI-3 CM greater than 0.8%, no further increment in proliferation was obtained with PMA. As little as 1/2 hour of exposure to phorbol esters was sufficient to cause translocation of protein kinase C from the cytosol to the membranes of DA-1 cells, and 1 hour of exposure to phorbol esters was sufficient to stimulate DNA synthesis. A protein kinase C inhibitor, H-7, at a concentration of 10 microM inhibited phorbol ester-induced stimulation of DA-1 cell proliferation. When DA-1 cells were exposed to the calcium ionophore A23187 in addition to both a phorbol ester and IL-3, their proliferation was enhanced over that stimulated by only the phorbol ester and IL-3. The data indicate that stimulation of proliferation of IL-3-dependent cells involves the activation of protein kinase C.     

Stimulation of protein kinase C modulates insulin-like growth factor-1-induced akt activation in PC12 cells

Activation of protein kinase C (PKC) plays an important role in the negative regulation of receptor signaling, but its effect on insulin-like growth factor-1 (IGF-1) receptor signaling remains unclear. In this study, we characterized the intracellular pathways involved in IGF-1-induced activation of Akt and evaluated the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on the Akt activation by IGF-1. IGF-1 induced a time- and concentration-dependent activation of Akt. The effect of IGF-1 was blocked by the phosphatidylinositide 3-kinase (PI3K) inhibitors LY294002 (50 micrometer) and wortmannin (0.5 micrometer), but not by the MEK inhibitor PD98059 (50 micrometer) or the p70 S6 kinase pathway inhibitor rapamycin (50 nm), suggesting that the stimulation of Akt by IGF-1 is mediated by the PI3K pathway. Interestingly, cotreatment with PMA (400 nm) attenuated IGF-1-induced activation of Akt. The attenuation was blocked completely by the PKC inhibitor GO6983 (0.5 micrometer), but only partially by the MEK inhibitor PD98059 (50 micrometer), indicating that MAPK-dependent and -independent pathways are involved. PMA induced the activation of PKC in PC12 cells, and this induction was blocked by GO6983. These data further support the role of PKC in the effect of PMA. Moreover, PKCdelta is likely involved in the action of PMA on the basis of data obtained using isoform-specific inhibitors such as rottlerin. PMA also decreased IGF-1-induced tyrosine phosphorylation of insulin receptor substrate-1 and its association with PI3K. Taken together, these results suggest, for the first time, that stimulation of PKC modulates IGF-1-induced activation of Akt.